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INTRODUCTION |
Fibroblast growth factor receptors are members of the
receptor-tyrosine kinase family (1). In contrast to other growth factor
receptors such as those for
EGF1 and PDGF, FGF receptors
are poorly auto-phosphorylated upon ligand binding. Instead, a 90-kDa
protein called SNT1 or FGF receptor substrate-2 (FRS2) (2, 3) is
phosphorylated at multiple tyrosine sites. FRS2 has also been reported
to be serine/threonine-phosphorylated in FGF-treated cell lysates (4).
SNT2, a recently identified isoform of FRS2, has about 50% identity to
FRS2 (5) mainly at the N- and C-terminal ends. FRS2 and SNT2 possess a
myristoylation site and a PTB domain at the N terminus. The PTB domain
is responsible for the direct interaction of FRS2 and SNT1 with the
juxtamembrane region of the FGFR in a phosphotyrosine-independent
manner. Deletion of the PTB domain of both proteins abrogates the
association and tyrosine phosphorylation of FRS2 and SNT2 by FGF
receptors (5, 6).
FRS2 and SNT2 substitute for their receptors as docking proteins, a
role similar to that of insulin receptor substrate (IRS) in insulin
signaling (7). To date, two important signaling proteins, Grb2 and
SHP-2, have been reported to bind directly to tyrosine-phosphorylated
FRS2 (2, 8). Grb2 is an adapter protein best known for its role in
linking receptor tyrosine kinases to the Ras pathway via the guanine
nucleotide-releasing factor Sos (9). The binding of Grb2 to FRS2 occurs
via the interaction of the SH2 domain of Grb2 with some or all of the
potentially phosphorylated tyrosine residues at Tyr-196, Tyr-306,
Tyr-349, and Tyr-392 on FRS2. Mutational studies showed that when the
tyrosine residues at all 4 sites were changed to phenylalanine, the
downstream MAP kinase activation was significantly reduced (2). SHP-2 is a tyrosine phosphatase whose activity has been proposed to be
necessary for cell growth and proliferation (10, 11). When cells are
stimulated with growth factors such as PDGF, SHP-2 is tyrosine-phosphorylated and binds to the SH2 domain of Grb2 (12). SHP-2
also binds to the activated receptors via its own SH2 domain (12). As a
result, SHP-2 functions not only as a phosphatase but also serves as an
adapter protein recruiting Grb2 to the receptors. Recently, SHP-2 has
been reported to bind directly to tyrosine-phosphorylated FRS2 through
its N-terminal SH2 domain (8). The association of SHP-2 with FRS2 and
the activation of SHP-2 are essential for a sustained MAP kinase
response as well as for the potentiation of FGF-induced neurite
outgrowth in PC12 cells (8). Hence, by recruiting Grb2 and SHP-2, FRS2
plays a crucial role in linking the FGF receptors to the Ras/MAP kinase pathway.
Apart from the Grb2 and SHP-2 proteins, the activity of the atypical
PKCs (aPKCs) is necessary for mitogenic signaling via the MAP kinase
cascade (13, 14). There are two members in the aPKC subfamily, PKC 
and PKC 
, and they share more than 75% identity. PKCs have been
subdivided into 3 subfamilies, and they are distinguished by their
lipid activation profiles. Conventional PKCs (cPKCs e.g.
, 
, and 
) are activated by diacylglycerol and calcium; novel
PKCs (nPKCs e.g. 
, 
, 
, and 
) do not respond to
calcium but require diacylglycerol for their activation; and aPKCs are
not activated by either diacylglycerol or calcium. It has been shown
that MAP kinase and MEK are activated in vivo by an active
mutant of PKC , and a kinase-defective dominant negative mutant of
PKC impairs the activation of both MEK and MAP kinase by serum and
tumor necrosis factor (14). However, whereas Grb2 and SHP-2 lie
upstream of Ras, PKC can bind to and act as a direct effector of
Ras (15). This is consistent with the observation that expression of a
dominant negative mutant of Ras (Asn-17) severely impairs the
activation of PKC by mitogens such as PDGF in mouse fibroblasts
(15).
A few groups of proteins that are either regulators or substrates of
aPKCs bind to the members in this subfamily. In the first group, a
protein called Zeta-Interacting
Protein (ZIP) binds specifically to the regulatory domain
of PKC (16), whereas Lambda-Interacting Protein (LIP) binds specifically to the regulatory domain
of PKC resulting in an activation of the kinase (17). The Par-4
protein also binds to the regulatory domain of PKC and PKC but
inhibits their activity (18). The second group comprises proteins like heterogeneous ribonucleoprotein A1 protein that has been found to bind
to the kinase domain of PKC in yeast two-hybrid screening and is a
specific substrate of the aPKCs (19).
We have been studying p75, a phosphotyrosine protein that is
dephosphorylated and dissociates from Grb2 upon growth factor stimulation (27). In our attempt to identify p75 by immunoprecipitating phosphotyrosyl proteins that are about 75-kDa in molecular mass, we
observed that a 90-kDa tyrosine-phosphorylated protein, p90, associates
specifically with members of the aPKC subfamily but not with other PKC
family members. In this report, we identified the p90 protein as FRS2.
We have also characterized the factors that regulate its association
with the aPKCs. We propose that FRS2 plays an important role in the
targeting of activated PKC or to the plasma membrane. Thus FRS2
may constitute a third group of proteins that bind to the aPKCs and
localize them in specific subcellular compartments. The recruitment of
aPKCs by FRS2 to the cell-surface membrane may be an important event
contributing to the regulation of the aPKC activity.
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EXPERIMENTAL PROCEDURES |
Reagents--
Monoclonal antibodies against phosphotyrosine
(PY20), Grb2, SHP-2, and PKC were purchased from Transduction
Laboratories (Lexington, KY). Polyclonal antibodies against PKC ,
, and PKC / were from Santa Cruz Biotechnology (Santa Cruz,
CA). Polyclonal antibodies against FRS2 (A872) were raised against
amino acids (residues 491-506) and produced by Neosystem Laboratoire
(Strasbourg, France). Secondary anti-mouse and anti-rabbit antibodies
conjugated to horseradish peroxidase were from Sigma, and protein A/G
plus agarose was from Santa Cruz Biotechnology. Anti-activation domain and anti-binding domain antibodies are from
CLONTECH (Palo Alto, CA). Recombinant human EGF and
PDGF were from Sigma, and basic FGF (bFGF) was from Roche Molecular
Biochemicals (Mannheim, FRG). PKC , PKC , PKC , and PKA
purified enzymes were from Life Technologies, Inc.
Cell Lines, Cell Stimulation, and Lysis--
Swiss 3T3
fibroblasts (ATCC CCL92, Rockville, MD) were grown and maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum (HyClone Laboratories, Logan, UT), 2 mM
glutamine, 10 mM HEPES, pH 7.4, and 100 units/ml penicillin and streptomycin. Human 293T kidney epithelial cells were grown in
150-mm culture dishes with RPMI medium supplemented with 10% fetal
bovine serum (HyClone Laboratories), 2 mM glutamine, 10 mM HEPES, pH 7.4, and 100 units/ml penicillin and
streptomycin. When the cells were about 80-90% confluent, the medium
was aspirated, and the cells were washed and maintained for another
18-24 h in serum-free medium. Various growth factors were added to the
quiescent cells prior to aspiration of the medium. The cells were then
washed rapidly in cold phosphate-buffered saline and lysed in 500 µl of lysis buffer containing 0.5% Nonidet P-40, 20 mM
Tris-HCl, pH 7.3, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM sodium
orthovanadate, and a mixture of protease inhibitors (Roche Molecular
Biochemicals) added according to the manufacturer's instructions. The
cell lysates were spun at 11,000 × g for 5 min at
4 °C, and the supernatants were used for subsequent analyses. The
protein concentrations of all cell lysates were normalized after
estimation of their protein content using a BCA protein assay kit from Pierce.
Construction of Plasmids--
PKC /
cDNA and HA-tagged
PKC in pCDNA3 were kind gifts from Dr. Jorge Moscat
(Universidad Autonoma de Madrid, Spain). PKC II cDNA and PKC cDNAs were from Dr. Alexandra Newton (University of California, San
Diego) and Dr. Li Weiqun (National Cancer Institute, Bethesda),
respectively. cDNAs encoding the full-length PKC , PKC fragment A (amino acids 1-239), PKC fragment B (amino acids
240-586), PKC fragment B (amino acids 240-592), PKC fragment
B (amino acids 354-701), and PKC II fragment B (amino acids
345-673) were obtained by PCR. These inserts were introduced into
pGEX4T1 vector for the expression of GST fusion proteins in bacterial
cells. FRS2 cDNA was obtained first by reverse transcription from
mRNA extracted from Swiss 3T3 cells. PCR was then carried out with
the following primers, which were designed based on the published
sequence of FRS2 (2), to obtain the full-length cDNA as follows:
(forward) 5' CGC GGA TCC GCG ATG GGT AGC TGT TGT AGC TGT CC 3' and
(reverse) 5' CG GCGG CCGC TCA CAT GGG CAG GTC AGT ACT ATT G 3'. The
BamHI/NotI insert was introduced into pGEX4T1 and
pXJ40HA for the expression of GST fusion protein in bacteria cells and
HA-tagged proteins in mammalian cells, respectively. The expressed
proteins were partially microsequenced and shown to be authentic. The
FRS2 fragments X (amino acids 1-152), Y (amino acids 153-300), Z
(amino acids 301-508), XY (amino acids 1-300), and YZ (amino acids
153-508) were obtained by PCR using the full-length FRS2 cDNA as
template. All the inserts were cloned into pGEX4T1 for the expression
of GST fusion proteins. Human SNT2 cDNA was a kind gift from Dr.
Mitchell Goldfarb (Mount Sinai School of Medicine, New York). cDNA
encoding the fragment Z (amino acids 281-492) of SNT2 was obtained by
PCR and cloned into pGEX4T1 for the production of GST fusion proteins.
The cDNA for human FGF receptor 1 (Flg) was a kind gift from Dr.
Lena Claesson-Welsh (Ludwig Institute for Cancer Research, Uppsala,
Sweden). cDNA encoding the cytoplasmic domain (amino acids
398-822) of Flg was obtained by PCR and cloned into pXJ40Flag for the
expression of Flag-tagged cytosolic Flg in mammalian cells.
For yeast two-hybrid screening, cDNAs encoding the full-length
fragment A (amino acids 1-239) and fragment B (amino acids 240-586)
of PKC were obtained by PCR as described above and introduced into
pAS vector suitable for yeast transformation and expression of
Gal4-binding domain fusion protein. SHP-2 cDNA was a kind gift from
Dr. Tony Pawson (Mount Sinai Hospital, Ontario, Canada). Full-length
FRS2 and SHP-2 were subcloned into pACT vector for yeast expression of
Gal4 activation domain fusion proteins. cDNAs for the tandem SH2
domains (amino acids 1-213) and PTP catalytic domain (amino acids
214-603) of SHP-2 were obtained via PCR, and the inserts were also
cloned into the pACT vector.
Mutagenesis--
Mutation of alanine to glutamate A120E in the
pseudo-substrate site of PKC was carried out using the QuickChange
mutagenesis kit from Stratagene (La Jolla, CA) according to the
manufacturer's instruction. The template used was wild-type
full-length PKC in pGEX4T1 and pXJ40HA. The primers used were as
follows 5' CCG GAG AGG GGA ACG CCG TGG GAG 3' and 5' CTC CAC CGG CGT
TCC CCT CTC CGG 3'. The products were sequenced and verified to be correct.
Transfections--
Human 293T kidney epithelial cells were grown
in 100-mm culture dishes as described above. Cells that were about 90%
confluent were used for transfection. For single or co-transfections,
15 µg of each DNA followed by 4.5 µl/µg DNA of TfX 50 from
Promega (Madison, WI) were added to 6 ml of serum-free RPMI and
incubated at room temperature for 15 min. The transfection mix was then added to cells prewashed with serum-free medium and left at 37 °C
for 1 h. After this, 12 ml of RPMI supplemented with 10% fetal bovine serum was added, and the cells were left to recover for 48 h. The cells were lysed with RIPA buffer (50 mM Tris-HCl,
pH 7.3, 150 mM NaCl, 0.25 mM EDTA, 1% sodium
deoxycholate, 1% Triton X-100, 1 mM sodium orthovanadate,
and a mixture of protease inhibitors from Roche Molecular Biochemicals)
and processed as described under "Cell Lines, Cell Stimulation, and
Cell Lysis."
GST Fusion Proteins--
All the constructs for the production
of GST fusion proteins were transformed into DH5 cells. The
transformed cells were grown in 1 liter of LB + ampicillin (50 µg/ml)
medium and incubated at 37 °C with shaking (220 rpm) to an
A600 of about 0.3. These cells were then induced
with 0.1 mM
isopropyl-1-thio--D-galactopyranoside at room
temperature overnight. The cells were spun down and frozen at
80 °C. The cell pellet was then left to thaw on ice, and 10 ml of
lysis buffer (phosphate-buffered saline, 1% Triton X-100, 1 mM dithiothreitol, and a mixture of protease inhibitors
from Roche Molecular Biochemicals) was added to the cell. The cell suspension was subsequently sonicated for a total of 12 pulses of
15 s with a 30-s pause between each pulse. The lysates were centrifuged and supernatants were incubated with glutathione beads overnight at 4 °C to purify the GST fusion proteins.
Immunoprecipitations, in Vitro Binding Assays--
Quiescent or
activated cells were lysed as described above, and an equal volume of
2× precipitation buffer (20 mM Tris-HCl, pH 7.4, 300 mM NaCl, 2% Triton X-100, 2 mM EDTA, 2 mM EGTA, and 1% Nonidet P-40) was added to the cell
lysate. For immunoprecipitation, 2.5 µg of the appropriate antibodies
were added to the diluted cell lysate and incubated for 1 h or
overnight at 4 °C. 2.5 µg of secondary antibodies conjugated to
agarose was added to capture the immunocomplex for 1 h or
overnight at 4 °C. In the depletion studies, the immunoprecipitation
described above was repeated 5 times, each for 1.5 h. After
washing, the immunoprecipitates were pooled together and resolved by
SDS-PAGE.
For in vitro binding assays with GST fusion proteins, 10 µg of the GST fusion proteins were incubated with the lysates for 1 h or overnight at 4 °C. All the beads were washed 3 times
with 1× precipitation buffer, and the bound proteins were eluted with 2× Laemmli buffer before separation by SDS-PAGE.
Yeast Two-hybrid Analysis--
The various constructs of PKC (full length, fragment A, and fragment B) in pAS were sequenced and
verified to be correct before they were introduced into yeast strain
190 using the yeast transformation kit from
CLONTECH (Palo Alto, CA). The transformed yeast
were grown in selective media SD-Trp at 30 °C until colonies appeared. Single transformants then underwent a second round of transformation with the pACT vectors containing full-length FRS2, full-length SHP-2, SH2 domains, or PTP catalytic domain of SHP-2. Successful dual transformants were selected on SD-Trp/Leu, and LacZ
blue assays were carried out, according to the manufacturer's instructions, to detect for protein interactions in the yeast. Yeast
transformed with pCL1 expressing functional Gal4 protein turned blue
between 0.5 and 1 h. This serves as a positive control for the
LacZ assay. Colonies turning blue after 8 h were considered negatives according to the manufacturer's instruction. The dual transformants were also analyzed for the expression of the various proteins by first growing them in SD-Trp/Leu liquid medium. The yeast
was lysed according to the transformation kit manufacturer's instructions, and the lysates were separated on SDS-PAGE. Following Western blotting, the various proteins were detected by probing with
the appropriate antibodies.
In Vitro Kinase Assay--
For activation studies of aPKCs by
growth factors, Swiss 3T3 cells were either untreated or stimulated
with bFGF at 20 ng/ml for 10 min. After the cells were lysed,
immunoprecipitations of aPKC and subsequent in vitro kinase
assays were carried out as described elsewhere (4). In vitro
kinase assays were also carried out either with purified PKA, PKC ,
PKC , or PKC enzyme purchased from Life Technologies, Inc.
HA-tagged A120E PKC mutant and HA-tagged PKC fragment B
(containing the kinase domain) were also used as a source of kinase
activities. In cases where the HA-tagged PKC proteins were used,
immunoprecipitations using HA antibodies were carried out before the
kinase assays were performed. GST full-length FRS2 or GST-FRS2 fragment
Z was tested as substrate for aPKCs, and hnRNPA1, a gift from Dr. Jorge
Moscat (Universidad Autonoma de Madrid, Spain), and MBP were used as
positive controls. All aPKCs reactions were carried out at 30 °C for
30 min in 20 µl of buffer (35 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.5 mM EGTA, 0.1 mM CaCl2, and 1 mM phenylphosphate)
containing 50 ng of enzymes, 2 µg of substrate, 5 µCi of
[-32P]ATP (Amersham Pharmacia Biotech), and 50 µM of ATP. The reactions for the PKC and PKC were
carried out in 20 µl of buffer (20 mM HEPES, pH 7.4, 1.5 mM CaCl2, 1 mM dithiothreitol, and
10 mM MgCl2) containing 50 ng of enzymes, 50 µg/ml sonicated phosphatidylserine, 2 µg of substrates, 5 µCi of
[-32P]ATP (Amersham Pharmacia Biotech), and 50 µM of ATP. The reaction was stopped by boiling with an
equal volume of 2× Laemmli buffer, and the proteins were separated on
SDS-PAGE. The gel was dried and subjected to autoradiography.
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RESULTS |
FRS2 Co-precipitates with PKC in Response to FGF
Stimulation--
Our laboratory has been characterizing p75 and its
association with Grb2. In quiescent cells, p75 is
tyrosine-phosphorylated and binds to the SH2 domain of Grb2. Upon
stimulation with growth factors including FGF, p75 is dephosphorylated
and dissociates from Grb2 (27). We were keen to identify p75 and
decided to test existing phosphotyrosyl proteins that are about 75 kDa
for dephosphorylation upon FGF treatment. One of the candidates that we
selected was PKC , a member of the nPKC subfamily which is about 78 kDa and is the only PKC member currently known to be tyrosine-phosphorylated (33). We therefore set out to investigate whether PKC is tyrosine-phosphorylated in quiescent cells.
Representative members, namely PKC and PKC , from the cPKC and
aPKC subfamilies, respectively, were also included for comparison.
Preliminary studies in our laboratory have shown that two
FGF-responsive cell lines, Swiss 3T3 and PC12 cells, expressed all the
three PKCs of interest. Swiss 3T3 cells were chosen for further experiments because they respond better to bFGF than PC12 cells. To
examine whether PKC , PKC , or PKC could be the p75 that undergoes dephosphorylation upon growth factor stimulation,
immunoprecipitation of the various PKCs was carried out on lysates on
Swiss 3T3 cells that were either untreated or stimulated with bFGF. The
immunoprecipitates were resolved by SDS-PAGE and Western blotted. The
membrane was probed with phosphotyrosine antibodies to detect for the
presence of tyrosine-phosphorylated PKCs. None of the PKCs was
tyrosine-phosphorylated in the lysates of quiescent cells (Fig.
1A, upper panel). The blot was
stripped and re-probed either with PKC , PKC , or PKC antibodies to show that the individual PKCs had been immunoprecipitated (Fig. 1A, lower panel). We conclude that none of the PKCs
tested are likely to be p75.
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Fig. 1.
A, co-immunoprecipitation of a
tyrosine-phosphorylated 90-kDa protein with PKC . Quiescent Swiss
3T3 cells were either not stimulated or stimulated with 10 ng/ml bFGF
for 10 min. The cells were lysed and the lysates subjected to
immunoprecipitation (IP) of PKC , PKC , or PKC as
described under "Experimental Procedures." Upper panel,
the immunoprecipitates were separated by SDS-PAGE and immunoblotted
(IB) with phosphotyrosine antibodies (PY20). The
arrows indicate the positions of p90 and p75. Lower
panel, the blot was stripped and re-probed with PKC , PKC ,
or PKC antibodies to reveal the amount of the various PKCs
immunoprecipitated. B, multiple immunoprecipitations of PKC
, PKC , and PKC . Swiss 3T3 cells were stimulated with bFGF
(10 ng/ml) for 10 min and lysed. The lysates were subjected to 5 successive rounds of immunoprecipitation of PKC , PKC , and PKC
as described under "Experimental Procedures" before the
immunoprecipitates for each PKC were pooled, resolved by SDS-PAGE, and
Western blotted. Top panel, the membrane was probed with
phosphotyrosine antibodies. Middle panel, the blot
containing the immunoprecipitates was cut into strips and re-probed
either with PKC , PKC , or PKC antibodies. These individual
strips were then re-aligned and the respective PKCs detected using the
ECL reagents to reveal the proteins immunoprecipitated. Bottom
panel, the immunoprecipitation efficiency for each of the PKCs was
assessed by probing the lysates before (Pre) or after
(Post) successive rounds of immunoprecipitation with the
respective antibodies. The arrows indicate the positions of
the various PKCs. C, association of FRS2 with PKC in
Swiss 3T3 cells. Serum-deprived cells were either non-stimulated or
stimulated with 10 ng/ml bFGF and the lysates subjected to
immunoprecipitation with PKC antibodies. Immunoprecipitates were
separated on SDS-PAGE and Western blotted. Top panel, the
blot was first probed with PY20 phosphotyrosine antibodies.
Middle panel, the blot was stripped and probed with FRS2
antibodies. Bottom panel, the blot was stripped a third time
and probed with PKC antibodies.
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In these experiments, we noted that a 90-kDa tyrosine-phosphorylated
protein, similar to an FGF-specific p90 protein that has been reported
to bind Grb2 (34), was co-immunoprecipitated with PKC (Fig.
1A, upper panel). Neither PKC nor PKC co-precipitated this tyrosine-phosphorylated protein significantly when
compared with PKC in the lysates from bFGF-stimulated cells. We
therefore decided to investigate this apparently specific association.
It is possible that in the experiments carried out above, differential
amounts of the various PKCs were immunoprecipitated by the antibodies
due to the different affinities of the individual antibodies for their
respective PKCs. The apparently larger amount of p90
co-immunoprecipitated with PKC may due to higher amounts of PKC immunoprecipitated compared with the other PKCs. Hence, it was
necessary to ensure that the majority (>80%) of each PKC was
immunoprecipitated. Preliminary optimization showed that five successive rounds of immunoprecipitation were enough to deplete 80% or
more of the various PKCs (data not shown). Therefore, five rounds of
immunoprecipitation of PKC , PKC , and PKC (as described under "Experimental Procedures") were carried out on lysates from Swiss 3T3 cells that have been stimulated with bFGF. The
immunoprecipitates were pooled, resolved by SDS-PAGE, and transferred
to a polyvinylidene difluoride membrane. The membrane was then probed
with phosphotyrosine antibodies to detect p90. Fig. 1B, top
panel, shows that p90 co-immunoprecipitated only with PKC . The
blot was stripped and cut between lanes and probed for the various PKCs
immunoprecipitated (Fig. 1B, middle panel). The amounts of
the various PKCs present in the lysates before and after multiple
immunoprecipitations were assessed by Western blot analyses. Fig.
1B, bottom panel, shows that more than 80% of PKC , PKC
, or PKC were immunoprecipitated. Therefore, the
co-immunoprecipitation of p90 with PKC is not due to a relatively larger proportion of PKC being immunoprecipitated compared with PKC
or PKC .
The molecular mass and the gel migration pattern of the 90-kDa
tyrosine-phosphorylated protein resembled that of FRS2, a protein that
our laboratory is currently studying. To determine whether the p90
protein was FRS2, lysates from Swiss 3T3 cells that were untreated or
stimulated with bFGF were subjected to immunoprecipitation of PKC .
The immunoprecipitates were processed as described above. The blot was
first probed with phosphotyrosine antibodies revealing the 90-kDa
tyrosine-phosphorylated protein co-precipitating with PKC upon bFGF
stimulation (Fig. 1C, top panel). The blots were stripped
and re-probed with A872, a polyclonal antibody raised against FRS2. As
shown in Fig. 1C, middle panel, FRS2 co-precipitated with
PKC from lysates of bFGF-stimulated but not non-stimulated cells.
It is noted that phosphotyrosine signal for p90 that
co-immunoprecipitated with PKC (Fig. 1C, top panel) is
greater than that of FRS2 co-immunoprecipitated with PKC (Fig.
1C, middle panel). This is attributed to the observation
that FRS2 is a multiply tyrosine-phosphorylated protein with at least 6 tyrosine phosphorylation sites. By comparing the amount of FRS2 in the
total lysate with the amount co-immunoprecipitated with PKC in
other independent experiments (data not shown), it is estimated that
the amount of FRS2 co-immunoprecipitated with PKC was approximately
5%. Probing this blot with PKC antibodies showed equal amounts of
PKC being immunoprecipitated from both lysates (Fig. 1C,
bottom panel). The experiment in Fig. 1C has also been
repeated by probing the blot first with FRS2 antibodies followed by
anti-phosphotyrosine antibodies. Similar results were obtained (data
not shown). This demonstrated that FRS2 and PKC exist in a complex
following bFGF stimulation of Swiss 3T3 cells. The association of FRS2
with PKC in FGF-stimulated cells might be 1) mediated by other
proteins, 2) dependent on the activation of PKC , 3) dependent on
the tyrosine phosphorylation of FRS2, or 4) a combination of some or
all the above factors. We therefore set out to address these possibilities.
FRS2 Binds to the Catalytic Domain of PKC --
Although FRS2
co-immunoprecipitated with PKC , it is possible that the association
of PKC with FRS2 is mediated through other proteins in the
immunoprecipitated complex. We have shown previously that SHP-2
associates with FRS2, and it is possible that PKC associates
directly with SHP-2 and not FRS2. Grb2 also binds to FRS2 but
experiments showed that Grb2 was not present in complexes containing
FRS2 and PKC (data not shown). We employed the yeast two-hybrid
technique to investigate whether FRS2 is likely to bind to PKC directly. The PKC protein exists in a "closed," catalytically
inactive state in non-stimulated cells. In addition to full-length PKC
, two other fragments that contained the regulatory domain (fragment
A) or the catalytic domain (fragment B) of PKC were generated.
These fragments would theoretically "expose" potential regions in
the protein that are normally masked. PKC fragment A contains amino
acids 1-239 and encompasses the N terminus extension, pseudo-substrate
site, zinc finger, and part of the hinge region; fragment B contains
amino acids 240-586 and includes part of the hinge, the kinase domain,
and carboxyl tail. Plasmids containing these proteins were transformed
into yeast as described under "Experimental Procedures." To study
the interaction of FRS2 with the various fragments of PKC , yeast expressing the various PKC proteins underwent another
transformation with an expression vector encoding full-length FRS2. In
addition, the SHP-2 full-length, the tandem N- and C-terminal SH2
domains, and the PTP catalytic domain of SHP-2 were also separately
introduced to assess their binding to PKC . In all cases, colonies
expressing all combination of proteins were obtained (data not shown)
and were subjected to LacZ assays. Yeast transformed with pCL1
expressing functional Gal4 protein turned blue between 0.5 and 1 h. This serves as a positive control for the LacZ assay. Colonies
turning blue after 8 h were considered negatives according to the
manufacturer's instruction. In the above assays, only yeast expressing
FRS2 and PKC fragment B (encompassing the kinase domain) turned
blue (at 2.5 h), indicating a strong interaction (Table
I). Of particular note was the
observation that full-length PKC and fragment A containing the PKC
regulatory domain did not interact with FRS2. Thus, FRS2 interacts
with PKC through a region (amino acids 240-586) that is
predominantly the catalytic domain. This region is probably masked in
the non-activated, full-length molecule since the latter cannot
interact with FRS2. The observation that the full-length SHP-2, the
tandem SH2 domains, and the PTP catalytic domains of SHP-2 did not
interact with any of the PKC polypeptides indicates that the
interaction between FRS2 and PKC is both specific and most likely
direct. FRS2 is not likely to be an activator of PKC since the well
characterized activators of the kinase bind to the regulatory region of
the kinase (16, 17).
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Table I
The catalytic domain of PKC interacts with FRS2 in a yeast
two-hybrid assay
Following co-transformation of yeast strain Y190 with a combination of
plasmids shown in the table, dual transformants were selected on
SD-Trp/Leu-selective media. Colonies that grew were subjected to LacZ
assay as described under "Experimental Procedures" to test for the
induction of -galactosidase activity that results from interaction
between two proteins expressed in the yeast. Blue colonies indicate a
positive interaction, and white indicates a negative
interaction.
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To verify the yeast two-hybrid results, GST-PKC fragment A and
GST-PKC fragment B were tested for their ability to bind to FRS2.
Since FRS2 co-immunoprecipitated with PKC only upon FGF
stimulation, Swiss 3T3 cells were stimulated with bFGF and the lysates
incubated with either GST-PKC fragment A or fragment B. An aliquot
of the lysate was also incubated with GST as a control. The
precipitates were resolved by SDS-PAGE and Western blotted. The
presence of FRS2 in the precipitates was detected by probing the
membrane with phosphotyrosine antibodies rather than FRS2 antibodies
because of the ease of detection as well as the avoidance of the high
nonspecific background signal encountered when using FRS2 antibodies.
Consistent with the yeast two-hybrid results, Fig.
2A shows that fragment B of
PKC is responsible for binding to FRS2. The amounts of
tyrosine-phosphorylated FRS2 seen to bind to PKC fragment A and GST
represent background signal.
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Fig. 2.
FRS2 binds to the catalytic domain of
PKC and but not
PKC II. A, quiescent Swiss 3T3
were stimulated with 10 ng/ml bFGF for 10 min and the cells lysed. The
lysates were incubated with 10 µg of GST fragment A or fragment B
(containing the catalytic domain) of PKC as described under
"Experimental Procedures." The precipitates were washed, eluted,
and separated on SDS-PAGE followed by immunoblotting (IB)
using phosphotyrosine antibodies. B, lysates from
bFGF-stimulated cells were incubated with the fragment B of PKC ,
PKC , or PKC II as described above. The precipitates were
resolved by SDS-PAGE, Western blotted, and the membrane probed with
phosphotyrosine antibodies (upper panel). The amounts of the
various GST fusion proteins used for this precipitation experiment are
shown by Coomassie Blue staining (lower panel).
PD, pull-down.
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To determine whether the binding of FRS2 to PKC fragment B is
specific, various GST fusion proteins were prepared for in vitro binding assays. Lysates of bFGF-stimulated Swiss 3T3 cells were incubated with the fragment B of PKC or, for comparison, fragment B of PKC II (a member of the cPKC family) and PKC (the
other member of the aPKC). The precipitates were resolved by SDS-PAGE
and immunoblotted with phosphotyrosine antibodies to detect for
tyrosine-phosphorylated FRS2. Fig. 2B, top panel, shows that
the fragment B of PKC but not that of PKC II precipitated tyrosine-phosphorylated FRS2. Fragment B of PKC , the other member of the atypical PKCs (aPKCs), also bound tyrosine-phosphorylated FRS2.
This is not surprising given the observation that the fragment B
containing the kinase domains of PKC have more than 85% identity with PKC . On the other hand, the kinase domain of PKC II did not
bind significant amounts of FRS2 compared with the aPKCs. The fragment
B of PKC also did not bind FRS2 significantly (data not shown).
This reflects a lack of affinity in the fragment B of cPKCs and nPKCs
for FRS2. Together, the above results demonstrate that only the members
of the atypical PKC subfamily interact with FRS2 through a region that
encompasses the catalytic domain, and this interaction is most likely
to be direct.
PKC Binds to a Region in the C Terminus of FRS2--
To define
the region on FRS2 that binds to PKC , GST fusion proteins
containing various fragments of FRS2 were produced (Fig. 3A). Lysates of human 293T
cells grown in 10% serum were incubated with each of the GST-FRS2
polypeptides overnight at 4 °C. The precipitates were washed and
resolved by SDS-PAGE and immunoblotted with PKC monoclonal
antibodies (Fig. 3B). Fragment Z (amino acids 301-508) of
FRS2 precipitated a comparatively larger amount of PKC compared
with fragments X and Y. Consistent with this observation, full-length
FRS2 and fragment YZ but not fragment XY could also bind PKC .
However, full-length FRS2 and FRS2 fragment YZ bind slightly lesser PKC
than fragment Z, suggesting that other parts of FRS2 may interfere
with the binding of PKC . It is also possible that the GST-FRS2
full-length or fragment YZ may not have been folded properly during the
preparation. As a control, the GST-FRS2 fragment Z alone was resolved
by SDS-PAGE, Western blotted, and probed with PKC antibodies. No
signal was obtained (data not shown) indicating that the PKC signal
from fragment Z in Fig. 3B was not intrinsic to the GST
fusion proteins.
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Fig. 3.
A, diagram showing the various GST
fusion proteins of FRS2 produced. The tyrosine residues Y196,
Y306, Y349, and Y392 are potential Grb2-binding sites
and Y436 and Y471 are potential SHP-2-binding
sites. B, PKC binds to a C-terminal region of FRS2. 293T
cells were grown in 10% serum and lysed when 95% confluent. The
lysates were incubated with 10 µg of GST fusion proteins of FRS2
full-length (FL), fragment X, fragment Y, fragment Z,
fragment XY, and fragment YZ as described under "Experimental
Procedures." The precipitates were washed, eluted, and the proteins
separated on SDS-PAGE. After the Western blotting, the membrane was
probed with PKC antibodies. C, PKC also binds to a
C-terminal region of SNT2. 293T cells were grown in 10% serum and
lysed when they were 95% confluent. The lysates were incubated with 10 µg of the GST fusion proteins of full-length FRS2 (FL),
FRS2 fragment Z, and SNT2 fragment Z. The precipitates were processed
as described in B, and the presence of PKC was examined
by immunoblotting (IB) with PKC antibodies.
PD, pull-down.
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To define further the binding region on FRS2, fragment Z of FRS2 was
subdivided into smaller fragments, and in vitro binding assays similar to those described above were performed. PKC showed
essentially equal binding to each of the subfragments of fragment Z
(data not shown), indicating that binding occurs at multiple points
within this peptide sequence.
Next, we investigated whether SNT2, an isoform that has 50% identity
to FRS2, can bind to PKC . A GST fusion protein of SNT2 fragment Z
(amino acids 281-492), whose sequence was aligned with that of FRS2
fragment Z, was produced and assessed for its binding to PKC . The
fragment Z from FRS2 and SNT2 share about 50% identity. GST
full-length FRS2 and GST fragment Z of FRS2 were included for
comparison, and a similar experiment to that described above was
carried out. Fig. 3C shows that fragment Z of SNT2 could
bind endogenous PKC as well as fragment Z from FRS2.
"Activated" PKC Binds to FRS2--
By having demonstrated
that the fragment B of PKC binds to the carboxyl portion of FRS2,
we proceeded to investigate whether activation of PKC was necessary
for its association with FRS2. First, we examined the activation status
of PKC by FGF. Swiss 3T3 cells were either not stimulated or
stimulated with bFGF. The cells were lysed, and immunoprecipitation of
PKC was performed on these lysates. Subsequently, the
immunoprecipitated PKC was tested for in vitro kinase
activity toward hnRNPA. hnRNPA1 is the only convincing aPKC substrate
identified so far (19). Fig. 4A showed that bFGF stimulated
the activity of PKC by about 3-fold. This level of activation is
comparable to that obtained by stimulating NIH3T3 cells with PDGF (19).
The opening of the otherwise closed PKC protein as a result of
activation by FGF may expose sites that are necessary for its
association with FRS2. This is consistent with the yeast two-hybrid
results (Table I) where only fragment B of PKC binds to FRS2.
Apparently, the masking of this fragment in the inactive, full-length
PKC contributed to its inability to bind FRS2.
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Fig. 4.
A, PKC is activated by bFGF in Swiss
3T3 cells. Swiss 3T3 cells were either non-stimulated or stimulated
with bFGF at 20 ng/ml for 10 min before the cells were lysed. The
lysates were subjected to immunoprecipitation using PKC antibodies.
After the immunoprecipitates were washed, they were used for in
vitro kinase assays using eluted hnRNPA1 as substrate as described
under "Experimental Procedures." After the reaction, the proteins
were resolved by SDS-PAGE. The gel was dried and exposed to x-ray film
(Fuji). The phosphorylated protein bands obtained were quantitated
using a densitometer (Bio-Rad), and the relative activity of PKC is
shown. The values shown represent the average ± half the range.
B, constitutively active PKC A120E mutant binds more
HA-tagged FRS2 than wild-type inactive PKC . 293T cells were
co-transfected with HA-tagged FRS2 (HA-FRS2), and the
cytosolic domain of Flg (Flg-cyto). The cells were lysed
after 48 h of recovery and the lysates incubated with 10 µg of
GST fusion proteins of PKC A120E mutant (mt), wild-type
(wt) PKC , PKC II fragment B, and PKC fragment B
as described under "Experimental Procedures." The precipitates were
washed, eluted, and separated on SDS-PAGE. After Western blotting the
blot was probed with phosphotyrosine antibodies to reveal the amounts
of tyrosine-phosphorylated FRS2 precipitated by the various fusion
proteins. C, GST-FRS2 fragment Z binds with a higher
affinity to HA-tagged PKC A120E mutant than the HA-tagged wild-type PKC . Upper
panel, 293T cells were transfected with either HA-tagged PKC A120E mutant or wild-type PKC or were not transfected. The cells
were lysed after 48 h of recovery, the lysates separated on
SDS-PAGE, and Western blotted. The blot was then probed with HA
antibodies to reveal the expression of the various tagged proteins.
Lower panel, the lysates from 293T cells transfected with
either HA-tagged PKC mutant or wild-type PKC were incubated
with 10 µg of the GST-FRS2 fragment Z as described under
"Experimental Procedures." The precipitates were washed, eluted,
and separated on SDS-PAGE. After Western blotting, the blot was probed
with HA antibodies to reveal the amount of HA-tagged proteins that were
precipitated. The arrow indicates the positions of the
HA-tagged wild-type PKC or PKC A120E mutant. IB,
immunoblotting; PD, pull-down.
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We postulated that if the inactive PKC could be "artificially"
opened up, thus exposing the sites represented on fragment B,
full-length PKC might acquire the ability to bind FRS2. It has been
shown previously that mutation of the alanine residue in the
pseudo-substrate site of PKC to glutamate would switch the kinase to a
constitutively active form (21, 22). We therefore set out to
investigate the relative affinities of the constitutively active PKC
A120E mutant and the wild-type PKC for tyrosine-phosphorylated FRS2. Although the data from yeast two-hybrid analysis showed that
non-tyrosine-phosphorylated FRS2 can bind to fragment B of PKC ,
tyrosine-phosphorylated FRS2 was used in the assay because tyrosine
phosphorylation may enhance the binding to PKC (refer to Fig.
1C). To eliminate any potential contribution of SNT2, we
transfected 293T cells with HA-tagged FRS2 (HA-FRS2). We have previously noted that the overexpression of the cytosolic fragment of
FGFR1 (Flg) leads to the activation of the endogenous tyrosine kinase
activity without the addition of exogenous bFGF (data not shown).
Hence, to obtain tyrosine-phosphorylated HA-FRS2, 293T cells were
co-transfected with the cytosolic fragment of Flg (Flg-cyto) and
HA-FRS2. Total cell lysates were separated on SDS-PAGE and Western
blotted. Probing the blot with FRS2 (A872) antibodies showed that the
level of HA-FRS2 was much higher than the endogenous FRS2, which was
present in very low abundance (data not shown). Immunoprecipitation
with HA antibodies showed that the expressed HA-FRS2 was
tyrosine-phosphorylated (data not shown).
To assess the binding of the constitutively active PKC mutant or
wild-type PKC to tyrosine-phosphorylated FRS2, 293T cells were
co-transfected with HA-FRS2 and the cytosolic fragment of Flg. The cell
lysates containing tyrosine-phosphorylated HA-FRS2 were then incubated
with equal amounts of GST-PKC A120E mutant or GST-wild-type PKC
. The GST fusion proteins containing the fragment B of PKC or
PKC II were also included for comparison. The precipitates were
separated by SDS-PAGE, Western blotted, and the membrane probed with
phosphotyrosine antibodies. Fig. 4B revealed that the
constitutively active PKC mutant binds much more
tyrosine-phosphorylated FRS2 protein than its wild-type counterpart.
The amount of FRS2 bound by wild-type PKC was about the same as
that bound by PKC II and is likely to represent the background level
of binding as we have shown previously that FRS2 does not associate
with this PKC isoform (Fig. 2B). In addition, the PKC fragment B is able to bind as much FRS2 as PKC A120E mutant
indicating that fragment B of the PKC molecule is sufficient for
binding FRS2 without the cooperation of other parts of the molecule. A
similar experiment was also carried out using lysates from Swiss 3T3
cells that were stimulated with FGF. The lysates were incubated with
GST-PKC A120E mutant or GST-wild-type PKC , and the precipitates
were probed with phosphotyrosine antibody to detect endogenous
tyrosine-phosphorylated FRS2. The results (data not shown) were
essentially the same as Fig. 4B.
The in vitro binding of mutant and wild-type PKC to FRS2
was also assessed using GST-FRS2 fragment Z, and the lysates of 293T
cells were transfected with HA-tagged wild-type or constitutively active mutant PKC . The transfected cells expressed equivalent amounts of HA-tagged mutant and wild-type PKC (Fig. 4C, top panel). Following incubation of the cell lysates with GST-FRS2 fragment Z, the precipitated proteins were separated on SDS-PAGE, Western blotted, and probed with HA antibodies. As shown in Fig. 4C (bottom panel), more mutant compared with the
wild-type PKC was bound to GST-FRS2 fragment Z. This strengthens
the observation that constitutively active PKC A120E mutant has a
stronger affinity for FRS2 than the inactive wild-type PKC and
indicates that activation of PKC is required to bind to FRS2.
Tyrosine Phosphorylation of FRS2 Is Not Required for Association
with aPKC--
Although the activation of PKC can account for the
induced association of the two proteins, we cannot exclude the
possibility that tyrosine phosphorylation of FRS2 is also a factor
regulating this association. Therefore, we set out to investigate the
role of tyrosine phosphorylation in the FRS2/PKC interaction. 293T cells were transfected with either HA-FRS2 alone or with cytosolic Flg,
allowing expression of nonphosphorylated or tyrosine-phosphorylated HA-FRS2. The cells were lysed and the lysates incubated with equal amounts of GST fusion proteins containing the constitutively active PKC
mutant or wild-type PKC . An equivalent amount of
agarose-conjugated GST protein was included as a control. The
precipitates were separated by SDS-PAGE and immunoblotted with
phosphotyrosine antibodies to detect FRS2. As expected,
tyrosine-phosphorylated HA-FRS2 was precipitated by mutant PKC .
Very low levels of tyrosine-phosphorylated FRS2 were seen with
wild-type PKC and GST alone, and these represented background
signals (Fig. 5, top panel).
When the blot was stripped and re-probed with FRS2 (A872) antibodies
(Fig. 5, bottom panel), equivalent amounts of FRS2 were
shown to be precipitated by the PKC A120E mutant regardless of
whether FRS2 was tyrosine-phosphorylated or not. Very low and equal
amounts of FRS2 signal were detected in the all the lanes containing
wild-type PKC or GST only after prolonged exposure of the blot
(data not shown) and is unlikely to be significant. The association of
PKC with FRS2 is therefore independent of tyrosine
phosphorylation.
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Fig. 5.
Binding of PKC to
FRS2 is not dependent on tyrosine phosphorylation of FRS2.
Upper panel, 293T cells were either singly transfected with
HA-FRS2 or co-transfected with the cytosolic fragment of Flg
(Flg-cyto). After recovery for 48 h, the cells were
lysed and the lysates incubated with 10 µg of GST fusion proteins of
PKC A120E mutant or wild-type PKC or GST alone, as described
under "Experimental Procedures." The precipitates were separated on
SDS-PAGE, Western blotted, and probed with phosphotyrosine
(PY20) antibodies. Lower panel, the blot was
stripped and re-probed with FRS2 antibodies to reveal the amount of
FRS2 precipitated. IB, immunoblotting; PD,
pull-down.
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FRS2 Is Not an in Vitro Substrate of aPKCs--
FRS2 has been
reported to be serine/threonine-phosphorylated (4). We have also shown
here that stimulation of Swiss 3T3 cells with FGF leads to the
activation of PKC and the binding of FRS2 or SNT2 to regions in PKC
and PKC that contain the catalytic domain. All the above
evidence suggests that FRS2 is a likely substrate of PKC . We
therefore addressed the enzyme-substrate relationship between PKC
/ and FRS2. We used hnRNPA1 and/or myelin basic protein (a
general substrate for PKC) as positive controls. GST full-length FRS2
and/or FRS2 fragment Z were tested as substrates for the aPKCs.
In vitro kinase assays, as described under "Experimental
Procedures," were carried out using purified PKC or HA-tagged PKC
fragment B (containing the kinase domain) or HA-tagged PKC A120E constitutively active mutant as a source of kinase activities.
Fig. 6A (top left
panel) revealed that hnRNPA1 and MBP are phosphorylated by
purified PKC , but full-length FRS2 is not. In addition, although
the HA-tagged PKC kinase domain and PKC A120E constitutively
active mutant were active against MBP, they did not phosphorylate
full-length FRS2 or FRS2 fragment Z, which is known to bind more
strongly to PKC than the full-length FRS2 (Fig. 6A, lower
panels). To investigate whether post-translational modification of
FRS2 was necessary for it to be a substrate of the aPKCs, FRS2 was
immunoprecipitated from Swiss 3T3 cell lysates that had been either
non-stimulated or stimulated with bFGF. In vitro kinase
assays with PKC enzyme or with enzymatically active HA-tagged PKC
proteins were carried out with the immunoprecipitated
tyrosine-phosphorylated or non-phosphorylated FRS2. Again, the aPKC
enzymes failed to phosphorylate either form of FRS2 (data not
shown).
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Fig. 6.
A, FRS2 is not a substrate of PKC
/ in vitro. GST full-length FRS2 was tested as a
substrate for PKC or PKC . MBP and hnRNPA1 were used as positive
controls. In vitro kinase assays as described under
"Experimental Procedures" were carried out by incubating the
GST-FRS2, MBP, and hnRNPA1 with purified PKC enzyme (top left
panel) or PKA enzyme (top right panel). The HA-tagged
PKC A120E constitutively active mutant (bottom left
panel) or HA-tagged PKC kinase domain (bottom right
panel) was expressed in 293T cells, immunoprecipitated using HA
antibodies, and used as a source of kinase activity during in
vitro kinase assays. The potential phosphorylation of MBP, GST
full-length FRS2, and GST-FRS2 fragment Z by PKC were tested in the
washed immunoprecipitates as described under "Experimental
Procedures." The arrows indicate the position of the
various substrates tested. B, FRS2 is not a substrate for
the cPKC and nPKCs. In vitro kinase assays, as described
under "Experimental Procedures," were carried out to investigate
whether GST full-length FRS2 or GST-FRS2 fragment Z are substrates for
PKC (left panel) and PKC (right panel).
MBP was used a positive control. The arrows indicate the
positions of the various substrates tested.
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To show that the GST-FRS2 proteins could be an authentic substrate
in vitro, GST full-length FRS2 was tested as a substrate for
protein kinase A (PKA). FRS2 contains potential PKA phosphorylation sites (Ser-366 and Ser-429), and preliminary studies had shown that it
was a likely substrate of that kinase. The hnRNPA1 protein was added as
a positive control as it can also be phosphorylated by PKA (20). Myelin
basic protein was also included in the assay. Fig. 6A
(top right panel) shows that full-length GST-FRS2 could be
phosphorylated by PKA to a greater degree than hnRNPA1. We have also
performed in vitro kinase assays with purified PKC and
PKC to determine whether FRS2 might be a substrate for PKCs from
other subfamilies. Fig. 6B shows that whereas both PKC (left panel) and PKC (right panel) were able
to phosphorylate MBP, they either failed to phosphorylate FRS2 (PKC
) or they phosphorylated FRS2 only very weakly (PKC ). Taken
together, these results demonstrate that whereas FRS2 was
phosphorylated by PKA, it is not an in vitro substrate and
hence is an unlikely in vivo substrate for the PKCs.
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DISCUSSION |
The concept of non-receptor, tyrosine-phosphorylated proteins
serving as dockers was conceived when the IRS family of proteins was
discovered and characterized. Gradually, more proteins were identified
that fall under this description. Like IRS proteins, Gab-1 and Dos
possess modules such as pleckstrin homology or phosphotyrosine-binding (PTB) domains that help in their membrane localization (7). More
recently, another docker protein named FRS2 has been identified and
cloned (2). It has a myristoylation site for membrane association, an
N-terminal phosphotyrosine-binding domain (PTB) for protein-protein interactions, and multiple tyrosine residues that are targets for
phosphorylation by the FGFRs upon ligand binding. FRS2 has been
reported to bind to the SH2 domains of Grb2 (2) and SHP-2 in a tyrosine
phosphorylation-dependent manner (8), and these associations exert a significant influence on the MAP kinase cascade. Docker proteins, including FRS2, have been shown to play important roles as initiation centers for diverse signaling pathways.
Identification of proteins that bind to FRS2 will therefore contribute
to the understanding of the signal transduction of FGF in cells.
In our studies, we found that about 5% of total FRS2
co-immunoprecipitated specifically with PKC , a member of a
subfamily of the family of PKC kinases called the atypical PKCs
(aPKCs). The interaction between FRS2 and PKC was shown to be
mediated by a region (fragment B) in the aPKCs that encompasses the
catalytic domain. We have also shown that activation of the aPKCs is
necessary for its association with FRS2. It can be construed that the
absence of co-immunoprecipitation of FRS2 with the cPKCs or nPKCs may be due to the inability of FGF to stimulate the various cPKCs and
nPKCs. However, the B fragments of cPKC (e.g. PKC II) and nPKC (e.g. PKC ) did not have the affinity to bind FRS2
and are unlikely to bind FRS2 even if they were activated. The sequence identities between the fragment B of PKC and those of other members
were 86% for PKC and 44-55% for other PKC members. The greater
amino acid sequence homology between the aPKCs is sufficient to provide
specificity for their binding to FRS2 but not for the more distantly
related members of the PKC family.
The association between aPKCs and FRS2 is not that of an
enzyme-substrate relationship. Only one strong in vivo
substrate of the aPKCs has so far been identified. hnRNPA1 protein was
identified in a yeast two-hybrid screen using the PKC kinase domain
as bait (19). The optimal peptide sequence, determined by peptide library screening, for phosphorylation by aPKC is
RRFKRQGS(P)FFYFF (where boldface
indicates the motif required for phosphorylation and boldface italic
indicates phosphorylation at serine) (23). This is similar to the motif
on hnRNPA1 that surrounds the phosphorylated serine residue
SQRGRSGS(P)GNFGG. It is crucial
to have the basic and hydrophobic amino acid residues to the N and C
terminus of the core sequence RXGS,
respectively. Such a sequence was not found in FRS2, and this validates
our experimental data. Although SNT2 seems to possess one such
potential motif PLTRRGS(P?)PRVFNFDF, it is only
very weakly phosphorylated by the aPKCs (data not shown).
FRS2 is located at the plasma membrane of cells and associates with FGF
receptors in a tyrosine phosphorylation-independent manner (5, 6). It
is possible that FRS2 may recruit the aPKCs to substrates in the
vicinity of the FRS2-receptor complex at the cell-surface membrane.
Proteins that are associated with FRS2 would therefore be potential
substrates. SHP-2 has a potential PKC phosphorylation site,
AGIGRTGT(P?)TFIVI (where P? indicates a potential threonine phosphorylation site). In vitro phosphorylation studies, however, showed that SHP-2
is not a substrate of PKC . Furthermore, yeast two-hybrid assays did
not show any association between PKC and SHP-2. The FGF receptor
was excluded as a plausible substrate because it does not possess any
potential aPKC phosphorylation sites in the cytosolic region. The
identification and characterization of additional FRS2-associated
proteins may lead to the identification of novel PKC substrates.
The association of aPKCs or SHP-2 with FRS2 has a common feature. Both
the aPKCs and SHP-2 are enzymes but their binding partner, FRS2, is not
a substrate for either the kinase or the phosphatase. On the other
hand, the aPKCs and SHP-2 show differences in their manner of binding
to FRS2. The binding of SHP-2, via its N-terminal SH2 domain, to FRS2
is dependent on tyrosine-phosphorylated residues in FRS2, and this
interaction is necessary for the activation of the phosphatase (24). In
contrast, the binding of PKC to FRS2 is not dependent on tyrosine
phosphorylation of FRS2 but dependent on the activation of PKC .
Hence, although FRS2 is both an activator and locator protein for
SHP-2, it is likely to be only a locator protein for the activated
aPKCs. However, we cannot rule out other possible functions FRS2 plays
in the regulation of aPKCs such as post-translational modification.
Preliminary experiments suggested that FRS2 is unlikely to be an
inhibitor of aPKC activity since incubation of a reaction mixture
containing PKC and hnRNPA1 with FRS2 did not block phosphorylation of hnRNPA1 (data not shown). Although we cannot totally exclude the
possibility that FRS2 is an inhibitor for aPKCs, we suspect that the
role of FRS2 in binding the aPKCs is similar to that of a group of
proteins called RACKs (Receptors for Activated C Kinase). The RACKs
have been proposed to anchor PKC at specific locations in the cell (25,
26). Like FRS2, these proteins have been shown to bind to the active
conformation of the PKC but are themselves not substrates for the
PKC.
All PKCs appear to be activated at the plasma membrane with
phosphatidylserine being an important co-activator for all members of
the greater family. In addition, the aPKCs are activated by particular
inositol phospholipids (29). PKC has also received considerable
recent attention as a target for PI-3 kinase (31, 32). Two reports show
that the phosphoinositide-dependent protein kinase 1 (PDK-1), which binds with high affinity to the PI 3-kinase lipid
product phosphatidylinositol 3,4,5-trisphosphate, phosphorylates and
potently activates PKC along with two other substrates, also
kinases, Akt/PKB and p70S6K. PKC and PDK-1 are associated in
vivo, and membrane targeting of PKC renders it constitutively active in cells. The association between PKC and PDK-1 reveals extensive cross-talk between enzymes in the PI 3-kinase pathway (28,
30). Evidence has been presented previously indicating that the IRS
protein associates with enzymes involved in the PI 3-kinase pathway
(7). It is possible that the strategic membrane locations of the IRS
and FRS2 docker proteins may see them playing a central role in the PI
3-kinase pathway as well as the MAP kinase pathways and the likely
interactions between the two pathways.
In conclusion, the association of activated aPKCs to FRS2 may serve to
target the aPKCs to specific sites on t
TOP
ABSTRACT
INTRODUCTION
