From the ¶ Lymphocyte Activation Laboratory and the
Protein Analysis Laboratory, Cancer Research UK London Research
Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn
Fields, London WC2A 3PX, United Kingdom, the
Lymphocyte
Signalling and Development Laboratory, The Babraham Institute,
Cambridge CB2 4AT, United Kingdom, and the
** Institute of Medical Microbiology, Immunology and Hygiene,
81675 Munich, Germany
Received for publication, November 4, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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In the present report we describe the properties
of a novel phospho-specific antiserum that has opened a route to the
characterization of antigen receptor-activated serine kinase pathways
in lymphocytes. The basis for the present work was that Ser-21
in glycogen synthase kinase 3 The T cell antigen receptor
(TCR)1 is the key to T cell
activation and controls the specific immune response. An immediate consequence of triggering the TCR is activation of cytosolic
protein-tyrosine kinases of the Src, Syk, and Tec families (1).
These tyrosine kinases phosphorylate an array of adapter molecules that
initiate a cascade of signaling pathways (2, 3). The key events are the
activation of Ras and Rho family GTPase signaling networks and the
metabolism of inositol phospholipids, which regulate both intracellular
calcium and the activity of diverse serine/threonine kinases. The
serine kinases triggered by antigen receptors include members of the
protein kinase C (PKC) and D (PKD) kinase family, phosphatidylinositol
3-kinase (PI3K)-controlled serine kinases such as protein kinase
B (PKB) and the Raf-1/Mek/Erk1,2/Rsk kinase cascades (4-7).
T cell activation requires a period of sustained signaling, and it is
during this sustained response to antigen receptor engagement that
serine kinase pathways are active (8-10). The action of antigen receptor-regulated serine kinases is essential for T cell activation, but there is little information about their substrates in T cells that
can explain their actions. Some progress in understanding this
action may be gained by looking for the existence of evolutionarily conserved serine kinase pathways in T cells. For example, Erk1/2 phosphorylation of the ternary complex transcription factors Elk1 (11),
PI3K/PKB- or PKC-mediated phosphorylation and inactivation of glycogen
synthase kinase 3 In the last 10 years, some useful biochemical and genetic techniques
have been developed to search for the downstream targets of serine
kinases. These are largely based on the use of purified serine kinases
to find substrates in cell lysates (16) or in cDNA expression
library screening (17). Biochemical strategies require relatively pure
serine kinase preparations of high specific activity and have the
limitation that they examine the substrate specificity of a kinase free
of any spatial restraints that might limit substrate availability in an
intact cell. A recent breakthrough in the analysis of serine kinase
networks came with the production of specific antisera against defined
serine or threonine phosphorylated peptides that could then be used to
map phosphorylation of known proteins as cells respond to external stimuli.
A particular protein kinase will have a theoretical optimal substrate
that is determined by its shape in relation to the reaction pocket of
the kinase. Because this is partly determined by the local residues
around the target serine, it has been possible to work out optimal
amino acid sequences for protein kinases using in vitro
assays of a degenerate peptide library. From this candidate, substrate
sites of serine/threonine kinases have been identified and used to
establish a protein data base motif-scanning program termed Scansite
(18-20). There are thousands of potential serine/threonine kinase
sites within the proteome, and it is still very difficult to predict
and map pathways of serine phosphorylation by data base analysis alone.
In vivo experimental data is still needed to narrow down
potential targets to a reasonable level. In this context, one useful
way to obtain biochemical data to facilitate computer-based searches
for serine kinase targets is to use antibodies raised against
phosphorylated peptide candidate sites (21).
The immunoreactivity of phospho-specific antisera is determined
not only by the presence of a phosphorylated amino acid but also by the
charges and hydrophilic or hydrophobic properties of surrounding amino
acids. Phospho-specific antisera thus have the potential to cross-react
with proteins that contain a phosphorylated peptide that is not
necessarily identical in terms of linear sequence but that is
structurally similar. The potential cross-reactivity associated with
the use of phospho-specific antisera is a feature that can be exploited
to delineate serine kinase networks in cells. In this context,
antibodies reactive with phosphorylated PKB and PKC substrate sequences
have been used recently to define new sites of serine phosphorylation
in intact cells (21-23).
The present study describes the properties of an antiserum termed PAP-1
(phospho antibody for
proteomics-1) that recognizes a tier of
proteins regulated by serine phosphorylation in activated cells. PAP-1
was raised against the phospho-peptide RARTSpSFAEP corresponding to
Ser-21 in glycogen synthase kinase 3 Antibodies and Reagents--
Phospho-peptides
synthesized by Dr. Nicola O'Reiley and colleagues (Peptides
Laboratory, Cancer Research UK) were coupled to keyhole limpet
hemocyanin (Calbiochem, Nottingham, UK) and injected into rabbits
at Murex (Dartford, UK). PAP-1 antiserum was raised against
RARTSpSFAEP, where pS denotes a phospho-serine.
PAP-1 was diluted in an equal volume of glycerol and stored at
DNA Constructs--
The cDNA encoding myc-tagged SLY in the
pSecTag vector was kindly provided by Sandra Beer (Institute of Medical
Microbiology, Munich, Germany). The phosphorylation sites SLY (Ser-27)
was mutated to alanine, following the manufacturer's recommendation
using the QuikChange kit from Stratagene. The point mutations were
confirmed by sequencing.
Cells and Cell Stimulation--
T lymphocytes were prepared from
peripheral blood mononuclear cells isolated from healthy donors. Human
T lymphoblasts were generated as described previously (24) and
maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal
calf serum (FCS), 2 mM glutamine, penicillin, streptomycin,
and IL-2 (20 ng/ml) (Chiron, Harefield, UK) at 37 °C and 5%
CO2. After 2 weeks of growth, lymphoblasts were rested by
washing in medium and allowing to rest in RPMI 1640 with 10% FCS in
the absence of IL-2 for 48 h. The mouse B lymphoma cell line A20
were cultured in RPMI 1640 supplemented with 5% FCS, 2 × 10
The PKB-ER retinal pigment epithelial (RPE) cells
(Clontech, Cowley, Oxford, UK) were kindly provided
by Dr. Julian Downward and Dr. Subham Basu (Cancer Research UK London
Research Institute). The cells were cultured in Dulbecco's modified
Eagle's medium:Ham's F-12 supplement with 2 mM
L-glutamine, 0.348% sodium bicarbonate, and 10% FCS.
Rested T-lymphoblasts were stimulated at 2 × 107
cells per milliliter in RPMI for 10 min, unless otherwise mentioned,
with either 50 ng/ml PDBu (Calbiochem, Nottingham, UK) or 10 µg/ml of the anti-CD3 antibody UCHT1. Similarly, A20 cells were
stimulated with either 10 µg/ml F(ab')2 rabbit anti-mouse
antibody (Zymed Laboratories Inc., Cambridge, UK) or a
pervanadate solution of 100 µM NaVO3, 1 mM peroxide. Cells were washed in cold PBS pelleted and
frozen at Transient Transfection--
A20 were electroporated as
previously described (25). Briefly, in 4-mm electroporation cuvettes
107 cells and the indicated quantity of plasmid DNA were
resuspended in 500 µl of RPMI medium and submitted to one pulse of
310 mV at 960 microfarads in a Bio-Rad electroporator yielding a time constant of 19-21 ms. A20 cells were then resuspended in complete medium at 106 per ml and left to express the transfected
protein at 37 °C in a 5% CO2 incubator for 12-17 h.
Cells Extracts and Immunopurification--
T-cells
(107), A20 cells (107), or RPE cells (2 × 106) were lysed in 0.5 ml of lysis buffer containing 1%
Nonidet P-40, 150 mM NaCl, 50 mM HEPES, pH 7.4, 10 mM NaF, 10 mM iodoacetamide, 20 mM Western Blot and Peptide Competition--
Proteins were
separated by SDS-PAGE using 7.5% acrylamide/0.2% bisacrylamide gels
and transferred onto Immobilon P membranes. Proteins were detected by
Western blot with the indicated primary antibodies diluted 1:1000 in
PBS-1% bovine serum albumin and 0.05% sodium azide. All secondary
antibodies were used at 1/10,000. Immunoreactive bands were visualized
with the chemiluminescence Western blotting system (Amersham
Biosciences). For peptide competition assays, the diluted primary
antibodies were mixed 5 min prior using with 10 µg/ml of the
indicated peptide.
ELISA--
The ELISAs were performed using standard methods.
Briefly, 10-amino acid peptides were coated onto ELISA plates
(Nalge NUNC, Roskilde, Denmark) at 50 ng per well in pH 9 carbonate
buffer. After blocking with PBS/0.05% Tween 20/5% FCS, the indicated
dilution of PAP-1 was incubated for 1 h, and the bound
immunoglobulins were assayed by a further incubation with a horseradish
peroxidase-conjugated goat anti-rabbit antibody (DAKO Ltd., Ely, UK)
and O-phenylene diamine tablets (Sigma-Aldrich,
Gillingham, UK) as a colorimetric substrate. The values of the optical
density measured in wells not incubated with PAP-1 but with diluent
only, were subtracted.
Chromatography and Protein Purification--
Pellets of 9 × 109 lymphoblasts stimulated with 50 ng/ml PDBu were
defrosted in 10 ml of lysis buffer (50 mM MES at pH 5, 150 mM NaCl, 10 mM NaF, 40 mM
Mass Spectrometry--
Tryptic peptides were analyzed on a
Tofspec 2E (Micromass, Manchester, UK) matrix-assisted laser desorption
time-of-flight (MALDI-TOF) mass spectrometer with saturated
A polyclonal rabbit antiserum was raised against the
phospho-peptide RARTSpSFAEP, where pS is a
phospho-serine corresponding to the GSK3 is robustly phosphorylated following
antigen receptor triggering. We predicted accordingly that antigen
receptors would also stimulate phosphorylation of other proteins with a
similar sequence. To test this idea we raised an antibody against the phospho-peptide RARTSpSFAEP, where pS is a phospho-serine
corresponding to the glycogen synthase kinase 3
Ser-21 sequence. The
resulting antiserum was called phospho antibody
for proteomics-1 (PAP-1). The present
study describes the properties of PAP-1 and shows that it can reveal
quite striking differences in the phospho-proteome of different cell
types and is able to pinpoint new targets in important signal
transduction pathways. PAP-1 was used to map protein phosphorylations
regulated by the antigen receptor in T cells. One of these
PAP-1-reactive proteins was purified and revealed to be a previously
unrecognized target for antigen receptor signal transduction, namely an
"orphan" adapter SLY (Src homology 3 (SH3) domain-containing
protein expressed in lymphocytes). The use of sera detecting specific
phosphorylation sites is thus proved as a powerful method for the
discovery of novel downstream components of antigen receptor signals in
T cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and 3
(GSK3
/
) (9, 12), and
PI3K/PKB-regulated phosphorylation of Forkhead family transcription factors (13-15) are all conserved responses that have been described in T cells. However, such an approach ignores the possibility that
ubiquitous kinases might have unique T cell-restricted substrates. Lack
of knowledge about serine kinase substrates is a bioinformatic problem
common in many cell systems. Phospho-amino acid analysis of the
proteome shows that serine phosphorylation is one of the most common
post-translational events. However, reliable identification of serine
kinase targets in intact cells has proved problematic and is a major
rate-limiting step in furthering our knowledge and understanding of
signal transduction pathways in eukaryotic cells.
, a known substrate for Akt/PKB.
PAP-1 recognizes a subset of PKB substrates and can be used to identify
novel components of PKB signaling. However, the immunoreactivity of
PAP-1 is quite distinct to that of antisera raised against the PKB
substrate sequence RXRXXpS/T. In T lymphocytes, the T cell antigen receptor is known to regulate the
activity of diverse serine kinases, but analysis of the complex patterns of serine phosphorylation in activated T cells has been a
laborious and slow process. In the present work, PAP-1 was used to map
protein phosphorylations regulated by the antigen receptor in T cells.
One of these PAP-1-reactive proteins was purified and revealed to be a
previously unrecognized target for antigen receptor signal
transduction, namely an "orphan" adapter SLY (Src homology 3 (SH3)
domain-containing protein expressed in lymphocytes). PAP-1 is thus able
to identify novel downstream components of antigen receptor signals in
T cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. Antibodies that recognized phospho-Ser473 PKB, pan PKB,
phospho-Ser21/9 GSK3
/
, phospho-Ser-256 FKHR, and the
phospho-substrate antibodies (PKB phospho-substrate, phospho-14-3-3 binding motif, and phospho-Ser/Thr-Phe) were purchased from New England
BioLabs (Beverly, MA). The pan GSK3 and phospho-Thr24 FKHRL1 antibodies
were purchased from Upstate Biotech Inc. (Lake Placid, NY). The IL-16
antibody was from R&D Systems (Abingdon, UK). The phospho-ERK1/2
antibody was from Promega (Southampton, UK). The Hybridoma
development unit of Cancer Research UK purified the anti-CD3 UCHT1 and
9E10 (anti-Myc) antibodies. Horseradish peroxidase-coupled donkey
anti-rabbit and sheep anti-mouse antibodies were obtained from Amersham
Biosciences (Little Chalfont, UK). Silver staining was performed using
the Owl kit (Cambridge Biosciences). Materials for protein
purification, including all columns and electrofocusing reagents, were
obtained from Amersham Biosciences.
5 M
2-mercaptoethanol,
penicillin, and streptomycin.
80 °C before being lysed. When indicated, cells were
preincubated with one of the following inhibitors for 30 min, either
PD098059 (New England BioLabs) or LY294002 (BIOMOL Research
Laboratories, Plymouth Meeting, PA). RPE cells were grown in 100-mm
tissue culture dishes and treated with 100 ng/ml tamoxifen for 4 h
or 50 ng/ml PDBu for 5 min, washed in cold PBS, and lysed in 1 ml of
lysis buffer per dish.
-glycerophosphate, 1 mM EDTA, 100 µM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and the small peptide inhibitors
aprotinin, leupeptin, pepstatin A, and chymostatin, all at 1 µg/ml.
Whole cell lysates were cleared at 14,000 rpm for 10 min, and acetone
was precipitated before being resuspended in reducing sample buffer as
previously described (26). For immunoprecipitation, the cleared lysates
were transferred to a fresh tube containing 20 µl of protein
G-agarose beads (Amersham Biosciences) and precleared at 4 °C for 15 min before being centrifuged at 5000 rpm for 5 min. The supernatant was
then transferred to a fresh tube containing 10 µl of anti-IL-16
antibody at 0.5 µg/µl in phosphate-buffered saline. After 1 h
of gentle agitation at 4 °C, 20 µl of protein G-agarose beads was
added to each sample and incubated for a further 2 h. The tubes
were then centrifuged as before, and the supernatant acetone was
precipitated. The bead complexes were then washed three times in 0.5 ml
of lysis buffer before being resuspended in 20 µl of reducing sample buffer.
-glycerophosphate, 1 mM EDTA, 1 mM PMSF,
0.1% Nonidet P-40, 1 µM small peptide inhibitors)
followed by a 10-fold dilution in low salt lysis buffer (20 mM Tris at pH 7.2, 10 mM NaF, 40 mM
-glycerophosphate, 1 mM EDTA, 1 mM PMSF, 1 µM small peptide inhibitors). The solution was first
cleared at 45,000 × g for 30 min. The supernatant was
then loaded onto a 200-ml heparin-Sepharose column equilibrated in low
salt buffer. The column was developed at 2 ml/min with a linear
gradient to 1 M NaCl. Ninety fractions of 13 ml were
collected, and the 55- and 85-kDa protein-positive fractions were
identified by Western blotting 40-µg aliquots from each fraction with
PAP-1. The fractions rich in PAP1p85 and PAP1p55 were pooled from three
runs of the heparin-Sepharose column and run through a gel-desalting
column equilibrated in low salt lysis buffer. The desalted samples were chromatographed on an 8-ml Mono Q column equilibrated in low salt buffer. The column was developed at 2 ml/min with a linear gradient to
1 M NaCl. Twenty-six fractions of 6 ml were collected.
Western blotting identified the fractions rich in PAP1p55 and p85 as
before using 4-µg aliquots. After this purification the PAP1p55 and
p85 proteins were separated into different groups of fractions, which were pooled separately. The two sets of pooled fractions were precipitated in acetone and resuspended in two-dimensional PAGE rehydration buffer (urea 9 M, CHAPS 2%, dithiothreitol
0.28%, immobilised pH gradient buffer bought from Amersham Biosciences buffer 0.5% (v/v) and a trace of bromphenol blue). Samples were then run, following manufacturer's instructions on an Igphor
isoelectric focusing system using a 13-cm, pH 4-7 linear strip. The
second dimension was resolved by standard SDS-PAGE. For each sample, a
small aliquot (40 µg of protein) was run in parallel with the main
sample (400 µg of protein). The two-dimensional PAGE from the aliquot
was transferred onto Immobilon membranes and probed with PAP-1, whereas
the main sample was stained for total protein using brilliant blue-G
colloidal stain (Sigma-Aldrich, Gillingham, UK). Protein spots that
co-localized with PAP-1-identified bands on Western blotting were cut
out and identified by mass spectrometry.
-cyanocinnic acid as the matrix. The mass spectrum was acquired in
the reflector mode and was internally mass-calibrated. The tryptic
peptide ions obtained were used to search a non-redundant data base
compiled by the National Centre for Biotechnology (NCBI) using the
MS-Tag program of protein prospector (University of California, San
Diego, CA). To confirm the MALDI-TOF data tryptic digests, spots of
interest were analyzed by nano-LC (LC Packings, Camberley, UK) coupled to a hybrid electrospray quadrupole-time-of-flight mass analyzer (Q-TOF) (Micromass, Manchester, UK). Separation was performed using a
75-µm × 150-mm C18 column at 200 nl/min. Q-TOF was
operated in data-dependent switching mode and used
with automated collision-induced dissociation on multiple charged
precursor ions of interest. Resultant fragmentation ion spectra were
scanned against the NCBI data base using MS-Tag.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ser-21 sequence. In initial
experiments to explore the reactivity of the resultant antiserum PAP-1,
we carried out Western blot analyses of cell lysates isolated from quiescent and pervanadate (PV)-activated T lymphocytes. Pervanadate is
used to pharmacologically induce a potent stimulation of
tyrosine kinases and their downstream effector pathways in lymphocytes and, hence, is a very useful tool for synchronously activating a broad
range of serine kinases in these cells. PAP-1 recognized a single
160-kDa protein in the unstimulated T cells but was strongly reactive
with a number of proteins across a wide molecular weight range isolated
from pervanadate-activated cells (Fig.
1A). These have molecular
masses of 50, 55, 75, 80, 85, 250, and 400 kDa, respectively. It
is important to establish whether PAP-1 can recognize proteins
phosphorylated in response to physiologically relevant stimuli, and,
hence, the immunoreactivity of PAP-1 with T cell lysates isolated from
antigen receptor activated T cells was examined. PAP-1 was strongly
reactive with several proteins in cell lysates isolated from stimulated
but not quiescent cells; the predominant proteins recognized by PAP-1
in TCR-activated cells were those with molecular masses of 50, 55, 85, and 250 kDa (Fig. 1B).
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Fig. 1.
Characterization of PAP-1 recognition pattern
in primary human T cell lysates. A, T cells were left
unstimulated (0) or were treated for 5 min with 100 µM pervanadate (PV). Total proteins from whole
cell lysates were subjected to Western blot analysis using PAP-1.
B, T cells were stimulated through their antigen receptor,
or not (0), for 5 min with 10 µg/ml of the anti-CD3
cross-linking antibody UCHT1. Whole cell lysates were subjected to
Western blot analysis with either PAP-1 (top panel),
anti-P-Ser-256-FKH (middle panel), mixed together with 10 µg/ml of the following competing oligopeptides. Lanes 1 and 2, non-phosphorylated sequence from GSK3
(RARTSSFAEP); lanes 3 and 4, P-Ser-21-GSK3
(RATSpSFAEP); lanes 5 and 6, P-Ser-256-FKHR
(RRRAApSMONN); lanes 7 and 8, P-Thr-23-IKK
(RERLGpTGGFG). Membranes were stripped and reprobed for GSK3.
C, T cells were left non-stimulated (0) or
treated with 10 µg/ml UCHT1 for 5 min in the presence of 1:1000
Me2SO (vehicle control). The indicated inhibitors were
added 30 min before stimulation. Whole cell lysates were subjected to
Western blot analysis with PAP-1 or anti-P-Ser473-PKB and anti P-ERK
1/2 antibodies. Membranes were stripped and reprobed with anti-pan
GSK3
/
.
The phospho-peptide immunogen used to generate PAP-1 was based around
Ser-21 in GSK3. The p50 protein recognized by PAP-1 in activated T
cells co-migrates on SDS-PAGE gels with GSK3
, and its identity as
GSK3
was confirmed by its co-elution during protein purification on
heparin-Sepharose and Mono Q columns (see below). The identity of the
other proteins recognized by the antiserum was unknown. Ser-21 in
GSK3
is a substrate for PKB (27). However, the immunoreactivity of
PAP-1 with these additional proteins was competed by the immunizing
phospho-peptide phospho-Ser-21 GSK3
(RARTSpSFAEP) and not by other
phosphorylated PKB substrate peptides, phospho-Ser-256 FKHR
(RRRAApSMONN) (28) or phospho-Thr-23 IKK
(RERLGpTGGFG) (29).
A commercial antiserum that recognizes phosphorylated FKHR serine 256 is available. This antiserum recognized a single protein of 97 kDa in
quiescent T cells and two proteins of 85 and 97kDa in antigen
receptor-activated T cells (Fig.
2B, middle panel). These did not co-migrate with any of the PAP-1-reactive proteins. Moreover, the immunoreactivity of the phospho-Ser-256 FKHR antiserum was competed by the phospho-Ser-256 FKHR peptide but not the
PAP-1-immunizing phospho-peptide or the phospho-Thr-23 IKK peptide.
The phospho-peptide competitions show that phospho-antisera are highly
selective and indicate that PAP-1 reacted with proteins that contain a
sequence structurally similar to GSK3
, Ser-21.
|
The TCR regulates GSK3 serine phosphorylation via PI3K-dependant
pathways (9). The PI3K inhibitor LY294002 prevents the PKB activation
response seen in TCR-activated cells. It also suppresses activation
of the MAPKs Erk1 and -2 (Fig. 1C). TCR induction of the phosphorylation of GSK3
(PAP-1p50), PAP1 p55, p80/85, and p250
was inhibited by Ly294002. PAP-1-reactive proteins, like GSK3
, are
regulated by PI3K pathways (Fig. 1C).
PAP-1: A Phospho-peptide-specific Antiserum That Recognizes a
Subset of PKB Substrates--
A critical question was whether the
PAP-1 antiserum would be a pan PKB substrate antiserum with the
potential to identify novel substrates for this kinase. Moreover, an
interesting question was whether the PAP-1 phospho-map shown in
activated T cells was unique or seen in other cell lineages. To probe
this issue we analyzed PAP-1 immunoreactivity with a human epithelial
cell line, RPE, which expressed a tamoxifen-inducible PKB mutant. In
quiescent RPE cells there is low basal activity of PKB and some basal
phosphorylation of GSK3 on Ser-21 (see Fig. 4 below). PKB activity
is induced by tamoxifen treatment of the cells with a concomitant
increase in GSK3
Ser-21 phosphorylation
(30).2
The data (Fig. 2) show that PAP-1 recognizes two main proteins in quiescent RPE cells, a p50 protein and an unknown 200-kDa protein. In tamoxifen-activated RPE cells, PAP-1 recognizes two additional proteins of 65 and 120 kDa. These were not seen in RPE cells activated with phorbol esters that activate PKC but not PKB pathways. The immunoreactivity of PAP-1 was competed by the immunizing PAP-1 phospho-peptide. It is of note that the immunoreactivity pattern of PAP-1 with lysates from PKB-activated RPE cells was different from that seen in antigen receptor-activated T cells.
During the course of these experiments, Cell Signaling Technology
(Beverley, MA) raised an antiserum against a mixture of phosphorylated
PKB substrate peptides. This antiserum recognizes the PKB substrate
sequence RXRXXpS/T, where X
is any amino acid and is referred to as a CST-pan PKB phospho-substrate
antiserum (21, 22). An important question was whether PAP-1 is also a
pan PKB substrate antiserum with an overlapping immunoreactivity with
this commercial antiserum. Accordingly, the immunoreactivity of PAP-1
and the CST pan PKB phospho-substrate antiserum on RPE cells that
express a tamoxifen-inducible PKB was also compared (Fig. 2,
A versus B). Key points were that the CST-pan PKB
phospho-substrate antiserum had a completely different pattern of
reactivity to PAP-1 in RPE cells. The pan PKB substrate antiserum did
not react with a p50 protein corresponding to GSK3. Nor did the pan
PKB substrate antiserum recognize the PAP-1-reactive p65 or p120
proteins; rather, it recognized a major protein, p70, in
tamoxifen-activated cells and p70 plus an additional p97 protein in
phorbol ester-activated cells. The pan PKB substrate antiserum was also
strongly reactive with a number of proteins migrating around p200 that
were not seen with PAP-1. Strikingly, the pan PKB phospho-substrate
antiserum was strongly reactive with proteins from PDBu-activated RPE
cells, whereas PAP-1 was not.
These results indicate that PAP-1 has the potential to recognize novel
PKB substrates. However, its different pattern of immunoreactivity to
the commercial pan PKB substrate antisera indicates that PAP-1 is not
broadly reactive with all PKB substrates. One further indication that
PAP-1 does not recognize all PKB substrates is given by the T cell data
in Fig. 1. Thus both GSK3 and the faster migrating GSK3
are known
PKB substrates, and commercial antisera selectively reactive with
phospho-GSK3
and
thus recognize a doublet of proteins. In
contrast, PAP-1 only detected the 50-kDa GSK3
protein and not the
GSK3
/
doublet (Fig. 1, B and C).
To explore the selectivity of PAP-1 further, the immunoreactivity of
this antiserum was examined by ELISA. Fig.
3A shows that PAP-1 displayed
a strong immunoreactivity with the immunizing phospho-peptide Ser-21 in
GSK3 but did not recognize a range of other phosphorylated PKB
substrate peptides, including phospho-Ser-256 in FKHR (28),
phospho-Thr-32 in FKHRL1 (31), phospho-Thr-23 in IKK (29), and
phospho-Ser-136 in BAD (32). To conclude, PAP-1 is not a pan PKB
substrate antiserum but rather a phospho-peptide specific antiserum
with the potential to recognize a subset of PKB substrates.
|
The previous data show that PAP-1 can recognize a subset of PKB
substrates, but another question is whether it has a broader specificity. In this context, GSK3 Ser-21 phosphorylation can be
regulated by PKB or via a PKC-mediated response (33, 34). It would be
predicted that the PAP-1-reactive proteins phosphorylated by
PI3K-mediated pathways in antigen receptor pathways might also be
phosphorylated in response to activation of PKC with phorbol esters.
Fig. 3B examines the effects of activation of T cells with
the phorbol ester; phorbol 12,13-dibutyrate (PDBu) on PAP-1-recognized proteins. PDBu mimics diacylglycerol and triggers protein kinase C and
D (PKC/PKD)-mediated responses without PKB activation. T cells induce
high levels of PAP1p50 (GSK3
) phosphorylation when activated with
phorbol esters. PAP1p85 and p55 were also seen in phorbol ester-treated
cells. The reactivity of CST-pan PKB antiserum with antigen receptor or
PDBu-activated T cells was also examined (Fig. 3C). This
antiserum also recognized proteins in activated T cells, but its
pattern of immunoreactivity was different to that of PAP-1. The CST-pan
PKB antiserum was strongly reactive with a 90-kDa protein in quiescent
and activated T cells. This band was not seen in PAP-1 Western blots.
PAP-1 was strongly reactive with p55 and p85, and these were not seen
with CST-pan PKB. Conversely, CST-pan PKB recognizes p60 and p100
proteins in activated T cells, and these were not seen with PAP-1. The CST-pan PKB recognizes a p50 protein in phorbol ester but not TCR-activated T cells, whereas PAP-1 recognizes a p50 protein in TCR
and phorbol ester-activated cells.
Identification of PAP-1-reactive Protein p55-- PAP-1 was able to recognize a number of proteins phosphorylated via PI3K pathways in antigen receptor-activated T cells. It can also recognize proteins phosphorylated in T cells activated via a phorbol ester/PKC pathway. Its immunoreactivity is highly selective for the cognate immunizing phospho-peptide, but it is not a pan kinase substrate antiserum. It has very different patterns of immunoreactivity in different cell populations and on cells activated by different stimuli. It thus recognizes protein phosphorylation events at the level where there is convergence of signaling of multiple pathways. PAP-1 can therefore be used to pinpoint novel cell specific or stimulus specific phosphorylation events for future analysis.
Given the selectivity of PAP-1 for its cognate phospho-peptide, all of
these proteins are likely to contain a phosphorylated peptide sequence
structurally similar to GSK3 Ser-21. The next stage in this process
was to purify PAP-1-identified proteins. From the experiments described
above, the PAP-1-reactive protein p55 was selected for further work.
The rationale for this choice was that this was an antigen
receptor-regulated protein. Moreover, a comparison of the PAP-1
phospho-fingerprint of T cells and RPE cells indicated that p55 is
selectively expressed/regulated in T lymphocytes. This was confirmed by
extensive comparisons of the pattern of PAP-1 immunoreactivity with
cell lysates isolated from different cell populations (data not shown).
Initial experiments found that the PAP-1 antiserum could not
selectively immunoprecipitate proteins from activated T cells. Alternative experimental protocols were therefore devised to purify p55
to homogeneity for protein sequence analysis by mass spectrometry. Cell
lysates were prepared from 9 × 109 activated T
lymphoblasts, and the lysates were fractionated initially on
heparin-Sepharose columns; a second step purification on Mono Q columns
was performed followed by separation by two-dimensional SDS-PAGE gels.
At each stage the presence of p55 in different column fractions was
determined by Western blot analysis with PAP-1. Fig.
4 shows the elution profiles on
heparin-Sepharose (Fig. 4A) and Mono Q columns (Fig.
4B) of p50 and p55 PAP-1-reactive proteins. p50 co-eluted
with GSK3, whereas p55 had distinct elution patterns. Fig.
4C (left panel) shows silver-stained
two-dimensional SDS-PAGE gels of the final fractionation step; Fig.
4C (right panel) shows the corresponding PAP-1
Western blot analysis of the purified material. The protein
spots corresponding to p55 were then digested with trypsin, and the
tryptic peptides were analyzed by matrix-assisted laser desorption
time-of-flight (MALDI-TOF) mass spectrometry (35). MALDI-TOF analysis
identified a single oligopeptide from p55, and it proved impossible to
obtain an effective fingerprint of this molecule. However, protein
sequencing of p55 tryptic peptides identified that p55 contained the
peptide (K)FIYVDVLPEEAVGHARPSR(R) (Fig.
5A). This corresponded to
amino acid residues 226-244 of a very recently described molecule
known as SLY (Src homology 3 (SH3) domain-containing protein expressed
in lymphocytes) (36).
|
|
Ser-27 in SLY Is a Substrate for Antigen Receptor-activated Serine
Kinases--
To identify the phosphorylated residues that are
recognized by PAP-1, we examined the sequences of SLY for peptides
corresponding to the PAP-1-immunizing peptide. SLY comprises 380 amino
acids, and Ser-27 had the closest homology to the GSK-3 Ser-21
peptide used to produce PAP-1 (Fig. 5B). Accordingly, a
phosphorylated peptide corresponding to SLY Ser-27
(LQRSSpSFK) was synthesized. PAP-1 was reactive with this
phospho-peptide as judged by ELISA (data not shown). The ability of
this peptide to compete PAP-1 immunoreactivity with p55 was also
assessed. The results in Fig. 6A show that the
phospho-peptide corresponding to Ser-27 in SLY was able to efficiently
compete out PAP-1 immunoreactivity with p55.
|
No immunoprecipitating antiserum to SLY is available. Hence, to confirm SLY as a PAP-1-reactive protein, a Myc epitope-tagged SLY was expressed in A20 cells. These cells were then activated with pervanadate or via the B cell antigen receptor, and whole protein lysates were subjected to Western blot analysis with PAP-1 followed by reprobing with 9E10 anti-Myc to confirm expression. The data (Fig. 6B) show that SLY expressed in activated lymphocytes is immunoreactive with PAP-1, whereas SLY expressed in quiescent cells is not. These experiments also confirm that SLY is a substrate for antigen receptor-regulated serine kinases. To determine if Ser-27 in SLY is the sequence recognized by PAP-1, this residue was substituted with alanine. The SLY S27A mutant was also not reactive with PAP-1. These mutant studies thus establish Ser-27 in SLY as a substrate for antigen receptor-activated serine kinases.
The initial characterization of PAP-1 showed that this antisera could
recognize proteins phosphorylated via PKB or PKC pathways. In this
context, the SLY Ser-27 sequence, LSLQRSSSFKDFAKS, is not a good
consensus PKB phosphorylation site. Instead, analysis of SLY with
Scansite, a protein data base motif-scanning program (19), indicated
that Ser-27 is an optimum substrate sequence for protein kinase C
family kinases. The data in Fig. 3 show that SLY phosphorylation was
induced by phorbol ester activation of T cells. The data in Fig.
7A show also that SLY is not
phosphorylated when T cells are activated by the cytokine interleukin
2. This cytokine is a strong activator of PKB and MAPKs and induces
robust phosphorylation of GSK3 (Fig. 7A). However, IL-2
is unable to activate PKC, as judged by its inability to induce
phosphorylation of a known downstream target of PKC, protein kinase D
(10) (Fig. 7A). Furthermore, pretreatment of T cells with RO
31-8425, a broad specificity PKC inhibitor, prevents TCR-mediated
phosphorylation of SLY without attenuating activation of PKB (Fig.
7B). These data collectively show that the regulated
phosphorylation of SLY Ser-27 correlates with activation of PKC
pathways in T cells.
|
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DISCUSSION |
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Antisera broadly reactive with structurally related
serine-phosphorylated peptides are emerging as valuable tools with
which to unmask complex patterns of serine phosphorylation in cells. Serine phosphorylation is one of the most frequent post-translational modifications, but pathways of serine phosphorylation are difficult to
resolve because it is difficult to distinguish basal serine phosphorylations from extracellular signal-regulated events. The challenge has been how to resolve the huge background noise seen in
most forms of analysis. The present data describe a novel antiserum PAP-1 that can be used for this purpose. The rationale behind the
generation of PAP-1 was that phospho-peptide-specific antisera would
cross-react with proteins that share similar phosphorylated peptide
sequences. The cellular system used to exploit this strategy was
antigen receptor-activated human T lymphocytes. One evolutionary conserved pathway in T cells is the regulated phosphorylation of the
serine kinase GSK3 on Ser-21. The present results show that an
antiserum termed PAP-1, which was raised against a phospho-peptide RARTSpSFAEP, corresponding to GSK3
Ser-21, can be used to
define other substrates for antigen receptor-regulated kinases that are
phosphorylated in parallel to GSK3
and contain a sequence structurally similar to GSK3
Ser-21. PAP-1 thus recognizes a tier of
serine kinase substrates regulated by antigen receptors via
PI3K-mediated pathways.
There have been recent descriptions of pan-kinase substrate antibodies, i.e. antibodies that can be used to identify substrates for known serine kinases such as PKB (22, 23). The immunizing peptide for PAP-1 is a PKB substrate, but it should be emphasized that PAP-1 only recognizes a subset of PKB substrates. Moreover, the PAP-1-immunoreactive pattern in activated T cells and epithelial cells is very different from the immunoreactivity pattern of the recently characterized Cell Signaling Technology "pan PKB substrate antiserum" (CST-pan PKB) raised against the panel of peptides with the consensus RXRXXpS/T, where X is any amino acid. It should also be emphasized that neither PAP-1 nor CST-pan PKB antisera have immunoreactivity restricted to PKB substrates. Both recognize proteins phosphorylated via PKC pathways in T lymphocytes. Moreover, the CST-pan PKB antisera recognize proteins phosphorylated in response to PKC activation in epithelial cells, whereas PAP-1 does not. These antibodies are thus very useful for phospho-proteomic analysis of cell activation, but they are not useful as reporters for activation of a particular kinase pathway.
The PAP-1 antiserum had a very distinct pattern of immunoreactivity in
different cell types. From simple comparative analysis, it was possible
to identify proteins that were regulated by antigen receptors and
apparently unique to T cells. Hence, in activated epithelial cells
PAP-1 recognized GSK3 and proteins of 65 and 120 kDa, whereas in
activated T cells PAP-1 predominantly recognized GSK3
and p55 and
p85. Analysis with PAP-1 thus could pinpoint novel targets in pathways
of antigen receptor-regulated serine phosphorylation. Similarly, in
epithelial cells expressing an inducible active PKB, PAP-1 could
identify novel proteins phosphorylated by a PKB pathway. Interestingly,
the PAP-1-reactive proteins in T cell or epithelial cells were not
recognized by the CST pan PKB substrate antisera. CST pan PKB is
broadly reactive with a range of phospho-peptides that are substrates
for the ACG family of serine kinases, including PKB/Akt, but the
present data show it does not recognize all substrates for this kinase.
PAP-1 and CST-pan PKB thus appear to recognize non-overlapping subsets
of PKB substrates. Accordingly, it looks like it will be difficult to
get a complete analysis of protein phosphorylation pathways using a
single antisera broadly reactive with serine-phosphorylated peptides.
Instead, a combination of different antisera that are selective against
different peptides will be required.
One of the PAP-1-reactive proteins in antigen receptor-activated T cells, p55, was selected for further characterization because it was regulated by antigen receptors and apparently unique to T cells. p55 was then purified and identified as SLY, the prototypical member of a recently identified family of adapter proteins with restricted expression in hematopoietic cells (36). The analytical work with PAP-1 had pinpointed p55 as a lymphoid-restricted protein, and SLY indeed has restricted expression to lymphoid tissues. SLY was originally found "accidentally" as part of an expression screen for genes that could stimulate adhesion of T lymphoma cells to endothelial cells (36). The SLY gene product was not able to induce cell adhesion, but its isolation was reported because its structure suggested that it might function as a signaling adapter protein. Northern blot and in situ hybridization analysis showed a preferential expression in lymphoid tissues, and the SLY gene is located on the X chromosome in proximity to genes involved in various immune disorders. SLY was so named because it is an Src homology 3 (SH3) domain-containing protein expressed in lymphocytes. The protein also contains a sterile motif (SAM). SH3 domains are comprised of about 50 amino acid residues that bind to proline-rich sequences of proteins with the consensus PXXP. SAM domains mediate homodimerization and heterodimerization of proteins (37). This structure makes it very likely that SLY can act as an adapter, although there have been no studies of its interaction partners. Interestingly, other hematopoietic proteins closely related to SLY have been described recently, HACS1 (38) and NASH (39). SLY, HACS1, and NASH have a similar organization of SH3 and SAM domains and appear to be restricted in expression to hematopoietic cells. Serial analysis of gene expression identified NASH as a gene preferentially expressed in mast cells; HACS1 was found in a wide range of hematopoietic cells, whereas SLY is preferentially expressed in lymphoid cells. The structures and restricted tissue expression of SLY, HACS1, and NASH suggest that these proteins might have a specific role in hematopoietic cells. The present data provide the first insights into the position of these adapter proteins in the context of signal transduction in lymphocytes by demonstrating that Ser-27 in SLY is a physiological substrate for antigen receptor-activated serine kinases. Moreover, SLY phosphorylation seems to occur selectively in response to antigen receptor engagement via a PKC-mediated pathway that cannot be triggered by cytokines. Ser-27 in SLY is contained within the sequence LQRSSSFK, and this sequence is conserved in SLY in different species from fish to mammals. Moreover, it is striking that the serine at position 27 and the surrounding sequence (RSSSFK) is conserved in the SLY-related proteins HACS1 and NASH. The conservation between SLY, HACS1, and NASH in terms of the PAP-1-recognized phosphorylation site provides clear biological insight into where to position other members of this adapter protein family in signal transduction cascades in hematopoietic cells. The identification of SLY as an antigen receptor-regulated serine kinase target also shows that the phospho-proteomic approach with PAP-1 described herein can identify links between antigen receptors and novel molecules and give some insight as to when and where such proteins might be important in T cells.
In conclusion, the present study describes an antisera raised against a
defined phospho-peptide that can be used as a tool for
phospho-proteomic analysis of cells. Herein it has been used to probe
lymphocyte activation. PAP-1 and various commercial
phospho-peptide-specific antisera have unique patterns of
immunoreactivity with activated T cell lysates and can be used to make
a phospho-map of TCR-activated cells. Phospho-specific antisera
thus opens a window that can reveal the existence of previously
unknown targets for antigen receptor signal transduction as well as a
new dimension for signal transduction studies in lymphocytes.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
44-20-7269-3591; Fax: 44-20-7269-3479; E-mail:
doreen.cantrell@cancer.org.uk.
Published, JBC Papers in Press, January 5, 2003, DOI 10.1074/jbc.M211252200
2 S. Basu, unpublished.
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ABBREVIATIONS |
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The abbreviations used are: TCR, T cell antigen receptor; PDBu, phorbol 12,13-dibutyrate; PKB, protein kinase B; PKC, protein kinase C; PKD, protein kinase D; PI3K, phosphatidylinositol 3-kinase; GSK3, glycogen synthase kinase 3; PAP-1, phospho antibody for proteomics-1; SH3, Src homology 3; SLY, SH3 domain-containing protein expressed in lymphocytes; IL, interleukin; ERK, FCS, fetal calf serum; RPE, retinal pigment epithelial; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; ELISA, enzyme-linked immunosorbent assay; MES, 4-morpholineethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Q-TOF, quadrupole-time-of-flight; P, phospho-; MAPK, mitogen-activated protein kinase; FKHR, forkhead in rhabdomyosarcoma; SAM, sterile alpha motif.
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