Citation
Centaurin-Alpha 1

Material Information

Title:
Centaurin-Alpha 1 function and sequence analysis of an ap-1 pathway activator
Creator:
Littrell Miller, BobbiJo Rose
Publication Date:
Language:
English
Physical Description:
ix, 67 leaves : ; 28 cm

Subjects

Subjects / Keywords:
Nerve tissue proteins ( lcsh )
Cellular signal transduction ( lcsh )
Carrier proteins ( lcsh )
Carrier proteins ( fast )
Cellular signal transduction ( fast )
Nerve tissue proteins ( fast )
Centaurin-Alpha 1
AP-1 Pathway activator
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 63-67).
General Note:
Department of Integrative Biology
Statement of Responsibility:
by BobbiJo Rose Littrell Miller.

Record Information

Source Institution:
|University of Colorado Denver
Holding Location:
|Auraria Library
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
463452518 ( OCLC )
ocn463452518
Classification:
LD1193.L45 2009m L57 ( lcc )

Full Text
CENTAURIN-ALPHA 1:
FUNCTION AND SEQUENCE ANALYSIS OF AN
AP-1 PATHWAY ACTIVATOR
by
BobbiJo Rose Littrell-Miller
B.S., Metropolitan State College of Denver, 2006
B.A., Metropolitan State College of Denver, 2006
A thesis submitted to the
University of Colorado Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Biology
2009


This thesis for the Master of Science
degree by
BobbiJo Littrell-Miller
has been approved
by
1-JZ2-Ql
Date


Littrell-Miller, BobbiJo Rose (M S., Biology)
Centaurin-Alpha 1: Function and Sequence Analysis of an AP-1 Pathway Activator
Thesis directed by Assistant Professor Lisa K. Johansen
ABSTRACT
Centaurin-Alphal (Cental), a 43 kDa phosphatidylinositol 3,4,5-trisphosphate
(PIP3) binding protein, is a potent activator of the AP-1 pathway. Deregulation of the
AP-1 pathway results in oncogenesis. Cental possesses a zinc finger, ARF GAP
(ADP-ribosylating factor GTPase activating protein), and two PH (pleckstrin
homology) domains. We questioned the involvement of Centals structural domains
in the proteins ability to activate the AP-1 pathway and initiate cellular
transformation. Mutation of Centals two PH domains has previously been shown to
inhibit binding of PIP3. We analyzed the AP-1 and transformation capabilities of two
previously-constructed Cental variants possessing R149C and R273C substitutions,
one of which also contained an N-terminal myristoylation signal. A third Cental
variant lacked the entire zinc finger domain (125 amino acids), which has previously
been shown to prevent Cental from entering the nucleus. The ability of the three
Cental variants to initiate the AP-1 pathway and induce cellular transformation was
determined via cell culture reporter, growth curve, and soft agar colony formation


assays. All variants were unable to activate the AP-1 pathway. Cells transfected with
the PH variants exhibited a reduced degree of transformation compared to wild-type
Cental. We also examined Centals protein sequence in search of post-translational
modifications, specifically phosphorylations. Cell-free kinase assays have
demonstrated that Protein Kinase C phosphorylates Cental at Serine 87 and
Threonine 276. We treated HEK cells stably expressing Cental with strong
phosphatase inhibitors or with a PKC-activating agent, isolated Cental, and analyzed
the protein using high performance liquid chromatography (HPLC) and mass
spectrometry. Using an ETD ion trap and QTOF mass spectrometers, we performed
both MS and MS/MS analysis. We report the presence of a phosphate group at
Tyrosine 364. Our results contribute to the knowledge of a protein involved in critical
cell growth processes and possess possible implications for human cancer research.
This abstract accurately represents the content of the candidates thesis. I recommend
its publication.
Signed
Lisa K. Johansen


ACKNOWLEDGMENTS
I wish to thank Rick and Nichole Reisdorph, of the National Jewish Health
Proteomics and Genomics Core Facility, for their vast expertise in things both
proteomic and molecular, as well as their guidance, patience, and desire to help me
leam during my time there. Also, thanks go to Roger Powell for his endless patience
and willingness to help me improve my laboratory skills. Finally, I wish to thank
Mike, Carolyn, and Spencer for putting up with me, answering random questions, and
making things fun.
I also wish to acknowledge that this thesis continues work begun by Rick
Reisdorph and Roger Powell, and as such contains, with their permission, the
unpublished results of experiments they have performed.


TABLE OF CONTENTS
Figures....................................................................viii
Tables.....................................................................ix
Chapter
1. Introduction...........................................................I
1.1 AP-1 Pathway...........................................................1
1.2 Centaurin-Alphal Structure and Function................................3
1.3 Scope of Study.........................................................9
2. AP-1 Activity and Cellular Transformation.............................11
2.1 Introduction..........................................................11
2.2 Methods and Materials.................................................13
2.2.1 Molecular cloning.....................................................13
2.2.2 Cell Culture..........................................................16
2.2.3 Western Blot..........................................................19
2.2.4 Data Analysis.........................................................21
2.3 Results...............................................................21
2.3.1 Deletion of the Zinc Finger and Modification of Both PH Domains Inhibit
Cental-Mediated AP-1 Activation.......................................21
2.3.2 Two PH Variants Exhibit Decreased Potential for
vi


Cellular Transformation
23
2.4 Discussion..............................................................27
3. Proteomic Analysis of Post-Translational Modifications..................31
3.1 Introduction............................................................31
3.2 Methods and Materials...................................................33
3.2.1 Cell Culture............................................................33
3.2.2 Protein Isolation, Verification, and Sample Preparation.................34
3.2.3 Mass Spectrometry.......................................................37
3.2.4 Data Analysis...........................................................40
3.3 Results.................................................................41
3.3.1 Cental is Phosphorylated in Vivo at Tyrosine 364........................41
3.4 Discussion..............................................................50
4. General Discussion and Summary..........................................53
Appendix
A Abbreviations...........................................................60
References....................................................................63
vii


LIST OF FIGURES
Figure 1 Diagram of Centaurin-Alpha 1 Protein...........................4
Figure 2 Activation of the AP-1 Pathway.................................7
Figure 3 Mutations in Centals PH Domains Inhibit AP-1 Activation.......8
Figure 4 Cental Structural Variants Used in AP-1 and Transformation Assays.13
Figure 5 Chapter 2 Methodologies.......................................15
Figure 6 Levels of AP-1 Activity in Mammalian Cells Transfected with Cental
Structural Mutants..............................................22
Figure 7 Growth of Avian Cells Infected with Cental Variants...........25
Figure 8 Colonies Formed by Infected DF-1 Cells in Soft Agar Media.....26
Figure 9 Effect of PI and PMA Treatment on Phosphorylation Status of ERK 1/2 ... 41
Figure 10 Phosphopeptide AVDRPmLPQEyAVEAHFK Identified in Targeted MS
and MS/MS Spectra...............................................46
Figure 11 Un-phosphorylated Peptide AVDRPmLPQEYAVEAHFK Identified in
Targeted MS and MS/MS Spectra...................................47
Figure 12 Phosphorylated and Un-phosphorylated Cental Peptides.........49
Figure 13 Cental Structure Containing Known Phosphorylation Sites......52
viii


LIST OF TABLES
Table 1 HPLC nano pump buffer B gradient for peptide elution prior to ion trap and
QTOF analysis..............................................................38
Table 2 Instrument parameters......................................................39
Table 3 Database search results for targeted MS/MS analysis of phosphatase
inhibitor-treated Cental samples...........................................48
IX


1.
Introduction
1.1 AP-1 Pathway
The lifecycle of a cell is tightly regulated by many intracellular signaling
pathways. These pathways, stimulated by extracellular molecules or events and
directed by cascades of intracellular interactions, drive responses that regulate normal
cell processes [1]. The end result is cell growth, division, movement, apoptosis, and
other key events. Of particular interest are the cellular processes that transform
healthy cells into cancerous ones, since understanding these pathways and their
mediators will result in better understanding of the progression of, and possible
treatments for cancer. One cell signaling pathway for which deregulation has been
linked to the development of breast, colon, bone, and liver tumors [2-5] is the AP-1
pathway, a regulatory pathway mediated by the Activator Protein 1 (AP-1)1
transcription factor.
Activation of the AP-1 pathway is considered a universal response, in that it is
initiated by a variety of stimuli, including growth factors, cytokines, and ultraviolet
radiation [6], Activation of the pathway results in expression and subsequent
activation of the AP-1 transcription factor. The dimeric AP-1 protein exists in
multiple forms via the homo- or hetero-dimerization of Jun, Fos, or ATF oncoproteins
1 A list of abbreviations and their definitions is included in Appendix A.


[7, 8]. Depending on dimer composition, the AP-1 protein then binds one of several
palindromic DNA consensus sequences called AP-1 sites [6, 8], These consensus
sequences are found in promoters throughout the genome, where binding of the AP-1
transcription factor regulates expression of genes involved in cell proliferation, cell
survival, or cell death [5, 9, 10],
In an attempt to identify molecules involved in AP-1 pathway signal
transduction, Chanda et al. screened mammalian genomic cDNAs for products
capable of activating the pathway [10]. The authors co-transfected mammalian cells
with cDNAs and a reporter construct under the control of an AP-1 DNA sequence.
From levels of reporter gene expression, 25 specific AP-1 pathway activators were
identified, nearly half of which were found to be involved in cell growth and
proliferation. Since previous works have shown successful AP-1 -mediated
transformation of avian cells [11]; Chanda et al. also used avian cells to determine the
transformative potential of their growth-related activators. Transfection of chicken
cells with several of these cDNAs resulted in morphologies associated with
transformed cells: specifically, increased cell proliferation and anchorage
independence. One AP-1 activator that was found to promote a strong transformation
response is the protein Centaurin-Alphal.
2


1.2 Centaurin-Alphal Structure and Function
Centaurin- Alpha 1, also known as PIPBP, p42IP4, AD API, or Cental, was
identified in 1999 as the human homologue of a phosphatidylinositol 3,4,5-
trisphosphate (PIP3) binding protein found in rats [12], Centaurin-Alpha proteins are
highly expressed in the brain, and are also expressed in the spleen, kidney, and lungs
[13], Over the past decade, Cental has been well-studied for its role in actin
rearrangement and GTPase regulation [14-17], Cental has also been indicated in
activation of extracellular signal-regulated kinases (ERKs) and their signaling
pathways, as well as in nuclear organization [18, 19], From Chanda [10], it is now
known that Cental is a potent activator of the AP-1 pathway, the activation of which
leads to cellular transformation and tumor development. Little is known about
Centals role in these events. The presence of multiple structural domains obscure
Centals exact role in the AP-1 pathway, necessitating study of the individual
domains in order to piece together a broader picture of its involvement. Elucidating
Centals functions will not only provide new information on the protein, this
information will conceivably allow better understanding of the AP-1 pathway and the
processes that turn a healthy cell into a tumor cell.
Like other signaling proteins, Cental possesses two pleckstrin homology (PH)
domains (Figure 1) for membrane binding. Each PH domain is approximately 100
amino acids in length and is folded into a conserved secondary structure that interacts
with various phosphatidylinositol lipids [20], The two PH domains found in Cental
3


are required for co-localization and binding of Cental to the membrane-bound lipid
PIP3 [21]. Mutations in one or both domains decrease or inhibit the protein-lipid
interactions necessary for membrane localization [17].
1-117/ 126
Zn Finger/ARF GAP
130-231_____________253-357
N PH C PH
Figure 1 Diagram of Centaurin-Alphal Protein
Cental contains an ARF GAP domain (residues 1-126), which encompasses a zinc finger motif (1-
117) and a possible, still undefined nuclear localization signal (not shown). Two structurally conserved
PH domains (130-231,253-357) are required for protein-lipid binding. Domain delineations are
reported in [12].
In addition to its PH domains, Cental contains a region found in ADP-
ribosylating factor GTPase-activating proteins (ARF GAPs). ADP-ribosylating
factors (ARFs) are G proteins typically involved in such cellular events as membrane-
vesicle trafficking and actin rearrangement [16, 22], In vivo ARF GAP activity has
been established for Cental in association with the membrane regulator ARF6;
Cental co-localizes to the plasma membrane with ARF6 and inactivates the G protein
through GTP hydrolysis [16]. As with similar ARF GAP proteins [17, 23], Centals
GAP domain consists primarily of a zinc finger (ZF) motif [24, 25]. While zinc finger
motifs are typically associated with protein-DNA binding [26], they also function in
protein-protein interactions [27], Association between Centals zinc finger and
KIF13B, a kinesin motor protein, facilitates membrane localization [28], while
mutation of conserved residues eliminate Centals ability to regulate ARF6 [16]. This
4


evidence suggests that the zinc finger is important for both Centals translocation to
the plasma membrane and its ARF GAP capabilities.
Consistent with its multiple activities, Cental is located throughout the cell,
including the nucleus. Currently, no definitive nuclear localization signal (NLS) has
been reported. Research has suggested that the first seven or nine amino acids may be
part of an NLS, but contradictory evidence precludes that classification [18, 29], The
evidence suggests, however, that there is some amino acid sequence or structure in
Centals N-terminal region that is required for nuclear entry. To date, the only
reported and reproducible method of inhibiting Centals nuclear localization is the
deletion of the first 125 N-terminal amino acids [28].
Together, the functions associated with Centals various structural domains
provide information regarding the proteins intracellular movements and ARF
regulation but fail to clarify Centals involvement in the AP-1 pathway. A recent
study involving the mitogen-activated protein kinase (MAPK) signaling pathway [ 19]
has allowed us to incorporate Cental into the known pathway, even if we do not yet
understand Centals exact involvement. Figure 2 presents a basic schematic of how
extracellular stimuli induce activation of the ERK kinases, which are two MAPK
mediators, and result in expression of the AP-1 transcription factor. In cells, over-
expression of both Cental and ERK 1/2 results in localization of Cental to the plasma
membrane and subsequent activation of the ERKs [19]. Centals role in activating the
ERKs is unknown, and as such the connection between the two is represented by a
5


dashed line in the figure. Once activated, the ERKs phosphorylate various
transcription factors, such as Elk-1, which enter the nucleus and induce expression of
the c-fos gene [30], The c-Fos protein then associates with a member of the same
oncoprotein family, or that of another family such as Jun or ATF, and forms one
version of an AP-1 dimer. The dimer binds a specific AP-1 DNA consensus
sequence, regulating activity of promoters containing the AP-1 sequence and
subsequent expression of a variety of genes. This scenario describes how extracellular
stimuli and phosphoinositide signaling events initiate the AP-1 pathway, but also may
explain how, through increased production of the AP-1 transcription factor subunits,
over-expression of Cental perpetuates AP-1 pathway activation.
6


Extracellular Stimuli
(UV, Growth factor, cytokines)
Figure 2 Activation of the AP-1 Pathway
Extracellular stimuli initiate cell signaling events which activate phosphoinositide 3-kinase (PI3K).
The result is increased production of the membrane lipid PIP3. Cental co-localizes to PIP3 at the
plasma membrane and through an unknown mechanism induces activation of ERK 1/2 (represented by
a dashed line). Activated ERKs phosphorylate and activate transcription factors, such as Elk-1, which
travel to the nucleus and induce expression of the c-fos gene. Other AP-1 subunits, such as Jun or ATF,
are expressed through similar mechanisms. The AP-1 subunits form a dimer, which then binds AP-1
DNA sites and regulate genes throughout the genome.
Recent work has sought to define the connection between Centals structure
and its ability to initiate the AP-1 pathway and induce cellular transformation [31].
Reisdorph and Powell questioned the involvement of Centals two PH domains in
Centals ability to activate the AP-1 pathway [31], A Cental variant in which both
PH domains are mutated (R149C and R273C) has previously been shown incapable
7


of binding PIP3 [17]. Using AP-1 reporter assays, Reisdorph and Powell showed that
this same mutant is also incapable of activating the AP-1 pathway (Figure 3). An
identical PH mutant with an additional N-terminal myristoylation signal was similarly
unable to activate the AP-1 pathway. While there are many activators of the AP-1
pathway, these reporter assays indicate that Centals PH domains, or the lipid-
binding capabilities they impart, are required for Cental to perform its AP-l-related
functions.
AP-1 Reporter Assay of Cental
PH Variants
Figure 3 Mutations in Centals PH Domains Inhibit AP-1 Activation
Reisdorph and Powell [31] co-transfected modified Cental cDNAs into mammalian cells along with
an AP-l/luciferase vector and an internal control vector. Normalized values of relative light units
(RLU) indicated levels of AP-1-driven expression of the luciferase gene. The experiment was
performed 3 times, each in triplicate. The asterisk indicates a significant difference between WT
Cental and all other columns, as determined via one-way ANOVA comparison tests. Error bars
represent standard deviation. Cental variants: WT: wild-type; Dbl PH: R149C and R273C
substitutions; Myr Dbl PH: R149C and R273C substitutions plus an N-terminal myristoylation signal;
GFP: green fluorescent protein negative control.
8


1.3 Scope of Study
This study continues the Cental structural analyses begun by Reisdorph and
Powell. Using both proteomic and cellular techniques, we analyzed Centals protein
sequence as well as the involvement of Centals structural domains in induction of
the AP-1 pathway and cellular transformation. Much of Centals known functions,
such as membrane binding and ARF regulation, occur within the cytoplasm or at the
plasma membrane. Therefore, we questioned whether Centals involvement in AP-1
activation was location-dependant. Three Cental variants were examined for their
effects on AP-1 activity and their potential for cellular transformation. Two variants
were previously used by Reisdorph and Powell and possessed mutations in both PH
domains; one of these variants possessed an additional N-terminal myristoylation
signal (consensus sequence: Gly2-X3-X4-X5-(Ser/Thr/Cys)6), the presence of which
directs protein myristoylation and membrane targeting [32, 33]. The third Cental
variant used in our assays was modeled after a 125-amino acid deletion previously
shown to prevent nuclear entry [28], By using these variants, we questioned the
extent to which protein-membrane binding and/or nuclear entry is required for
Cental-mediated AP-1 activation.
In addition to studying Centals intracellular activity, we examined potential
post-translational modifications (PTM) to Cental using high performance liquid
chromatography (HPLC) and mass spectrometry (MS). In vitro kinase assays have
shown that several isoforms of protein kinase C (PKC) are able to phosphorylate
9


Cental, and two phosphorylated residues have been reported: Serine 87 in Centals
zinc finger and Threonine 276 in the C-terminal PH domain [34], In addition to PKC,
Cental is known to associate with casein kinase 1 and potentially ERK 1/2 [14, 19,
34], and Cental is a known activator in the AP-1 signaling pathway. Therefore, we
hypothesized that Cental is phosphorylated in vivo, as are many other signaling
molecules. We sought to confirm the presence of the two reported phosphorylations
and identify any novel phosphorylations that might exist.
10


2. AP-1 Activity and Cellular Transformation
2.1 Introduction
The AP-1 pathway has been implicated in tumor development, yet Centals
role in this process remains unclear. To determine the relationship between Centals
structure, its localization, and the cellular changes Cental mediates, we assayed the
ability of three structural variants to activate the AP-1 pathway and induce cellular
transformation. A reporter assay, in which AP-1-driven expression of a reporter gene
was correlated to levels of AP-1 activity, was performed to determine the effects of
the structural modifications on Centals ability to activate the AP-1 pathway. To
identify the transformative potential of each Cental variant, two common tools for
measuring cellular transformation were used: growth curves and soft agar colony
formation assays. Growth curves illustrate both the increased rate of cell proliferation
and the loss of contact inhibition that are characteristic of tumor cells [35, 36], Soft
agar assays exploit the morphological trait of anchorage independence exhibited by
transformed cells, wherein suspended cells are able to form colonies and grow in a
soft agar medium without attachment to a solid matrix [37], Previous research has
established methods for successful transformation of avian cells [10, 11], whereas
mammalian cells have proven more difficult in obtaining consistent, reproducible
evidence of transformation. However, since this study primarily impacts human
11


cancer research, and the focus remains on the protein Cental, a combination of
mammalian and avian cells were used for AP-1 and transformation assays.
In addition to wild-type Cental, the cellular effects of three Cental variants
(Figure 4) were studied: 1) one variant with substitutions (R149C and R273C) in both
PH domains (Dbl PH); 2) the same PH mutant with an N-terminal myristoylation
signal (Myr Dbl PH); and 3) a variant lacking the 125 N-terminal amino acids
encompassing Centals zinc finger and ARF GAP domains (AZF). By studying these
Cental variants, we questioned whether Centals ability to activate the AP-1 pathway
and induce cellular changes is location-dependent.
Many of Centals known activities and interactions occur at the plasma
membrane or in the cytoplasm; therefore, we hypothesized that localization of Cental
to the nucleus is not required for Cental to perform its AP-1-related functions.
Deletion of the zinc finger prevents nuclear entry. If nuclear entry is not necessary for
Cental activity, we expect the AZF Cental variant to exhibit AP-1 and transformative
effects similar to wild-type Cental. The effects of the Dbl PH and Myr Dbl PH
Cental variants were also analyzed in order to determine whether Cental functions
free in the cytoplasm or in a membrane-bound state.
12


Wild-type C cnta 1 Zn Finger/ARFGAPi NPH C PH
R149C R273C
Double PH mutation (Dbl PH) Zn Finger/ARF GAP |!j|j N PH C PH
Double PH mutation + Myr signal (Myr Dbl PH) R149C R273C
| Zn Finger )ARF GAP HH NPH CPH

A125 amino acids (AZF) H NPH CPH
Figure 4 Cental Structural Variants Used in AP-1 and Transformation Assays
Wild-type Cental, AZF, Dbl PH, or Myr Dbl PH Cental structural variants were studied for their
effects on AP-1 activation and cellular transformation.
2.2 Methods and Materials
2.2.1 Molecular cloning
2.2.1.1 Cloning of AZF Cental into Mammalian and Avian Vectors
The truncated Cental (AZF) was created via polymerase chain reaction (PCR)
using a wild-type Cental cDNA template. Forward and reverse primers incorporated
an EcoRI and Xbal restriction site into the product, respectively. (Forward Cental-
specific primer: 5-GAGAATTCGCGGCCACCTACTC-3; reverse BGF1 primer: 5-
TAGAAGGCACAGTCGAGG-3) AZF Cental cDNA was ligated into the
mammalian vector pcDNA 3.1(-) (Invitrogen, Carlsbad, CA) with a 2:1 insert:vector
ratio, T4 DNA ligase, and appropriate buffer (Fisher Scientific, Pittsburgh, PA) for
one hour at room temperature. We transformed 10 pL of the ligation reaction into 50
pL competent XL1 Blue E. coli (Stratagene, La Jolla, CA) by first incubating the
reaction on ice for 30 minutes and then heat-shocking for 45 seconds at 42C. Next,
13


Luria Bertani (LB) Broth (Becton Dickinson, Franklin Lakes, NJ) was added to the
reaction, which was then shaken at 37C for one hour. The total transformation
volume was plated on agar containing 75 pg/mL ampicillin and incubated overnight
at 37C. Transformants were randomly picked and inoculated into overnight LB
cultures for screening.
We isolated plasmid DNA from the transformed cultures using the Qiagen
mini-prep kit (Qiagen, Valencia, CA) and digested the product with EcoRI and Xbal
restriction endonucleases (New England BioLabs, Ipswich, MA). The digest results
were visualized with agarose gel electrophoresis to identify which cultures contained
the AZF Cental insert. Plasmid preps containing the insert were submitted for DNA
sequencing with vector-specific and internal primers (T7, BGH, and a custom internal
forward primer: 5-TTCTGTTTGCCTGCGAGACG-3). Using a Qiagen plasmid
midi-prep kit, transfection-grade DNA was isolated for the AZF variant, and DNA for
the Dbl PH and Myr Dbl PH variants was isolated from frozen bacterial stocks
provided by Reisdorph and Powell.
To prepare the AZF cDNA for use in avian cell culture, the insert was digested
out of pcDNA with EcoRI and Xbal, separated via gel electrophoresis, and gel
purified. The insert was ligated as described into pBSfil, an adaptor plasmid
containing a multiple cloning site, and transformed into XL1 Blue E. coli.
Recombinant plasmid was isolated using the Qiagen mini-prep kit. AZF Cental was
then digested out of pBSfil with Sfil endonuclease (New England BioLabs),
14


separated via gel electrophoresis, and gel purified. Concurrently, whole RCASBP(A)
avian retroviral vector was also digested with Sfil. AZF Cental was ligated into the
RCAS vector with a 6:1 insert:vector ratio. Ten microliters of the ligation reaction
was transformed into 100 pL CaCl2-treated XL 10 Gold E. coli (Stratagene), a
bacterial strain suitable for large plasmid uptake. Transformed colonies were screened
for insert via plasmid isolation, Sfil digest, and gel electrophoresis, and were
subsequently used to inoculate 50 mL overnight cultures for transfection-grade
plasmid isolation. RCAS cloning for the two PH variants had been previously
performed by Reisdorph and Powell, and fresh plasmid preps were performed using
their frozen bacterial stocks. Figure 5 illustrates the key methodologies used in this
chapter.
Figure 5 Chapter 2 Methodologies
Cental cDNA variants were created via PCR, cloned into mammalian (pcDNA3.l) and avian (RCAS)
vectors, and transfected into cell culture. Transfected cells were assayed to determine the effects of the
variants on AP-1 activity and cellular transformation.
15


2.2.2 Cell Culture
2.2.2.1 Transfection of Human Embryonal Kidney Cells
Human embryonal kidney cells (HEK 293) were cultured in Dulbeccos modified
Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin (Mediatech, Inc., Manassas, VA) and were incubated at 37C
with 5% CO2. Cells were seeded in a 24-well tissue culture dish at 2 x 105 cells/well,
in antibiotic-free DMEM plus 10% FBS. Following Invitrogens recommended
protocol, confluent cells (60-80%) were covered with 200 pL serum-free (SF),
antibiotic-free media. DNAs were mixed with SF media in the following quantities:
1)10 ng/well CMV B-galactosidase vector; 2) 100 ng/well TRE-luciferase vector; and
3) 275 ng/well of pcDNA, GFPpcDNA, or wild-type, AZF, Dbl PH, and Myr Dbl PH
Cental in pcDNA. Four microliters Plus Reagent (Invitrogen) was added to each
reaction and incubated for 15 minutes at room temperature. A 1:1 ratio of
Lipofectamine solution (1 pL Lipofectamine per 25 pL SF DMEM) was then added
and the mixture incubated for 15 minutes at room temperature. One-third of the total
reaction volume was added to each well. Transfected cells were incubated at 37C for
three to five hours, after which a 1:1 ratio of antibiotic-free media containing 20%
FBS was added to each well.
2.2.2.2 AP-1 Reporter Assay
Twenty-four hours post-transfection, HEK 293 cells were harvested from each
well and lysed in 75 pL room temperature Glo Lysis Buffer (Promega, Madison, WI).
16


B-galactosidase and luciferase assays were performed on 30 pL cell lysate using the
B-galactosidase Enzyme Assay System and the Steady Glo Luciferase Assay System
(Promega), respectively. Light output representative of luciferase activity was
y
measured with a 10-second luminometry protocol on a Wallac Victor 1420 plate
reader. The CMV (3-galactosidase vector was used as an internal control to indicate
transfection efficiency; P-galactosidase activity was measured with the Wallac
instrument using a one-second photometry protocol at 405 nm. Luciferase values
were normalized to P-galactosidase values to determine AP-1 activity.
2.2.2.3 Transfection of Chicken Embryo Fibroblasts
DF-1 chicken embryo fibroblasts (CEFs) were cultured in DF-1 media (DMEM
containing 10% heat-inactivated chicken serum [Sigma, St. Louis, MO], 10% FBS,
and 1% penicillin/streptomycin) and incubated at 37C. Cells were seeded in DF-1
media in 60 mm culture dishes at a concentration of 2.5 x 105 cells/well. Once 50-
60% confluent, cells were supplied 800 pL SF, antibiotic-free DMEM and transfected
using Lipofectamine and Plus Reagent (Invitrogen). Each dish received 1000 ng of
each of the following DNAs: empty RCAS vector, wild-type Cental, AZF, Dbl PH,
or Myr Dbl PH Cental in RCAS, or c-Jun in RCAS. For each treatment, DNA was
added to 100 pL SF media and incubated with 6 pL Plus Reagent for 15 minutes at
room temperature. A 1:1 volume of Lipofectamine solution (4 pL Lipofectamine plus
100 pL SF media) was added to each DNA mixture and incubated for 15 minutes at
room temperature. The total reaction volume for each treatment was then added to
17


each respective dish. Treated cells were incubated at 37C for three to five hours,
after which they were washed with media containing 5% calf serum and covered with
2 mL DF-1 media. Transfected cells were incubated at 37C for 24-48 hours and then
sub-cultured into 100 mm dishes in DF-1 media. After three subsequent passages,
cells and the surrounding media were considered completely infected with the
recombinant RCAS virus.
2.2.2.4 DF-1 Growth Curve
Infected DF-1 cells were seeded into a 24-well tissue culture dish at 2 x 104
cells/well in media containing 5% FBS, 2% chicken serum, and 1%
penicillin/streptomycin. The dishes were incubated at 37C, and three wells were
harvested and the cells counted at each of the following time points: Day 0 (24 hours
after seeding), and Days 1, 3, 6, and 8. To remove cells from the tissue culture matrix,
200 pL IX trypsin EDTA (0.05% trypsin, 0.53 mM EDTA, Mediatech, Inc.) was
added to each well. One hundred microliters of the trypsin/cell suspension was
diluted with 20 mL cell culture diluent and counted on a Beckman Coulter Counter.
Counts for each infected cell type were averaged. The averages for each time point
were divided by Day 0 averages and the ratios plotted against time.
2.2.2.5 DF-1 Soft Agar Colony Formation Assay
Soft agar media (0.42%) was prepared using 4% sea-plaque agarose
(Cambrex, East Rutherford, NJ), 5% FBS, and 2% chicken serum in filtered IX
serum-free DMEM. The solid agar underlay was prepared using 1.5% sea-plaque
18


agarose, 5% FBS, and 2% chicken serum in sterile-filtered 2X serum-free DMEM
(Sigma). Agars were boiled and cooled to 42C, after which 1 mL of agar underlay
was poured into 6-well culture dishes and allowed to solidify. Infected DF-1 cells
were suspended in 42C soft agar media (2.5 x 104 cells/5 mL) and subsequently
diluted in soft agar media to obtain concentrations of 2.5 x 103, 5 x 103, and 1 x 104
cells/well. Soft agar cell suspensions were layered in each well over the agar
underlay. The agar was allowed to solidify at room temperature for 30 minutes, and
then the dishes were incubated for approximately three weeks at 37C. Cells were
supplied IX DMEM containing 5% FBS and 2% chicken serum every 1-2 weeks and
were periodically checked for colony formation.
2.2.3 Western Blot
To verify protein expression of Cental cDNAs in transfected cells, western
blot was performed on whole cell lysate. HEK 293 cells were transfected in 60 mm
dishes, with reagent volumes scaled up according to Invitrogen recommendations,
and harvested 24 hours later in 300 pL IX RIPA lysis buffer (Santa Cruz
Biotechnology, Santa Cruz, CA) and IX Complete Protease Inhibitor (Roche).
Infected DF-1 cells were passaged 3-4 times in 100 mm dishes, after which they were
harvested in 600 pL RIPA plus Complete Protease Inhibitor. Forty micrograms of
protein from each lysate was boiled in 2X Laemmli sample loading buffer (Bio-Rad,
Hercules, CA) and separated via SDS-PAGE on a 10.5-14% Tris-HCl gel (Bio-Rad)
for 120 minutes at 180V, using IX Tris/Glycine/SDS running buffer (Bio-Rad).
19


Proteins were transferred to a nitrocellulose membrane using a semi-dry
method at 15V for 30 minutes, prior to which filters, membrane, and gel were soaked
in transfer buffer for 15 minutes (IX Tris/Glycine transfer buffer [Bio-Rad] plus 20%
methanol [Fisher Scientific]). After transfer, the membrane was blocked in 5% non-
fat powdered milk in phosphate buffered saline plus Tween (PBST: 138 mM NaCl,
2.7 mM KC1, 10.1 mM Na2HPC>4, 1.7 mM KH2PO4, pH 7.1, and 0.05% Tween) for
one hour at room temperature. After blocking, the membrane was washed twice in
PBST and then cut as necessary for probing with appropriate antibodies. Primary
antibodies were diluted in PBST containing 1% bovine serum albumin (BSA) and
incubated with the appropriate membranes overnight at 4C (c-Jun antibody [Cell
Signaling Technology, Danvers, MA] was diluted 1:1000; Cental-specific antibodies2
[Affinity BioReagents, Rockford, IL] were each diluted 1:20,000). The next day,
membranes underwent three ten-minute washes in PBST and were incubated with
secondary antibody in PBST and 5% non-fat powdered milk for one hour at room
temperature (1:2000 dilution of an HRP-linked anti-rabbit antibody [Cell Signaling
Technology]). The membranes were washed three more times in PBST. Protein
visualization was achieved using an electrochemiluminescence (ECL) kit (Promega)
and exposure of the membrane to film.
2 Cental antibodies are custom-designed and are specific to epitopes GALKYFNRNDAKEPK. and
KAVDRPMLPQEYAVE.
20


2.2.4 Data Analysis
AP-1 activity and DF-1 growth curve results were analyzed using Prism 3.0
(GraphPad Software, San Diego, CA) with one-way ANOVA tests and a Tukey
multiple comparison post-test.
2.3 Results
2.3.1 Deletion of the Zinc Finger and Modification of Both PH Domains Inhibit
Cental-Mediated AP-1 Activation
To determine the involvement of Centals zinc finger and PH domains in
activation of the AP-1 pathway, an AP-1 reporter assay was performed on
mammalian cells transfected with structurally-modified Cental cDNAs. We created a
Cental variant lacking 125 N-terminal amino acids (AZF) and examined the ability of
it and two PH variants (Dbl PH and Myr Dbl PH) to initiate the AP-1 pathway. Cells
received the above cDNAs, wild-type Cental, or negative controls pcDNA3.1 and
GFPpcDNA3.1. Additionally, cells were co-transfected with a luciferase reporter
construct under control of an AP-1 sequence, and a P-galactosidase construct to
account for variability in transfection efficiency. To verify expression of the Cental
cDNAs, cell lysates were subjected to western blotting with Cental-specific
antibodies; we confirmed the expression of all Cental variants in pcDNA3.1.
Cell lysates were then assayed for both luciferase and P-galactosidase activity,
respectively quantified as light emission and optical density. The two values were
normalized to one another in order to produce a scale indicative of AP-1 activity.
21


Experimental averages are presented in Figure 6. Cells transfected with wild-type
Cental cDNA consistently exhibited the highest levels of AP-1 activity, as indicated
by AP-1-driven luciferase expression. Cells transfected with the AZF, Dbl PH, and
Myr Dbl PH Cental variants all exhibited significantly reduced AP-1 pathway
activity compared to those transfected with wild-type (p<0.001). The GFP and
pcDNA3.1 negative controls exhibited a low-level of AP-1 activity, likely due to the
presence of other pathway activators within the cell lysate. Amongst themselves, the
Cental variants exhibited similar levels of activity; there were no significant
differences amongst them or between the variants and the negative controls.
Effects of Cental Structural Modifications on
AP-1 Activity in 293 Cells
*
Figure 6 Levels of AP-I Activity in Mammalian Cells Transfected with Cental Structural Mutants
HEK 293 cells were transfected with Cental cDNAs or negative controls, and the lysates were assayed
for luciferase and p-galactosidase enzyme activity. Measurements of luciferase activity were divided
by p-galactosidase activity and adjusted to a 100 scale. The averages of four experiments, each done in
triplicate, are presented, with error bars representing standard deviations. The asterisk indicates
significant difference between WT Cental and all other columns, as determined with one-way
ANOVA.
22


2.3.2 Two PH Variants Exhibit Decreased Potential for Cellular
Transformation
In addition to questioning whether Cental localization affects AP-1 pathway
activation, we also questioned the impact localization has on Centals ability to
induce cellular transformation. In an attempt to answer these questions, we examined
the effect of the above Cental variants on cell growth patterns. Cental or c-Jun
cDNAs were cloned into a replication-competent avian retroviral vector (RCAS) and
transfected into chicken embryo fibroblasts. After passaging the chicken cells several
times, they were considered infected. Using a viral vector to infect cells ensured
complete dissemination and expression of the exogenous cDNAs, maximizing the
chances of detecting any potential physiological effects. Physiological changes in
infected DF-1 cells were observed using growth curve and soft agar colony formation
assays, two common tools for the identification of transformed cells. To determine
the effects of each Cental variant or c-Jun on cell proliferation, infected cells were
seeded in 24-well dishes, harvested at various time points, and the number of cells
counted and averaged for each time point. To determine whether or not infected cells
were able to grow in the absence of a solid matrix, infected cells were seeded in soft
agar media and incubated for 2-3 weeks to allow the formation of suspended colonies.
Concomitant western blot analysis revealed that the AZF Cental cDNA, which was
successfully expressed as protein in the mammalian vector, was not expressed in the
23


RCAS vector. DNA sequencing revealed a loss of four base pairs downstream of the
Cental stop codon that may have occurred during the cloning procedure.
The above transformation assays were performed using secondary chicken
embryo fibroblasts, in which Cental-mediated growth effects have proven more
difficult to detect than in primary cells. For these reasons, our results are not reported
here. However, our preliminary data mirror those obtained by Reisdorph and Powell
[31], with the exception of the newly-tested £ZF Cental cDNA. In their previous DF-
1 growth curve results, cells were harvested and counted at 0, 1, 3, 6, and 8 days
(Figure 7). Growth of cells over-expressing c-Jun increased the fastest compared to
the other infected cell lines. Cells infected with the Cental variants and negative
RCAS control exhibited a slower rate of growth that began to increase at Day 6. Cell
counts were highly variable, as evidenced by the error bars at Day 6 and Day 8. Using
one-way ANOVA statistical tests to compare each cell line at different time points,
we determined that from Day 1 to Day 8, all infected cell lines exhibited a significant
difference in growth, with the exception of the negative RCAS control. Several cell
lines also exhibited significant differences in growth from Day 1 to Day 6 (c-Jun) and
Day 3 to Day 8 (Myr Dbl PH, c-Jun). One-way ANOVA comparing the cell lines to
each other revealed a significant difference in growth between c-Jun and all other cell
lines at Day 6.
24


Growth Curve of DF-1 Cells Infected with
Cental Variants
RCAS
Dbl PH
v Myr Dbl PH
-O- WT Cental
Jun
Figure 7 Growth of Avian Cells Infected with Cental Variants
Infected DF-1 cells were seeded at 2 x 104 cells/well and counted in triplicate at Day 0, 1,3, 6, and 8.
Cell counts were averaged and growth plotted as the ratio of Day X/Day 0. This graph represents the
averages of four experiments. Error bars represent standard deviation. The asterisk at Day 6 indicates a
significant difference between growth of Jun-infected cells and all other cell lines for that time point,
as determined with one-way ANOVA.
Soft agar colony formation assays performed by Reisdorph and Powell
provide a qualitative look at how structural modifications affect Centals ability to
induce cellular transformation. As expected, DF-1 cells infected with the empty
RCAS vector formed several small colonies in soft agar (Figure 8). Wild-type Cental
and Jun were used as positive controls, since they have previously been shown to
induce soft agar colony formation in chicken cells, and cells infected with both
Cental and Jun formed a large number of colonies (Jun not shown). Cells infected
25


with the Dbl PH and Myr Dbl PH variants were better able to form colonies in soft
agar compared to the RCAS control, but formed noticeably fewer colonies than cells
infected with wild-type Cental.
r*
RCAS
WT Cental
-.i*.


,
-

4 '
A* ,.l . % * 4 m *
* ft i;\* *' V' **' V
. * *

^ .**
Myr Dbl PH
.< * .
*\ t / V r
tjj
, jK. % *' ' / *, ' 4 v
dUBEfi^a^ii.. ^ , 5i ** '>#!*
Dbl PII
*
, *
> *

Figure 8 Colonies Formed by Infected DF-1 Cells in Soft Agar Media
DF-1 cells infected with RCAS retrovirus containing the Dbl PH, Myr Dbl PH, WT Cental, or Jun
cDNAs, were plated at varying concentrations and incubated for three weeks (concentration shown: 1
x 104 cells/well).
26


2.4 Discussion
Cental is a potent activator of the AP-1 pathway and cellular transformation
[10], but little is known about its role in these important cell processes. By studying
the relationship between Centals structure and function, we questioned the effects
that intracellular localization has on Centals ability to initiate these changes. We
assayed the ability of three Cental variants to initiate the AP-1 pathway and induce
cellular transformation. The Dbl PH variant contains R->C mutations in both PH
domains, preventing Cental from binding the membrane-bound lipid PIP3. The Myr
Dbl PH variant possesses the same PH mutations as well as an N-terminal
myristoylation signal, while the AZF Cental variant lacks 125 N-terminal amino
acids, rendering it incapable of entering the nucleus.
In AP-1 reporter assays, none of the above variants were capable of activating
the AP-1 pathway. Additionally, the Dbl PH and Myr Dbl PH variants also proved
less able to induce cellular transformation compared to wild-type Cental or Jun
proteins. Contact inhibition, the loss of which is characteristic of transformed cells,
was maintained in chicken cells infected with the two Cental PH variants. In soft
agar colony assays, cells over-expressing the PH variants formed fewer suspended
colonies than did cells over-expressing wild-type Cental. Since AP-1 pathway
activation is one, although certainly not the only, mechanism of inducing cellular
transformation, loss of AP-1 activity would logically result in loss of, or reduction in,
cellular transformation.
27


Most of Centals known functions, such as ARF regulation, occur outside of
the nucleus; therefore, we hypothesized that Centals AP-1-related activities occur
outside of the nucleus as well, and that lack of nuclear entry will not affect the AP-1
pathway. We tested the Dbl PH and Myr Dbl PH variants to identify whether Cental
functions in the cytoplasm or while membrane-bound. AP-1 activity results for the
AZF variant would indicate that Cental does in fact require nuclear localization to
function. However, use of the zinc finger deletion as an indicator of mechanism is
admittedly limiting. Clearly, the AZF Cental variant contains a large deletion,
encompassing Centals zinc finger and most of its ARF GAP domain. Multiple
interactions, AP-1 -related or not, may be affected by this deletion and may contribute
to observed differences in AP-1 function. Additionally, while loss of the zinc finger
prevents nuclear entry, this deletion also prevents the binding of Cental to the kinesin
motor protein KIF13B and transport to the plasma membrane [28], The loss of AP-1
activity observed with the AZF variant may be due to lack of nuclear entry or to loss
of transportation to the plasma membrane.
How Cental functions during activation of the AP-1 pathway is still unclear;
however, we can infer the relationship between Centals structural domains and AP-1
activation by examining information known about Centals localization. Cental has
been well-established as an ARF GAP [14-16]. In particular. Cental binds ARF6, a G
protein involved in membrane-endosome trafficking, via both the zinc finger and the
N-terminal PH domain [28]. Cental co-localizes with ARF6 to the plasma membrane
28


[15]. Deletion of the zinc finger and mutation of the N-terminal PH domain likely
interfere with these associations. The localization mediated by Centals zinc finger
and PH domains may prove critical in elucidating the proteins AP-1-related
functions as well. Since membrane-endosome trafficking is critical for nutrient intake,
and actin rearrangement for cell movement and growth, a decrease in these activities
due to Centals structural modifications may mitigate the physical changes associated
with cellular transformation. The loss of AP-1 activity and transformative potential
associated with the two PH domains may indicate that Cental, like the ARF proteins
it regulates, functions best when it is able to cycle between the plasma membrane and
cytoplasm.
The structure-function analyses performed here and in previous works address
the question of how intracellular localization affects Centals performance in AP-1
activation and cellular transformation. The AP-1 assay results for the AZF Cental
variant are ambiguous at best, and it is unclear whether the observed effect on AP-1
activation is due to loss of nuclear entry or a decrease in plasma membrane
localization, or other factors. As such, our initial hypothesis that Cental performs its
AP-1-related functions outside of the nucleus remains unconfirmed. However, we
used this deletion as a starting point to determine the effect that localization has on
Cental activity with the knowledge that future work is required to correlate specific
protein regions to specific functions.
29


If Cental requires nuclear entry to perform its AP-1 functions, then one or
both of the PH variants which still possessed their nuclear localization signals -
might have exhibited some degree of function above background levels. Loss of the
zinc finger prevents nuclear entry, but also inhibits transport by KIF13B to the plasma
membrane. As indicated by the lack of AP-1 activity and transformation in cells
transfected with the two PH variants, Cental may require the freedom of membrane-
cytoplasm cycling in order to perform its elusive AP-1-related functions. This
hypothesis is supported by the fact that Cental binds ARF6, a protein which does
cycle between the plasma membrane and endosomes, but confirmation is still
pending. Unless Cental requires both nuclear and membrane localization, our results
as well as circumstantial evidence suggest that nuclear entry is not critical for Cental-
mediated AP-1 activation. The zinc finger domain, however, may still be significant,
as the PH domains appear to be.
30


3.
Proteomic Analysis of Post-Translational Modifications
3.1 Introduction
Centals role in the AP-1 pathway is still under investigation, and our
knowledge continues to grow with regards to Centals movement, regulatory
functions, and intracellular associations. As an ARF GAP, Cental regulates the
activity of multiple ARF proteins [16], Additionally, Cental is involved in activation
of ERK kinases, proteins involved in the MAPK and AP-1 signaling pathways [19].
A common feature of regulatory proteins and cell signaling mediators in particular is
the presence of post-translational modifications, such as the addition of a phosphate
group to a serine, threonine, or tyrosine reside. Many signaling molecules, such as
those involved with PI3K functions (reviewed in [21]), are activated via
phosphorylation.
Cental may also be activated via phosphorylation. To date, no phosphorylated
Cental has been identified in vivo. A recent intracellular localization study presented
the western blot of a nuclear fraction with a potentially mass-shifted Cental, which
may have indicated the added mass of a post-translational modification [18].
Additionally, in vitro kinase assays have shown that protein kinase C (PKC)
phosphorylates Cental at both S87 and T276 [34]. These findings initiated our
sequence-level analysis of Cental. Considering Centals associations with PKC,
31


casein kinase 1, and possibly the ERKs, and the fact that Cental is an activator of a
major cell signaling pathway, we hypothesized that Cental is phosphorylated in vivo
as well as in vitro.
Mammalian cells stably over-expressing wild-type Cental were used as an
abundant source of potentially-phosphorylated Cental. The phosphorylation state of a
protein is determined in part by the availability of specific kinases as well as
intracellular phosphatase activity. Several cell culture treatments were utilized with
these considerations in mind. To induce phosphorylation of Cental, we treated cells
with phorbol 12-myristate 13-acetate (PMA), a PKC-activating agent. Not only does
PKC reportedly phosphorylate Cental, it is also an upstream mediator of the AP-1
pathway. An increase in activated PKC, as well as involvement of the AP-1 pathway,
would potentially create an environment suitable for Cental phosphorylation. In order
to retain any phosphate groups present on Cental, we also treated cells with Calyculin
A, a serine/threonine phosphatase inhibitor, and sodium pervanadate, a tyrosine
inhibitor. Potentially phosphorylated Cental was then isolated from cell culture,
simplified, and fractionated via liquid chromatography (LC). Centals protein
sequence was then analyzed with tandem mass spectrometry (MS/MS) to obtain the
exact location of potential phosphate groups.
32


3.2 Methods and Materials
3.2.1 Cell Culture
HEK 293 cells stably expressing wild-type Cental were cultured in DMEM
containing 10% FBS, 1% penicillin/streptomycin, and 500 ng/pL of the selective
agent G418 (Invitrogen) at 37C under 5% CO2. Prior to treatment, cells were split
into 100 mm dishes and grown to -70-80% confluency.
3.2.1.1 Calyculin A and Sodium Pervanadate Treatment
Confluent cells were treated with Calyculin A (Sigma) and sodium
pervanadate as previously described [38]. Prior to treatment, confluent cells were
washed once in 5 mL 37C phosphate buffered saline (PBS: 150 mM NaCl, 10 mM
Na2HP04, pH 7.2) and covered in 5 mL growth media. To the media, 50 mM sodium
pervanadate (100 mM activated sodium orthovanadate [Sigma] mixed 1:1 with 0.36%
H2O2) was added to a final concentration of 1 mM, along with 500 ng Calyculin A.
Cells were incubated at 37C for 30 minutes, rinsed twice in ice cold PBS, and lysed
in 600 pL IX RIPA lysis buffer (Santa Cruz) containing protease inhibitors (sodium
ortho vanadate, phenylmethylsulphonyl fluoride (PMSF) [Santa Cruz]) and
Phosphatase Inhibitor Cocktails 1 and 2 (Sigma). The cell lysate was centrifuged for
10 minutes at 4C and the supernatant retained.
3.2.1.2 Phorbol 12-Myristate 13-Acetate Treatment
Confluent cells were serum starved for 24 hours prior to treatment.
Immediately prior to harvest, cells were stimulated with 10% FBS and 100 pM
33


phorbol 12-myristate 13-acetate (PMA [Sigma]) to a final concentration of 100 nM
[39]. Stimulated cells were incubated for 30 minutes at 37C and harvested with IX
RIPA and protease/phosphatase inhibitors as described above.
3.2.1.3 Combined Treatment
Cells were serum starved for 24 hours. Immediately prior to treatment, cells
were washed once in 37C PBS, and then 5 mL serum-containing media was added.
Sodium pervanadate and Calyculin A were added as described above, and the cells
incubated for 30 minutes. PMA was added to a concentration of 100 nM, and after
another 30 minute incubation, cells were harvested as described.
3.2.2 Protein Isolation, Verification, and Sample Preparation
3.2.2.1 Immunoprecipitation
Cental and phosphorylated ERK (pERK) immunoprecipitations were
performed on PMA-treated and phosphatase inhibitor (Pl)-treated cell lysates.
Cental or pERK-specific antibodies were incubated with cell lysate for one hour at
4C (Cental: 17.2 pg of antibody specific to GALKYFNRNDAKEPK and 17.6 pg of
antibody specific to KAVDRPMLPQEYAVE; pERK: 1:100 dilution of an antibody
specific to singly phosphorylated ERK 1 and 2 [Cell Signaling Technology]). To each
lysate sample, we added 20 pL Protein A agarose beads (Santa Cruz), and incubated
the mixture at 4C overnight. The next morning, the beads were centrifuged and
washed four times with 500 pL RIPA buffer containing all protease/phosphatase
inhibitors. After washing, the beads were boiled in 2X Laemmli sample loading
34


buffer (Bio-Rad) and resolved using SDS-PAGE with a 10.5-14% gel (Bio-Rad) for
120 minutes at 180V. Gels were stained using Sypro Ruby Protein Gel Stain
(Invitrogen) following the manufacturers protocol for protein visualization.
3.2.2.2 De-phosphorylation with Calf Intestinal Phosphatase
Untreated HEK 293 Cental-expressing cells were harvested in IX RIPA
containing PMSF as previously described. After immunoprecipitation, beads were
washed once in RIPA and suspended in 150 pL Buffer 3 (New England BioLabs).
The suspension was incubated with 20 units calf intestinal phosphatase (CIP) (New
England BioLabs) for 45 minutes at 37C [40], after which the beads were washed
with RIPA buffer and separated via SDS-PAGE as previously described.
3.2.2.3 Western Blot
To determine if PM A and/or sodium pervanadate and Calyculin A resulted in
phosphorylation of Cental, western blot was performed against Cental and pERK
1/2. For Cental, 20 pg treated whole cell lysate was separated via SDS-PAGE for 120
minutes at 180V (gel composition described above). Proteins were transferred to
nitrocellulose using a semi-dry method for 30 minutes at 15V, after which the
membrane was blocked in 5% milk-PBST for one hour at room temperature as
described previously in Chapter 2. The membrane was subsequently probed with
Cental-specific antibodies. Primary antibody incubation took place in PBST-1% BSA
at 4C overnight with dilutions of 1:20,000 of each antibody. After washing in PBST,
membranes were incubated in 5% milk-PBST with 1:2000 dilution of an appropriate
35


HRP-linked secondary antibody for one hour at room temperature. The membranes
were rinsed three times in PBST and visualized via ECL and film exposure, as
described in Chapter 2. For pERK 1/2, western blot was performed against the
immunoprecipitated sample. Three microliters of immunoprecipitate was resolved via
SDS-PAGE for 120 minutes at 180V. All subsequent steps were as described above,
using a 1:2000 dilution of pERK 1/2 specific antibody.
3.2.2.3 Protein Digest
Protein gels were stained with Sypro Ruby Protein Gel Stain and visualized
with an ultraviolet (UV) light source. Resulting protein bands were compared to
molecular weight standards, and Cental was initially identified by its predicted size
(43 kDa). Cental bands were excised from the gel and digested prior to LC/MS/MS
analysis according to an established protocol [41]. Briefly, gel slices were cut into
small pieces, covered with a solution of 50% HPLC-grade acetonitrile (ACN) and 25
mM ammonium bicarbonate (ABC) (both from Fisher Scientific), and shaken at room
temperature for 10 minutes. Gel fragments were then dehydrated in 100% ACN and
proteins reduced in 9.7 mM dithiothreitol (Bio-Rad) in 25 mM ABC for 30 minutes.
After reduction, denatured proteins were alkylated in 54 mM iodacetamide (Bio-Rad)
in 25 mM ABC for 30 minutes in the dark. Next, gel pieces were briefly washed in 50
mM ABC and then twice more in 25 mM ABC-50% ACN. After dehydration and
drying, gel pieces were covered with 10 ng/pL Endoproteinase Lys-C (Roche) in 25
mM ABC. Digests were incubated at 37C for 18 hours, after which the enzyme was
36


deactivated with 1 jj,L 10% formic acid (FA). Peptides were extracted from the gel
fragments using 60% ACN and 0.1% FA, speed-vacuumed to dryness, and
resuspended in 3% ACN and 0.1% FA for mass spectrometric analysis. Depending on
the downstream application, some digests were pooled at the peptide extraction step
to generate a concentrated peptide sample. Digests were also performed using Trypsin
(25 ng/pL, 37C [Promega]), Glu-C (25 ng/pL, 25C [Roche]), and Proteinase K (25
ng/pL, 37C [Promega]).
3.2.3 Mass Spectrometry
3.2.3.1 Ion Trap
Samples were resuspended in 3% ACN and 0.1% FA and separated via
reverse-phase mode on an Agilent 1200 Series HPLC equipped with a capillary pump
(pump A"), and a nano pump (pump "B") (Agilent Technologies, Santa Clara, CA).
Eluted peptides were analyzed on an Agilent ETD ion trap (model 6340) mass
spectrometer with an HPLC-chip interface and electrospray ionization source. The
buffer for pump A (the capillary pump) contained 3% ACN, 97% HPLC grade water,
and 0.1% FA. Pump B (the nano pump) utilized two buffers: buffer A, which was
comprised of 0.1% FA in HPLC-grade water, and buffer B, which was comprised of
90% ACN, 10% HPLC-grade water, and 0.1% FA. HPLC gradient conditions for
pump B are listed in Table 1. Typically 5-7 pL of each peptide sample were injected
onto the enrichment column for concentration/purification and then loaded onto and
eluted from the analytical column over an increasing gradient. Peptides were scanned
37


without fragmentation (MS) and abundant peptides were automatically selected and
trapped for further fragmentation (MS/MS). Fragmentation utilized either collision
induced dissociation (CID) or electron transfer dissociation (ETD). Mass
spectrometer instrument parameters are listed in Table 2.
Table I HPLC nano pump buffer B gradient for peptide elution prior to ion trap and QTOF analysis
Ion Trap Time (min) 0 15 16 20 22
% B 3 45 80 80 3
QTOF Time (min) 0 1 10 13 16
% B 3 3 40 80 3
3.2.3.2 QTOF
Resuspended samples were separated with an Agilent 1200 Series HPLC.
Capillary pump (A) and nano pump (B) buffer compositions were as described above.
The buffer gradient for pump B is listed in Table 1. Eluted peptides were analyzed
with an Agilent QTOF (model 6510) mass spectrometer with an HPLC-chip interface
and electrospray ionization. For MS-level analysis, peptides were scanned without
fragmentation. Using MassHunter BioConfirm software (Agilent Technologies), the
peptide masses detected in the MS scan were matched to peptide masses predicted by
an in silica digest of Centals protein sequence. Drawing from the literature and
38


preliminary ion trap data, potential phosphoresidues were incorporated into the
BioConfirm database. Peptides matching the in silica digest were selected based on a
5 ppm error margin. For matching peptides, m/z values for all charge states were
obtained. Thirteen matching peptides were selected for targeted MS/MS analysis
based on their retention time, m/z ratio, charge state, and presence in the raw MS
data. For MS/MS analysis, all HPLC parameters were as described above. The m/z
values of the 13 selected peptides were targeted for fragmentation three times with
three different collision energies. QTOF parameters are listed in Table 2.
Table 2 Instrument parameters
Parameter Ion Trap MS/MS QTOF MS QTOF MS/MS
Nano flow rate (pL/min) 0.44 0.55 0.45
Capillary flow rate (pL/min) 4 3 2
Temperature (C) 350 310 310
Drying gas (L/min) 5 3.5 3.5
Acquisition range (m/z) 300-1800 290- 3200 MS: 290-1225 MS/MS: 100-3000
Scan rate 26,000 m/z 1 1
Ion polarity Positive Positive Positive
39


3.2.4 Data Analysis
For ion trap experiments, raw MS/MS data were extracted and analyzed using
SpectrumMill database searching software (Rev A.03.03.080 SRI) (Agilent
Technologies). Raw spectral data were searched against the SwissProt Homo sapiens
database or a Cental sequence database, with carbamidomethylation as a fixed
modification, and oxidized methionine, phosphorylated serine, threonine, or tyrosine
as variable modifications. Data were searched for enzyme cleavage depending on the
enzyme used. A maximum of two missed cleavages were allowed. Precursor mass
tolerance was +/- 2.5, product mass tolerance was +/- 0.7, and maximum ambiguous
precursor charge was equal to 3. Spectra for Cental peptides identified by
SpectrumMill as possessing a phosphorylation were manually validated by comparing
observed b- and y-ion masses to predicted ion fragment masses.
For QTOF experiments, MS data were searched against the Cental protein
sequence using BioConfirm database software (Agilent Technologies). Matches
between detected peptides and predicted peptides were within 5ppm error. Raw MS
spectra were searched for m/z values of matched peptides to verify their presence.
MS/MS data were searched against SpectrumMill with the above parameters, and
both software-generated spectra and raw spectra were manually searched for b- and y-
ion fragments matching predicted ion fragments masses.
40


3.3
Results
3.3.1 Cental is Phosphorylated in Vivo at Tyrosine 364
HEK cells over-expressing wild-type Cental were treated with either PMA, or
the phosphatase inhibitors Calyculin A and sodium pervanadate, or both. Through
western blot detection of phosphorylated ERK 1/2, we confirmed that PMA, an
activator of PKC, induces phosphorylation of ERK 1/2 (Figure 9). Additionally, we
confirmed that phosphatase inhibitor (Pl)-treatment prevents de-phosphorylation of
ERK 1/2. Untreated samples did not contain detectable levels of pERK; either ERK
was not phosphorylated, or phosphate groups were lost due to intracellular
phosphatase activity. Western blot analysis proved inconclusive in determining the
effects of cell treatments on Cental; an 80 Da mass shift in potentially
phosphorylated Cental bands was not detected, although the absence of a shift does
not preclude the existence of a modification.
+P1 -PI +PMA -PMA
12 3 4
kOii
50 IgCi
I ! pE/RK]
12 pE!RK2
Figure 9 Effect of PI and PMA Treatment on Phosphorylation Status of ERK 1/2
Phosphorylated ERK 1/2 was immunoprecipitated from PMA- or Pi-treated cell lysate. Three
microliters of the immunoprecipitate was subjected to western blot analysis with a pERK-specific
antibody. In Lanes 1 and 3, both phosphorylated ERK proteins were detected. Phosphorylated ERK
proteins were not detected in untreated samples.
41


Cental was isolated from treated cells, subjected to SDS-PAGE, and the
resulting protein band was digested with endoproteinases. Various enzymes were
tested, with Lys-C digestion resulting in larger peptides and an increased potential for
internal phosphate groups. PMA- or Pi-treated samples were analyzed on a nano-ESI
ion trap mass spectrometer and fragmented with either collision-induced dissociation
(CID) or electron transfer dissociation (ETD). CID fragmentation yielded no phospho
data. ETD fragmentation resulted in the detection of 30 potential phosphopeptides,
encompassing 33 unique phosphoresidues. However, when searched and summarized
with SpectrumMill database-searching software, these potential phosphopeptides
were not considered high confidence hits. All of the detected peptides possessed
protein scores < 8, and most possessed scored-peak intensities (SPI) < 75%. A protein
score > 20 and an SPI > 80% indicate with high confidence that a reported peptide
was detected and matches its predicted mass.
Samples were also analyzed using a nano-ESI quadrupole time of flight
(QTOF) mass spectrometer to confirm alleged phosphopeptides detected on the less
accurate ion trap. In order to increase the probability of detecting low-abundance
peptides, multiple Cental immunoprecipitations were performed on cells treated with
PMA, PI, or PI+PMA, and the digested peptides combined. As a de-phosphorylated
control, Cental immunoprecipitate was treated with calf intestinal phosphatase (CIP).
Samples were analyzed following a protocol of MS scanning, database matching, and
targeting selected peptides for MS/MS fragmentation.
42


We report here the presence of a higher abundance peptide with a phosphate
group at Y364. The un-phosphorylated form of the peptide
AVDRPmLPQEYAVEAHFK3 was detected on the ion trap, but the QTOF identified
triply- and quadruply-charged states of the phosphorylated version,
AVDRPmLPQEyAVEAHFK. This phosphopeptide was detected in PI-, PI+PMA-,
and PMA-treated samples, but was most abundant, and generated the highest quality
data, in the concentrated Pi-treated sample. Manual calculation of m/z values
confirmed that the difference in mass between the two peptides was equal to the mass
of a single phosphate group. This calculation was performed by adding the masses of
the peptide plus its charges, oxidized methionine, and phosphate group, and dividing
the sum by the peptides charge state (2100 Da + 4 Da + 16 Da + 80 Da / 4 = 550
Da). Performing the same calculation without the 80 Da phosphate group yields a
predicted m/z value of approximately 530 for the un-phosphorylated peptide.
Examination of the raw MS spectra confirmed the presence of the quadruply-
charged phosphopeptide (predicted m/z = 550.0135; observed m/z = 550.2620).
(Figure 10A, page 46). The peptide was then fragmented, forming smaller b- and y-
ions (Figure 10B). The y8 peak corresponds to the phosphopeptide fragment
yAVEAHFK. The predicted mass of this singly-charged, un-modified fragment is
964.4887 Da. The observed mass of the singly-charged y8+ fragment was 1044.4502
Da. The 80 Da difference between the predicted and observed masses supports the
J Upper case Y indicates an un-modified tyrosine residue, and a lower case y indicates the presence
of a phosphate group.s
43


presence of a phosphate group on the observed peptide fragment. A second, doubly-
charged ion fragment (bl 1++) was also detected in Pi-treated samples, corresponding
to the m/z of the doubly-charged phosphorylated fragment AVDRPmLPQEy
(predicted m/z = 698.8020); however, the bl 1++ peak is obscured by surrounding
peaks and is not visible in Figure 10.
Similar analyses were performed on CIP-treated Cental samples, in which the
de-phosphorylated Cental peptide was targeted first in MS and then in MS/MS mode
(Figure 11, page 47). The MS scan confirmed the presence of the quadruply-charged,
un-phosphorylated peptide (predicted m/z = 530.0135, observed m/z = 530.2724)).
MS/MS fragmentation reduced the parent peptide to b- and y-ions. The same singly
charged y8+ ion fragment detected in Pi-treated samples was also detected in de-
phosphorylated samples (Figure 1 IB), providing a useful reference point for mass
comparison. The observed y8+ fragment possessed an m/z = 964.4860, closely
matching the predicted mass of 964.4887. The difference in y8 masses between
samples is 80 Da, again confirming the presence of a phosphate group in Pi-treated
samples and the absence of one in CIP-treated samples. The bl 1++ fragment
encompassing the un-phosphorylated tyrosine residue was also detected in CIP-
treated samples, but is not visible due to the surrounding spectra.
QTOF-generated MS/MS data were searched against human and Cental
protein databases using SpectrumMill. Matching the phosphopeptide to database
sequences yielded high confidence hits with consistently high protein scores, high SPI
44


values, and error margins within 5 ppm (Table 3, page 48). Software-generated
spectra also identified y8 and bl 1 ion fragments; the masses of which matched the y8
and bl 1 ions observed in the raw spectra.
After the phosphorylated and un-phosphorylated forms of the peptide were
identified via targeted MS/MS analyses, we returned to the original MS data and
verified the presence of both peptide versions in these preliminary, un-targeted scans.
Extracted ion chromatograms (EICs) from MS spectra revealed a high abundance
peak with m/z = 550.0124 in Pi-treated samples (Figure 12A, page 49), as well as a
barely-detectable peak representing the un-phosphorylated peptide. In CIP-treated
samples, EIC analysis identified a high abundance peak with m/z = 530.0124 (Figure
12B), and a low abundance peak representing the phosphorylated peptide. These
chromatograms illustrate the ratio of phosphorylated to un-phosphorylated peptide in
each sample.
45


x10
4^
ON
A
B
x10:l
Counts vs. Mass-to-Charge (m/z)
Figure 10 Phosphopeptide AVDRPmLPQEyAVEAHFK Identified in Targeted MS and MS/MS Spectra
A) Targeted MS scan of Pi-treated Cental digest. The quadruply-charged phosphopeptide AVDRPmLPQEyAVEAHFK was detected at m/z =
550.2620, The difference between the detected m/z and predicted m/z (550.0135) is due to the additional mass of 13C in the sample. B) Targeted MS/MS
fragmentation of the above phosphopeptide resulted in the formation of the singly-charged ion fragment yAVEAHFK (peak y8+), encompassing a
phosphorylated tyrosine.


)0 >0 )0 SO )0 >0 )0 >0 )0 >0 )0 SO )0 SO )0 SO )0 SO 00
Counts vs. Mass-to-Charge (m/z)
Figure 11 Un-phosphorylated Peptide AVDRPmLPQEYAVEAHFK Identified in Targeted MS and MS/MS Spectra
A) Targeted MS scan of CIP-treated Cental digest. The quadruply-charged peptide AVDRPmLPQEYAVEAHFK was detected at m/z = 530.2724. The
difference between the observed and predicted m/z (530.0135) is due to the additional mass of 13C in the sample. B) Targeted MS/MS fragmentation of
the above peptide resulted in the formation of the singly-charged ion fragment YAVEAHFK (peak y8+), possessing a phosphate group.


Table 3 Database search results for targeted MS/MS analysis of phosphatase inhibitor-treated Cental samples
Score SPI (%) Intensity Sequence Modifications RT (min) z m/z Detected Matched Mass Shift Error (ppm)
20.28 94.3 5.49E+04 (K)AVDRPmLPQEyAVEAHFK(H) m:Oxidized methionine 7.3 4 550.0119 2101.054 95.9722 5
y:Phosphorylated Y
18.73 92 3.38E+04 (K)AVDRPmLPQEyAVEAHFK(H) m:Oxidized methionine 7.31 3 733.0122 2101.054 95.9685 3.3
y:Phosphorylated Y
18.72 85.8 5.49E+04 (K)AVDRPmLPQEyAVEAHFK(H) m:Oxidized methionine 7.31 4 550.0119 2101.054 95.9722 5
y:Phosphorylated Y
7.25 66.7 5.45E+04 (K)AVDRPmLPQEyAVEAHFK(H) m: Oxidized methionine 7.54 4 550.0119 2101.054 95.9722 5
y:Phosphorylated Y


x10 4
+ESI EIC(550.0124) Scan Frag=170.0V centa_IP_MS_1ul.d Smooth (2)
7.285
5696
Phosphorylated
AVDRPmLPQEyAVEAHFK
m/z = 550.0124
z = 4
Un-phosphorylated
. i
.r,,...........
5 5 ' 5 i 5 ) 5 0 1.5
E
Figure 12 Phosphorylated and Un-phosphorylated Cental Peptides
The extracted ion chromatogram (EIC) of a preliminary MS scan confirms the presence of (A) the quadruply-charged phosphopeptide
AVDRPmLPQEyAVEAHFK in Pi-treated samples, and (B) un-phosphorylated AVDRPmLPQEYAVEAHFK in CIP-treated samples (B).


3.4 Discussion
Since Cental is a mediator of the AP-1 pathway, the probability that it is
phosphorylated, like so many other signaling molecules, seemed high. Prior in vitro
kinase assays have shown that PKC phosphorylates Cental at two residues, S87 and
T276 [34]. We attempted to verify whether or not Cental is phosphorylated in vivo as
well, and used several methods for maximizing detection of phosphorylations. First, a
mammalian cell line over-expressing wild-type Cental was used as a source of
Cental protein. While expression of endogenous Cental may produce sufficient
protein for detection via LC/MS/MS, the low ratio of phosphorylated protein to un-
phosphorylated protein would hinder detection and validation of these modifications.
Second, in an attempt to induce PKC-mediated phosphorylation of Cental, cells were
treated with PMA, a PKC-activating agent. Cells were also treated with Calyculin A
and sodium pervanadate to prevent de-phosphorylation by intracellular phosphatases.
Using ERK 1/2, known phosphorylated proteins that are also involved in the AP-1
pathway, we confirmed the efficacy of these cell treatments; PMA-treatment resulted
in an increase in phosphorylated ERK 1/2, and Pi-treatment prevented loss of
phosphate groups from the ERK proteins (Figure 9). We attempted to visually
identify the effects treatments had on Centals phosphorylation status as well, but
western blot results proved inconclusive. However, the ERK results indicated that
PMA- and Pi-treatments create an environment theoretically suitable for Cental
phosphorylation, and so we continued our sequence analyses.
50


Sequence and PTM data were obtained from digested and concentrated
Cental samples using LC/MS/MS. Preliminary data were obtained using an ion trap
mass spectrometer, and potential phosphopeptides were targeted using a QTOF
instrument. Raw data were searched against Cental or human protein databases, the
results of which identified the Cental peptide AVDRPmLPQEyAVEAHFK as
possessing a potential phosphate group at Tyrosine 364. To validate the database
results, raw MS and MS/MS spectra were manually searched for this peptide. The
phosphorylated form of the peptide (m/z = 550) was the dominant species in all cell
treatments. Compared to PMA- or PI+PMA-treated samples, Pi-treated samples
yielded the highest quality spectra for the phosphopeptide. This difference was likely
due to the fact that Pi-treated samples were five-fold more concentrated than the other
samples. In CIP-treated samples, the un-phosphorylated form of the peptide
dominated (m/z = 530). We were unable to verify the presence of the two previously
reported phosphoresidues, S87 and T276, in any sample; S87 was apparently
undetected while T276 was detected but was matched to the databases with low
confidence.
The sequence data obtained through MS analysis has confirmed the
hypothesis that Cental is phosphorylated in vivo. Tyrosine 364 is located just
downstream of Centals C-terminal PH domain (Figure 13). The implications of
Centals newly-discovered phosphorylation are grounds for future research.
Considering the body of knowledge that exists regarding Cental, one must now
51


question the involvement of this phosphate group in all of Centals known activities
and interactions. The location of Y364 may also be important, as many of Centals
activities involve binding of its PH domains to PIP3, and it remains to be seen if
phosphorylation of Y364 affects that interaction. Considering the focus of our work is
on Cental as an activator of the AP-1 pathway, determining how phosphorylated
Y364 affects Centals AP-l-related activities may provide key information for
understanding exactly how Cental functions in this pathway.
SS7
2n Finger/ARF GAP
T276
---1-
Y364
N PH
C PH
Figure 13 Cental Structure Containing Known Phosphorylation Sites
52


4. General Discussion and Summary
Centaurin-Alphal, or Cental, is an enigmatic protein. For the past decade,
Cental has been studied for its various roles in lipid- and protein-binding, ARF
regulation, actin rearrangement, intracellular localization, and tumor development.
Cental is located throughout the cell and as such associates with a variety of
molecules, including the membrane-bound lipid PIP3, various protein kinases, motor
proteins, ARF proteins, and even proteins involved in nuclear organization [16-18,
28, 34, 42]. Cental is also one of many activators of the AP-1 cell signaling pathway,
the deregulation of which has been linked to the development of bone, liver, breast
and colon tumors [2-5], Centaurin proteins have been linked to the development of
tumors in breast tissue, and may play a role in Alzheimers disease as well [13, 43,
44], Much has been discovered about some of Centals activities, such as ARF
regulation; however, the mechanism by which Cental activates the AP-1 pathway
remains largely unknown.
In these experiments, we attempted to ascertain the relationship between
Centals structural domains and the proteins ability to activate the AP-1 pathway
and initiate cellular changes. Cental possesses a zinc finger domain which binds the
kinesin motor protein KIF13B and assists in transport of Cental to the plasma
membrane [28], The zinc finger domain is incorporated into a larger ARF GAP
53


domain, through which Cental associates with and regulates G proteins involved in
actin rearrangement and intracellular transport [15, 16]. Cental also possesses two
PH domains with secondary structures that enable Cental to bind PIP3 [17]. Finally,
Cental contains a putative NLS. The individual functions of Centals structural
domains are well-characterized. Thus, in order to determine their role in Centals
activation of the AP-1 pathway, we also examined these domains individually.
Th's paper continues prior structure-function analyses begun by Reisdorph
and Powell. We examined the ability of three structurally-modified Cental variants to
activate the AP-1 pathway and the effects these modifications have on cellular growth
characteristics, such as contact inhibition and anchorage dependence. The three
structural variants tested included: 1) Cental containing R149C and R273C
substitutions in both PH domains (Dbl PH) [17]; 2) Cental containing the same two
PH mutations, with an additional N-terminal myristoylation signal (Myr Dbl PH); and
3) Cental lacking 125 N-terminal amino acids encompassing the zinc finger and most
of the ARF GAP domains (AZF). Deletion of the zinc finger has been shown to
definitively inhibit localization of cental to the nucleus [28]; therefore, the deletion
was used to determine the impact localization has on Centals ability to activate the
AP-1 pathway. Considering the fact that many of Centals known functions occur in
the cytoplasm or at the plasma membrane, it was hypothesized that Cental would be
able to activate the AP-1 pathway even if its nuclear entry was inhibited. The two PH
variants, one of which was unable to bind cell membranes and the other theoretically
54


anchored to cell membranes, were examined to determine where in the cytoplasm
Cental functions.
AP-1 activity reporter assays indicated that all three Cental structural variants
lost their ability to activate the AP-1 pathway (Figure 6). Prior transformation assays
were performed by Reisdorph and Powell using the two PH variants; the apparent
decrease in these variants transformative capacity (Figures 7-8) reinforces the
concept that the AP-1 pathway is a critical process in the transformation of a normal
cell into a tumor cell. The fact that neither PH variant was able to activate the AP-1
pathway suggests that Cental may function in both a membrane-bound and free state.
Since Cental co-localizes and associates with ARF6, a protein which cycles between
the plasma membrane and cytoplasm [16], this proposal seems probable. The loss of
AP-1 activity associated with the AZF variant may be due to loss of an NLS, but may
also be due to loss of transportation to the plasma membrane, a mechanism aided by
interaction of Centals zinc finger with KIF13B.
While not definitive, the above AP-1 activity results provide the basis for
some conjecture. Unlike the AZF variant, both PH variants retained a potential NLS.
Currently, the effects of double PH mutations on Centals ability to bind the nuclear
membrane remain unpublished. If Centals nuclear localization is in fact required for
AP-1 pathway activation, and the double PH mutations do not inhibit nuclear
membrane binding as they do plasma membrane binding, then one might assume that
55


the cytoplasmic Dbl PH variant would enter the nucleus and initiate AP-1 activation.
Instead, AP-1 activity was completely inhibited.
In order to clearly ascertain the impact of Cental localization on AP-1
pathway activation, additional work is required with both the PH and the AZF
variants. The first step would be to determine whether PH mutations affect Centals
nuclear localization. Second, mysristoylation of the Myr Dbl PH Cental variant must
be confirmed and the variants intracellular location identified. Furthermore, to assign
any type of significance to the observed effects of the zinc finger deletion, it must be
determined whether the deletion impacts more than just Centals ability to activate
the AP-1 pathway. Such a large deletion may, for example, prevent proper protein
folding and completely deactivate the protein. In order to attribute some significance
to the effect its loss has on AP-1 activity, the functionality of the AZF Cental variant
must first be ascertained. Next, smaller and smaller deletions within the zinc finger
domain should be tested in AP-1 and transformation experiments to pinpoint the
region of the protein responsible for Centals AP-1 functions.
In addition to this functional approach, we examined Centals protein
sequence in search of post-translational modifications. Specifically, we were
interested to learn if Cental, like many other cell signaling molecules, possessed any
phosphorylated residues. In vitro kinase assays have shown that PKC phosphorylates
Cental at S87 and T276 [34], but no published data exists on Centals in vivo
phosphorylation status. Based on the fact that Cental is known to associate with
56


several protein kinases, such as PKC, casein kinase 1, and the ERKs, and plays an
important role in the activation of a major cell signaling pathway, we hypothesized
that Cental is phosphorylated in vivo. Using proteomic tools including LC/MS/MS to
obtain sequence data, we sought to confirm the presence of the two reported
phosphorylations and searched for novel phosphorylations as well.
Phosphorylation is typically a transient modification that in signaling
pathways activates a variety of molecules. We employed several methods to stimulate
the addition, and retention, of phosphate groups on Cental. Mammalian cells over-
expressing Cental were treated with PMA to induce potential PKC-mediated
phosphorylation. Additionally, cells were treated with Calyculin A and sodium
pervanadate phosphatase inhibitors to prevent de-phosphorylation of serine,
threonine, and tyrosine residues. Potentially-phosphorylated Cental was isolated from
cell lysate via immunoprecipitation and SDS-PAGE, and digested with
endoproteinases prior to MS analysis. Cental peptides were fractionated via HPLC
and subsequently analyzed with a nano-ESI ion trap or nano-ESI QTOF mass
spectrometer.
MS and MS/MS results confirm that Cental is phosphorylated at Tyrosine
364. The phosphopeptide AVDRPmLPQEyAVEAHFK was not detected on the ion
trap, but was detected with the QTOF instrument in multiple charge states in PMA,
PI+PMA-, and Pi-treated samples. The un-phosphorylated version of the peptide was
detected in de-phosphorylated Cental samples, with a mass difference representative
57


of a single phosphate group. Database searching and manual validation of raw spectra
confirm the presence of this PTM, the first reported in vivo phosphorylation of
Cental. We were unable to confirm or deny the presence of phosphorylated S87 and
T276 in vivo.
The functional implications of phosphorylated Y364 will undoubtedly
constitute the basis of future research. The location of the phosphate group may prove
important to Centals various functions; its position just downstream of the N-
terminal PH domain may indicate a role in Cental-PIP3 binding as well as Cental-
ARF6 interactions. Research must now be performed to determine if and how
phosphorylated Y364 affects Centals intracellular localization, protein and/or lipid
interactions, ARF regulation, and AP-1 pathway activation. One method of obtaining
this information would be to perform function-specific assays using Cental variants
modified at Y364. These assays might utilize targeted mutagenesis to convert Y364
to aspartic acid, an amino acid which mimics a constitutively-phosphorylated residue
[45], or to a constitutively un-phosphorylated residue such as alanine. The effects of
the phosphorylated vs. un-phosphorylated variants on Centals various activities and
interactions within the cell may indicate a role for the phosphate group.
Considering that many cell signaling molecules are activated via
phosphorylation, it would be unsurprising if phosphorylated Y364 proved to be
involved in Centals AP-l-related activities. Additional work is also required to
determine which protein kinase is responsible for phosphorylating Cental. PKC-
58


mediated phosphorylation of S87 and T276 remain unconfirmed in vivo; their
presence in vitro may prove to be an artifact induced by experimental conditions or
close proximity to PKC. Finally, to determine whether phosphorylated Y364 is a
byproduct of stoichiometric imbalance, an attempt should be made to concentrate
endogenous Cental protein from normal cells and identify the same phosphate group
as has been identified in cells over-expressing Cental. Clearly, our discovery poses
numerous questions, and will hopefully stimulate further research. Ultimately, the
more information that is accumulated regarding Cental, the closer we become to
understanding how Cental functions within the cell, and how Cental can initiate
those critical processes that transform a normal cell into a cancer cell.
59


APPENDIX A
ABBREVIATIONS
ABC Ammonium bicarbonate
ACN Acetonitrile
AP-1 Activator Protein 1
ARF ADP-ribosylating Factor
ARF GAP ADP-ribosylating Factor GTPase-activating Protein
BSA Bovine Serum Albumin
CEF Chicken embryo fibroblasts
CID Collision induced dissociation
CIP Calf Intestinal Phosphatase
DBL PH Cental variant containing R149C and R273C mutations
DMEM Dulbeccos Modified Eagles Medium
ECL Electrochemiluminescence
ERK - Extracellular Signal-Regulated Kinase
ESI - Electrospray ionization
ETD Electron transfer dissociation
FA -Formic acid
FBS - Fetal bovine serum
60


GFP Green Fluorescent Protein
HEK Human embryonal kidney cells
HPLC High performance liquid chromatography
LC Liquid chromatography
MAPK Mitogen-activated Protein Kinase
MS Mass spectrometry
MYR DBL PH Cental variant containing R149C and R273C mutations and an N-
terminal myristoylation signal
NLS Nuclear localization signal
PBS Phosphate buffered saline
PBST Phosphate buffered saline + Tween
PCR Polymerase chain reaction
PH Pleckstrin homology
PI Phosphatase inhibitor
PIP3 Phosphatidylinositol 3,4,5-trisphosphate
PKC Protein Kinase C
PMA Phorbol 12-myristate 13-acetate
PMSF Phenylmethylsulphonyl fluoride
PTM Post-translational modification
QTOF Quadrupole time of flight
RCAS Replication-Competent ASLV long terminal repeat with a Splice acceptor
61


SF - Serum-free
SPI - Scored peak intensity
ZF - Zinc finger
AZF - Cental variant lacking 125 N-terminal amino acids
62


REFERENCES
1. Ozanne, B. W., Spence, H. J., McGarry, L. C., and Hennigan, R. F. (2007).
Transcription factors control invasion: AP-1 the first among equals. Oncogene
26, 1-10.
2. Ashida, R., Tominaga, K., Sasaki, E., Watanabe, T., Fujiwara, Y., Oshitani,
N., Higuchi, K., Mitsuyama, S., Iwao, H., and Arakawa, T. (2005). AP-1 and
colorectal cancer. Inflammopharmacology 13, 113-25.
3. Eferl, R., Ricci, R., Kenner, L., Zenz, R., David, J. P., Rath, M., and Wagner,
E. F. (2003). Liver tumor development. c-Jun antagonizes the proapoptotic
activity ofp53. Cell 112, 181-92.
4. Liu, Y., Ludes-Meyers, J., Zhang, Y., Munoz-Medellin, D., Kim, H. T., Lu,
C., Ge, G., Schiff, R., Hilsenbeck, S. G., Osborne, C. K., and Brown, P. H.
(2002). Inhibition of AP-1 transcription factor causes blockade of multiple
signal transduction pathways and inhibits breast cancer growth. Oncogene 21,
7680-9.
5. Shaulian, E., and Karin, M. (2001). AP-1 in cell proliferation and survival.
Oncogene 20, 2390-400.
6. Wisdom, R. (1999). AP-1: one switch for many signals. Exp Cell Res 253,
180-5.
7. Bussolino, D. F., Guido, M. E., Gil, G. A., Borioli, G. A., Renner, M. L.,
Grabois, V. R., Conde, C. B., and Caputto, B. L. (2001). c-Fos associates with
the endoplasmic reticulum and activates phospholipid metabolism. Faseb J
15, 556-8.
8. Olive, M., Krylov, D., Echlin, D. R., Gardner, K., Taparowsky, E., and
Vinson, C. (1997). A dominant negative to activation protein-1 (API) that
abolishes DNA binding and inhibits oncogenesis. J Biol Chem 272, 18586-94.
9. Ameyar, M Wisniewska, M., and Weitzman, J. B. (2003). A role for AP-1 in
apoptosis: the case for and against. Biochimie 85, 747-52.
63


10.
Chanda, S. K., White, S., Orth, A. P., Reisdorph, R., Miraglia, L., Thomas, R.
S., Dejesus, P., Mason, D. E., Huang, Q., Vega, R., Yu, D. H., Nelson, C. G.,
Smith, B. M., Terry, R., Linford, A. S., Yu, Y., Chim, G. W., Song, C.,
Labow, M. A., Cohen, D., King, F. J., Peters, E. C., Schultz, P. G., Vogt, P.
K., Hogenesch, J. B., and Caldwell, J. S. (2003). Genome-scale functional
profiling of the mammalian AP-1 signaling pathway. Proc Natl Acad Sci U S
A 100,12153-8.
11. Bos, T. J., Monteclaro, F. S., Mitsunobu, F., Ball, A. R., Jr., Chang, C. H.,
Nishimura, T., and Vogt, P. K. (1990). Efficient transformation of chicken
embryo fibroblasts by c-Jun requires structural modification in coding and
noncoding sequences. Genes Dev 4, 1677-87.
12. Venkateswarlu, K., and Cullen, P. J. (1999). Molecular cloning and functional
characterization of a human homologue of centaurin-alpha. Biochem Biophys
Res Commun 262, 237-44.
13. Martin, R. K., and Jackson, T. R. (2005). Centaurin beta4 in cancer. Biochem
Soc Trans 33, 1282-4.
14. Hawadle, M. A., Folarin, N., Martin, R., and Jackson, T. R. (2002).
Cytohesins and centaurins control subcellular trafficking of macromolecular
signaling complexes: regulation by phosphoinositides and ADP-ribosylation
factors. Biol Res 35, 247-65.
15. Thacker, E., Kearns, B., Chapman, C., Hammond, J., Howell, A., and
Theibert, A. (2004). The arf6 GAP centaurin alpha-1 is a neuronal actin-
binding protein which also functions via GAP-independent activity to regulate
the actin cytoskeleton. Eur J Cell Biol 83, 541-54.
16. Venkateswarlu, K., Brandom, K. G., and Lawrence, J. L. (2004). Centaurin-
alphal is an in vivo phosphatidylinositol 3,4,5-trisphosphate-dependent
GTPase-activating protein for ARF6 that is involved in actin cytoskeleton
organization. J Biol Chem 279, 6205-8.
17. Venkateswarlu, K., Oatey, P. B., Tavare, J. M., Jackson, T. R., and Cullen, P.
J. (1999). Identification of centaurin-alpha 1 as a potential in vivo
phosphatidylinositol 3,4,5-trisphosphate-binding protein that is functionally
homologous to the yeast ADP-ribosylation factor (ARF) GTPase-activating
protein, Gcsl. Biochem J340 ( Pt 2), 359-63.
64


18. Dubois, T., Zemlickova, E., Howell, S., and Aitken, A. (2003). Centaurin-
alpha 1 associates in vitro and in vivo with nucleolin. Biochem Biophys Res
Commun 301, 502-8.
19. Hayashi, H., Matsuzaki, O., Muramatsu, S., Tsuchiya, Y., Harada, T., Suzuki,
Y., Sugano, S., Matsuda, A., andNishida, E. (2006). Centaurin-alphal is a
phosphatidylinositol 3-kinase-dependent activator of ERK1/2 mitogen-
activated protein kinases. J Biol Chem 281, 1332-7.
20. Blomberg, N., Baraldi, E., Nilges, M., and Saraste, M. (1999). The PH
superfold: a structural scaffold for multiple functions. Trends Biochem Sci 24,
441-5.
21. Vanhaesebroeck, B., Leevers, S. J., Ahmadi, K., Timms, J., Katso, R.,
Driscoll, P. C., Woscholski, R., Parker, P. J., and Waterfield, M. D. (2001).
Synthesis and function of 3-phosphorylated inositol lipids. Annu Rev Biochem
70, 535-602.
22. Randazzo, P. A., Nie, Z., Miura, K., and Hsu, V. W. (2000). Molecular
aspects of the cellular activities of ADP-ribosylation factors. Sci STKE 2000,
RE1.
23. Hammonds-Odie, L. P., Jackson, T. R., Profit, A. A., Blader, I. J., Turck, C.
W., Prestwich, G. D., and Theibert, A. B. (1996). Identification and cloning of
centaurin-alpha. A novel phosphatidylinositol 3,4,5-trisphosphate-binding
protein from rat brain. J Biol Chem 271, 18859-68.
24. Cukierman, E., Huber, I., Rotman, M., and Cassel, D. (1995). The ARF1
GTPase-activating protein: zinc finger motif and Golgi complex localization.
Science 270, 1999-2002.
25. Gillingham, A. K., and Munro, S. (2007). The small G proteins of the Arf
family and their regulators. Annu Rev Cell Dev Biol 23, 579-611.
26. Pavletich, N. P., and Pabo, C. O. (1991). Zinc finger-DNA recognition: crystal
structure of a Zif268-DNA complex at 2.1 A. Science 252, 809-17.
27. Miller, J., McLachlan, A. D., and Klug, A. (1985). Repetitive zinc-binding
domains in the protein transcription factor IIIA from Xenopus oocytes. Embo
J 4, 1609-14.
28. Venkateswarlu, K., Hanada, T., and Chishti, A. H. (2005). Centaurin-alphal
interacts directly with kinesin motor protein KIF13B. J Cell Sci 118, 2471-84.
65


29. Tanaka, K., Horiguchi, K., Yoshida, T., Takeda, M., Fujisawa, H., Takeuchi,
K., Umeda, M., Kato, S., Ihara, S., Nagata, S., and Fukui, Y. (1999). Evidence
that a phosphatidylinositol 3,4,5-trisphosphate-binding protein can function in
nucleus. JBiol Chem 274, 3919-22.
30. Karin, M. (1995). The regulation of AP-1 activity by mitogen-activated
protein kinases. J Biol Chem 270, 16483-6.
31. Reisdorph, R., and Powell, R. Characterization of the mechanisms that
mediate centaurin-al-induced oncogenic transformation. Unpublished
manuscript
32. Martin, D. D., Vilas, G. L., Prescher, J. A., Rajaiah, G., Falck, J. R., Bertozzi,
C. R., and Berthiaume, L. G. (2008). Rapid detection, discovery, and
identification of post-translationally myristoylated proteins during apoptosis
using a bio-orthogonal azidomyristate analog. Faseb J 22, 797-806.
33. Utsumi, T., Kuranami, J., Tou, E., Ide, A., Akimaru, K., Hung, M. C., and
Klostergaard, J. (1996). In vitro synthesis of an N-myristoylated fusion
protein that binds to the liposomal surface. Arch Biochem Biophys 326, 179-
84.
34. Zemlickova, E., Dubois, T., Kerai, P., Clokie, S., Cronshaw, A. D.,
Wakefield, R. I., Johannes, F. J., and Aitken, A. (2003). Centaurin-alpha(l)
associates with and is phosphorylated by isoforms of protein kinase C.
Biochem Biophys Res Commun 307, 459-65.
35. Abercrombie, M., Heaysman, J. E., and Karthauser, H. M. (1957). Social
behaviour of cells in tissue culture. III. Mutual influence of sarcoma cells and
fibroblasts. Exp Cell Res 13, 276-91.
36. Macieira-Coelho, A. (2002). The biology of conformation in the regulation of
the senescent and transformed cell phenotypes. Semin Cancer Biol 12, 165-71.
37. Carney, D. N., Gazdar, A. F., and Minna, J. D. (1980). Positive correlation
between histological tumor involvement and generation of tumor cell colonies
in agarose in specimens taken directly from patients with small-cell carcinoma
of the lung. Cancer Res 40, 1820-3.
38. Thingholm, T. E., Larsen, M. R., Ingrell, C. R., Kassem, M., and Jensen, O.
N. (2008). TiO(2)-based phosphoproteomic analysis of the plasma membrane
and the effects of phosphatase inhibitor treatment. J Proteome Res 7, 3304-13.
66


39. Faoro, L., Loganathan, S., Westerhoff, M., Modi, R., Husain, A. N.,
Tretiakova, M., Seiwert, T., Kindler, H. L., Vokes, E. E., and Salgia, R.
(2008). Protein kinase C beta in malignant pleural mesothelioma. Anticancer
Drugs 19, 841-8.
40. Cory, G. O., Cramer, R., Blanchoin, L., and Ridley, A. J. (2003).
Phosphorylation of the WASP-VCA domain increases its affinity for the
Arp2/3 complex and enhances actin polymerization by WASP. Mol Cell 11,
1229-39.
41. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., and Mann, M. (2006). In-
gel digestion for mass spectrometric characterization of proteins and
proteomes. Nat Protoc 1, 2856-60.
42. Dubois, T., Kerai, P., Zemlickova, E., Howell, S., Jackson, T. R.,
Venkateswarlu, K., Cullen, P. J., Theibert, A. B., Larose, L., Roach, P. J., and
Aitken, A. (2001). Casein kinase I associates with members of the centaurin-
alpha family of phosphatidylinositol 3,4,5-trisphosphate-binding proteins. J
Biol Chem 276, 18757-64.
43. Reiser, G., and Bernstein, H. G. (2002). Neurons and plaques of Alzheimer's
disease patients highly express the neuronal membrane docking protein
p42IP4/centaurin alpha. Neuroreport 13, 2417-9.
44. Reiser, G., and Bernstein, H. G. (2004). Altered expression of protein
p42IP4/centaurin-alpha 1 in Alzheimer's disease brains and possible
interaction of p42IP4 with nucleolin. Neuroreport 15, 147-8.
45. Huang, W., and Erikson, R. L. (1994). Constitutive activation of Mekl by
mutation of serine phosphorylation sites. Proc Natl Acad Sci U SA 91, 8960-
3.
67