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(Received for publication, November 16, 1994) From the
Crk, a cellular homolog of v-crk, is an SH2 and SH3
domain-containing adaptor protein related to Grb2 and Nck, two proteins
which have been shown to be involved in growth factor signal
transduction. Crk proteins have recently been found to associate with
two guanine nucleotide releasing proteins, mSos and C3G, and thus
appear to lie on the Ras pathway. We investigated whether Crk is a
target for the insulin-like growth factor I (IGF-I) receptor tyrosine
kinase. We show that IGF-I stimulates tyrosine phosphorylation of Crk
II via stimulation of endogenous IGF-I receptors in both 293 cells and
NIH-3T3 cells. IGF-I stimulated tyrosine phosphorylation of Crk II in a
dose- and time-dependent manner. In 293 cells, which express both IGF-I
and insulin receptors, insulin also induced a dose-dependent tyrosine
phosphorylation of Crk II, but with somewhat reduced sensitivity,
compared to IGF-I. In NIH 3T3 cells, IGF-I also stimulated tyrosine
phosphorylation of a 45- kDa protein which co-immunoprecipitated with
Crk II. These findings indicate that Crk II is an endogenous substrate
of the IGF-I receptor tyrosine kinase and provide the first
demonstration that a mitogenic growth factor induces tyrosine
phosphorylation of endogenous c-Crk.
Crk proteins are the cellular homologs of the viral oncogene
v-crk, an SH (Src homology)2 and SH3 domain-containing
protein, encoded by the avian sarcoma virus CT10(1) . The Crk
family of proteins includes Crk II, a 40-kDa protein comprised of an
N-terminal SH2 domain followed by two SH3
domains(2, 3) ; Crk I, a 21-kDa protein that appears
to be an alternately spliced form of Crk II, with the second SH3 domain
deleted and most closely resembles v-crk(2) ; and
Crkl, a 36-kDa Crk-like protein with two SH2 and SH3 domains, that
shares Like other growth factor receptor
tyrosine kinases, IGF-I (
To determine whether Crk proteins are tyrosine-phosphorylated
in response to IGF-I, 293 cells were treated for 5 min with 100 nM IGF-I. Cleared whole cell lysates were immunoprecipitated for Crk
as described. Crk immunoprecipitates were then run on
SDS-polyacrylamide gel electrophoresis and blotted for phosphotyrosine.
As shown in Fig. 1A, stimulation of 293 cells with
IGF-I increased the phosphotyrosine content of two closely migrating
proteins of M
Figure 1:
IGF-I
induces tyrosine phosphorylation of Crk in 293 cells. Cells were either
unstimulated(-) or stimulated (+) with 100 nM IGF-I
for 5 min. A, lysates were immunoprecipitated with an anti-Crk
antibody and immunoblotted for phosphotyrosine. B, the same
blot shown in A was stripped and relabeled with an anti-Crk
antibody. The relative positions of phosphotyrosine and Crk
immunoreactivity were aligned as shown, by utilizing luminescent
markers (Stratagene) taped directly to the nitrocellulose
membrane.
Fig. 2shows a time course
of the effect of IGF-I on tyrosine phosphorylation of Crk II in 293
cells. Cells were stimulated with 3 nM IGF-I for various times
between 0 and 10 min. Lysates were immunoprecipitated for Crk and
immunoblotted for phosphotyrosine. A small amount of
tyrosine-phosphorylated Crk II was observed after 30 s of exposure to
IGF-I and continued to increase up to 5 and 10 min. After 5 min of
IGF-I stimulation, phosphorylation of band 2 appeared maximal, and
phosphorylation of band 1 continued to increase from 5 to 10 min. These
data were quantitated by densitometric analysis, as shown in the lower
portion of Fig. 2.
Figure 2:
Time course of the effect of IGF-I on
tyrosine phosphorylation of Crk in 293 cells. Cells were stimulated
with 3 nM IGF-I for 0, 0.5, 1, 3, 5, or 10 min as indicated.
Lysates were immunoprecipitated for Crk, then immunoblotted for
phosphotyrosine, as shown in the upper panel of the figure.
Quantitation of Crk phosphorylation by densitometric scanning is shown
in the lower portion of the figure, expressed as percent
change in phosphorylation from basal conditions (time = 0).
Similar results were obtained in a duplicate
experiment.
As 293 cells express both IGF-I and
insulin receptors, it was of interest to determine whether insulin
could also induce tyrosine phosphorylation of Crk II. Fig. 3shows a dose-response experiment, comparing the effects of
insulin and IGF-I on Crk II phosphorylation. Cells were stimulated for
5 min with various doses of IGF-I or insulin. Lysates were then
immunoprecipitated for Crk and immunoblotted for phosphotyrosine. Both
IGF-I and insulin induced tyrosine phosphorylation of Crk II in a
dose-dependent manner. However, the cells were somewhat more sensitive
to IGF-I than to insulin, in terms of induction of tyrosine
phosphorylation of Crk II. Scatchard analysis, using
Figure 3:
Dose-dependent effects of insulin and
IGF-I on tyrosine phosphorylation of Crk in 293 cells. Cells were
stimulated for 5 min with various doses of IGF-I and insulin, as
indicated. Lysates were immunoprecipitated for Crk, then immunoblotted
for phosphotyrosine. Data shown are representative results from two
experiments.
We also
studied the effect of IGF-I on Crk II in NIH-3T3 mouse fibroblasts,
another cell line that expresses endogenous IGF-I receptors
(approximately 12,000 receptors per cell, as determined by radioligand
binding, data not shown). Cells were stimulated with various doses of
IGF-I for 5 min. Lysates were then immunoprecipitated for Crk and
blotted for phosphotyrosine. Fig. 4A shows a dose-dependent
increase in tyrosine phosphorylation of Crk II by IGF-I. Interestingly,
in NIH-3T3 cells, only a single phosphotyrosine-containing band (band
1) was observed at 40 kDa, whereas in 293 cells,
tyrosine-phosphorylated Crk appeared as a doublet (see Fig. 1).
Furthermore, in NIH-3T3 cells, a 45-kDa tyrosine-phosphorylated
protein, which was also increased in phosphotyrosine content in
response to IGF-I, co-immunoprecipitated with Crk. As shown in Fig. 4B, where the blot was stripped and reprobed with an
anti-Crk antibody, the 40-kDa phosphotyrosine-containing band
corresponds to Crk II. In unstimulated cells, the majority of Crk
immunoreactivity was localized in the lower 40-kDa band (band 2). With
IGF-I stimulation, Crk immunoreactivity was progressively shifted into
a more slowly migrating band (band 1). Similar to what was observed in
293 cells, the lower band (band 2) was not immunolabeled by an
anti-phosphotyrosine antibody, indicating that it represents the
unphosphorylated form of the protein.
Figure 4:
Effects of IGF-I on tyrosine
phosphorylation of Crk in NIH-3T3 cells. Cells were stimulated for 5
min with various doses of IGF-I, as indicated. A, lysates were
immunoprecipitated for Crk, then immunoblotted for phosphotyrosine. B, the same blot shown in A was stripped and
relabeled with an anti-Crk antibody.
IGF-I receptor signaling involves interaction of the
activated tyrosine-phosphorylated receptor with various SH2
domain-containing proteins, including IRS-1, Shc, and the p85 subunit
of phosphatidylinositol
3-kinase(11, 15, 16, 19, 21) .
These proteins all become rapidly phosphorylated by the IGF-I receptor
upon exposure of cells to IGF-I. In this report, we identify Crk II as
a novel substrate of the IGF-I receptor. Upon treatment of either 293
kidney-derived cells or NIH-3T3 fibroblasts with IGF-I, Crk II becomes
rapidly tyrosine-phosphorylated in a dose- and time-dependent manner.
It has previously been shown that in PC12 cells overexpressing
v-crk, stimulation with nerve growth factor or EGF induces
tyrosine phosphorylation of v-crk(9) . Similarly, in
human carcinoma A431 cells overexpressing Crk II or a bacterially
expressed 31-amino acid N-terminal extended Crk protein, there was a
slight induction of tyrosine phosphorylation of these proteins by
EGF(25) . However, this is the first report of a mitogenic
growth factor strongly inducing tyrosine phosphorylation of endogenous
c-Crk. It should be emphasized that these studies were conducted in
cells expressing endogenous IGF-I receptors, at approximately 1 Using bacterially expressed chicken Crk constructs, it has recently
been shown that Crk II binds to and is tyrosine-phosphorylated by c-Abl
on Tyr-221(5) . This is a region located between the two SH3
domains and that is deleted in v-crk and Crk I, the cellular
21-kDa Crk protein(2, 3) . In 293 cells, IGF-I appears
to induce two states of tyrosine phosphorylation of Crk II, as
evidenced by an upward shift of mobility of Crk immunoreactivity into
two phosphotyrosine-containing bands. The uppermost
phosphotyrosine-containing band (band 1, Fig. 1) was
only very faintly recognized by the monoclonal Crk antibody used in
these studies. This could indicate either that the relative abundance
of this isoform is very low, and that multiple tyrosine
phosphorylations render the protein more easily detectable with a
phosphotyrosine antibody, or that the epitope recognized by the Crk
antibody is altered by multiple tyrosine phosphorylations, or a
combination of both. Thus, it is difficult to estimate the relative
abundance of the two tyrosine-phosphorylated isoforms of Crk II. An
alternative possibility is that there is a single tyrosine
phosphorylation of Crk II, and that the second shift in mobility is due
to another post-translational modification, such as serine or threonine
phosphorylation. In NIH-3T3 cells, IGF-I apparently induced only a
single state of tyrosine phosphorylation of Crk II, as only one
phosphotyrosine-containing Crk immunoreactive band was detected in Crk
immunoprecipitates of these cells. This differential phosphorylation
suggests that Crk II may function differently in various cell types. Treatment of 293 cells with insulin also resulted in tyrosine
phosphorylation of Crk, with somewhat lower sensitivity than that
produced by IGF-I. Whereas IGF-I induced strong tyrosine
phosphorylation of Crk at 10 Like other tyrosine
kinase growth factor receptors, the IGF-I and insulin receptors are
known to phosphorylate Shc
proteins(17, 18, 19, 20, 21) ,
which can then associate with the Grb2-mSos complex, leading to
activation of Ras and the subsequent Raf and mitogen-activated protein
kinase pathways(26, 27, 28) . A second
mechanism by which IGF-I and insulin receptors can activate Ras is via
IRS-1 association with the Grb2-mSos complex(29, 30) .
Recent findings have demonstrated SH3-mediated interactions of Crk with
the Ras family guanine nucleotide releasing proteins mSos and
C3G(7, 8) . Thus, Crk may participate in a third
mechanism by which the IGF-I receptor can signal Ras. It will be of
interest to understand the mechanism of interaction between the IGF-I
receptor and Crk. In preliminary experiments, IGF-I receptor
immunoreactivity was not detected in Crk immunoprecipitates, nor was
Crk detected in IGF-I receptor immunoprecipitates (data not shown).
While we cannot rule out the possibility that another intervening
tyrosine kinase is involved in coupling the IGF-I receptor to Crk, we
favor the hypothesis that Crk is a direct substrate of the IGF-I
receptor itself, based on the induction of Crk phosphorylation by IGF-I
at early time points and at low doses. One mechanism by which these two
proteins could associate is via interaction of the Crk SH2 domain with
phosphotyrosine residues on the IGF-I receptor, similar to the
mechanism of interaction of other phosphorylated growth factor
receptors with various SH2 domain-containing signaling
proteins(31) . However, the Crk SH2 domain strongly prefers a
proline in the +3 position relative to tyrosine(31) , and,
since the IGF-I receptor does not contain any Y-X-X-P
motifs(32) , this mechanism appears unlikely. Another possible
mechanism by which Crk can associate with other proteins is by
interaction of its SH3 domains with proline-rich sequences, such as
those found in mSos and C3G(7) . The IGF-I receptor sequence
also does not appear to be sufficiently proline-rich to confer SH3
binding(32, 33) . Thus, further studies are needed to
identify specific regions of the IGF-I receptor that are involved in
Crk association and phosphorylation and/or other associated proteins
that may be involved in such interactions. In NIH-3T3 cells, the as yet
unidentified Crk-associated protein pp45 could be involved in IGF-I
receptor association, as this protein was also tyrosine-phosphorylated
in response to IGF-I stimulation. In PC12 cells, Crk proteins have been
found to associate with Shc(9) . However, pp45 does not
co-migrate with Shc and was not recognized by an anti-Shc antibody
(data not shown). Studies are currently underway to identify and
characterize pp45. It will also be of interest in future studies to
determine what, if any, role Crk phosphorylation plays in Ras
activation.
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5187-5190
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
60% homology with Crk II(4) . Crk II and Crkl are
known to be tyrosine-phosphorylated by c-Abl; Crkl is also a substrate
for Bcr/Abl(5, 6) . It has recently been shown that
Crk proteins associate with two guanine nucleotide releasing proteins,
C3G and mSos(7, 8) . In addition, overexpression of
Crk proteins can modulate growth factor-induced differentiation and
activation of Ras in pheochromocytoma (PC12)
cells(8, 9) .
)receptor activation and
autophosphorylation is followed by tyrosine phosphorylation of various
substrate proteins involved in postreceptor signaling. To date, the
best characterized substrates of the IGF-I and insulin receptors are
IRS-1(10, 11, 12) , the p85 subunit of
phosphatidylinositol 3-kinase (13, 14, 15, 16) , and Shc
proteins(17, 18, 19, 20, 21) .
While these proteins clearly play important roles in IGF-I receptor
signaling, the intracellular pathways involved in coupling IGF-I and
insulin receptors to Ras have still not been completely defined. Based
on the fact that Crk proteins share homology with Grb2 and
Nck(2, 3) , two SH2 and SH3 domain-containing proteins
that are involved in insulin and IGF-I receptor signaling (22, 23, 24) , we were interested in
determining whether Crk proteins might also be involved in IGF-I
receptor signaling pathways.
Cell Culture
Both human embryonic kidney
carcinoma (293) cells and NIH-3T3 mouse fibroblasts were cultured in
Dulbecco's modified Eagle's medium (Biofluids) supplemented
with 10% fetal bovine serum (Upstate Biotechnology Inc., Lake Placid,
NY). Prior to growth factor stimulation, subconfluent cultures of cells
in 60-mm or 100-mm dishes were switched to serum-free Dulbecco's
modified Eagle's medium supplemented with 0.1% insulin-free
bovine serum albumin (Intergen, Purchase, NY) and 20 mM HEPES
(pH 7.5), for 18 h. Cells were treated with IGF-I (Genentech), insulin
(Sigma), epidermal growth factor (EGF, Upstate Biotechnology Inc.), or
platelet-derived growth factor (PDGF BB, Life Technologies, Inc.)
diluted in serum-free Dulbecco's modified Eagle's medium at
37 °C for various time points and concentrations, as indicated.Immunoprecipitations
After treatment with growth
factors, cells were washed twice with ice-cold phosphate-buffered
saline and harvested in a lysis buffer containing 50 mM HEPES
(pH 7.4), 2 mM sodium orthovanadate, 100 mM NaCl, 4
mM sodium pyrophosphate, 200 mM EDTA, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml
leupeptin, 2 µg/ml aprotinin, and 1% Triton X-100. Lysates were
incubated for 1 h at 4 °C, then centrifuged at 10,000 
g for 30 min at 4 °C to remove Triton-insoluble material.
Protein content of the lysates was determined by the Bio-Rad method.
600 µg of protein from each dish was immunoprecipitated overnight
at 4 °C with 3 µg of a monoclonal anti-Crk antibody
(Transduction Labs, Lexington, KY), followed by adsorption to 50 µl
of 10% Protein A-Sepharose beads (Pharmacia Biotech Inc.) for 5 h at 4
°C. Immunoprecipitates were washed three times with ice-cold
immunoprecipitation buffer containing 10 mM Tris (pH 7.4), 150
mM NaCl, 0.2 mM sodium orthovanadate, 1 mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl
fluoride, 1% Triton X-100, and 0.5% Nonidet P-40. All of the
immunoprecipitated samples were then boiled for 2 min in sample buffer
containing 50 mM Tris (pH 6.7), 2% SDS, 2%
-mercaptoethanol, and bromphenol blue as a marker. Samples were
then run on 9% SDS-polyacrylamide gel electrophoresis gels and
transferred to nitrocellulose membranes using standard electrophoresis
and electroblotting procedures.Immunoblotting
Nitrocellulose membranes were
blocked with either 3% insulin-free bovine serum albumin (for
phosphotyrosine blotting) or 3% nonfat dry milk in a PBST buffer
containing 10 mM sodium phosphate (pH 7.2), 140 mM NaCl, and 0.1% Tween 20. Blots were then immunolabeled overnight
at 4 °C for phosphotyrosine using a monoclonal antibody (RC20H,
1:2500, Transduction Labs) or Crk using a monoclonal antibody (1:500,
Transduction Labs). Immunolabeling was detected by enhanced
chemiluminescence (ECL, Amersham) according to the manufacturer's
conditions. Some blots were stripped and reprobed with another
antibody. Blots were stripped by incubation for 1 h at 50 °C in a
solution containing 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and
0.7% -mercaptoethanol. Blots were then washed for 1 h in several
changes of PBST at room temperature and probed with ECL to confirm that
antibodies had been completely removed. Blots were then reblocked and
immunolabeled as described above.
= 40,000, labeled as
bands 1 and 2. These proteins were also faintly detectable in
unstimulated Crk immunoprecipitates. This blot was then stripped and
reprobed with an anti-Crk antibody. Using luminescent markers taped
directly to the nitrocellulose membrane, the resulting films were
aligned as shown in Fig. 1. In unstimulated cells, a single band
of Crk immunoreactivity (labeled as band 3) was observed at 40
kDa, corresponding to the molecular mass of Crk
II(2, 3) . In stimulated cells, there was a partial
shift of Crk immunoreactivity into two more slowly migrating bands (Fig. 1B). Band 3, the most prominent
Crk-immunoreactive band in unstimulated cells, was not detected by the
phosphotyrosine antibody. In stimulated cells, the major shift of Crk
immunoreactivity corresponded to band 2, the more abundant
phosphotyrosine-containing species. In longer exposures, band 1, the
less abundant phosphotyrosine species, was also faintly detectable by
the Crk antibody (data not shown).
I-IGF-I and I-insulin radioligand binding,
indicated that these cells express roughly 11,000 and 9,000 IGF-I and
insulin receptors per cell, respectively (data not shown).
10
receptors per cell, as determined by radioligand
binding. This is in contrast to many of the studies which have
characterized Shc proteins as targets of the insulin and IGF-I receptor
tyrosine kinases, where cells have generally been engineered to
overexpress either the receptors (on the order of 1 10
receptors per cell(17, 20, 23) ) or Shc
proteins(21) . The fact that at low doses (10
M) IGF-I readily induces tyrosine phosphorylation of
endogenous Crk II via stimulation of endogenous receptors indicates
that Crk II is a sensitive in vivo substrate for the receptor. 
M, insulin was
equally effective only at 10
M. At high
doses, insulin is known to activate IGF-I receptors, and these data
could be interpreted to suggest that insulin is actually producing its
effects via IGF-I receptors. However, we have also found that EGF (50
ng/ml), and to a lesser extent, platelet-derived growth factor (50
ng/ml), also induce tyrosine phosphorylation of Crk in 293 cells (data
not shown). Thus, it would appear more plausible that insulin receptors
can also mediate this effect, albeit with less efficacy than IGF-I
receptors. This indicates that, similar to their common effects on many
other signaling pathways, insulin and IGF-I receptors share the ability
to induce tyrosine phosphorylation of Crk II.
)
We thank Drs. Simeon Taylor, Carol Renfrew-Haft, and
Vicky Blakesley for critical review of the manuscript and Keren Paz for
helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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V. A. Blakesley, D. Beitner-Johnson, J. R. Van Brocklyn, S. Rani, Z. Shen-Orr, B. S. Stannard, S. Spiegel, and D. LeRoith Sphingosine 1-Phosphate Stimulates Tyrosine Phosphorylation of Crk J. Biol. Chem., June 27, 1997; 272(26): 16211 - 16215. [Abstract] [Full Text] [PDF]
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A. G. Scrimgeour, V. A. Blakesley, B. S. Stannard, and D. LeRoith Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase Pathways Are Not Sufficient for Insulin-Like Growth Factor I-Induced Mitogenesis and Tumorigenesis Endocrinology, June 1, 1997; 138(6): 2552 - 2558. [Abstract] [Full Text] [PDF]
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C. S.S. Rani, F. Wang, E. Fuior, A. Berger, J. Wu, T. W. Sturgill, D. Beitner-Johnson, D. LeRoith, L. Varticovski, and S. Spiegel Divergence in Signal Transduction Pathways of Platelet-derived Growth Factor (PDGF) and Epidermal Growth Factor (EGF) Receptors. INVOLVEMENT OF SPHINGOSINE 1-PHOSPHATE IN PDGF BUT NOT EGF SIGNALING J. Biol. Chem., April 18, 1997; 272(16): 10777 - 10783. [Abstract] [Full Text] [PDF]
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S. Okada and J. E. Pessin Interactions between Src Homology (SH) 2/SH3 Adapter Proteins and the Guanylnucleotide Exchange Factor SOS Are Differentially Regulated by Insulin and Epidermal Growth Factor J. Biol. Chem., October 11, 1996; 271(41): 25533 - 25538. [Abstract] [Full Text] [PDF]
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S. Kuroda, T. Ohtsuka, B. Yamamori, K. Fukui, K. Shimizu, and Y. Takai Different Effects of Various Phospholipids on Ki-Ras-, Ha-Ras-, and Rap1B-induced B-Raf Activation J. Biol. Chem., June 21, 1996; 271(25): 14680 - 14683. [Abstract] [Full Text] [PDF]
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D. Beitner-Johnson, V. A. Blakesley, Z. Shen-Orr, M. Jimenez, B. Stannard, L.-M. Wang, J. Pierce, and D. LeRoith The Proto-oncogene Product c-Crk Associates with Insulin Receptor Substrate-1 and 4PS J. Biol. Chem., April 19, 1996; 271(16): 9287 - 9290. [Abstract] [Full Text] [PDF]
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V. Ribon and A. R. Saltiel Nerve Growth Factor Stimulates the Tyrosine Phosphorylation of Endogenous Crk-II and Augments Its Association with p130[IMAGE] in PC-12 Cells J. Biol. Chem., March 29, 1996; 271(13): 7375 - 7380. [Abstract] [Full Text] [PDF]
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C. Hernández-Sánchez, V. Blakesley, T. Kalebic, L. Helman, and D. LeRoith The Role of the Tyrosine Kinase Domain of the Insulin-like Growth Factor-I Receptor in Intracellular Signaling, Cellular Proliferation, and Tumorigenesis J. Biol. Chem., December 8, 1995; 270(49): 29176 - 29181. [Abstract] [Full Text] [PDF]
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E. L. K. Goh, T. Zhu, S. Yakar, D. LeRoith, and P. E. Lobie CrkII Participation in the Cellular Effects of Growth Hormone and Insulin-like Growth Factor-1. PHOSPHATIDYLINOSITOL-3 KINASE DEPENDENT AND INDEPENDENT EFFECTS J. Biol. Chem., June 2, 2000; 275(23): 17683 - 17692. [Abstract] [Full Text] [PDF]
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S. Jin, B. Zhai, Z. Qiu, J. Wu, M. D. Lane, and K. Liao c-Crk, a Substrate of the Insulin-like Growth Factor-1 Receptor Tyrosine Kinase, Functions as an Early Signal Mediator in the Adipocyte Differentiation Process J. Biol. Chem., October 27, 2000; 275(44): 34344 - 34352. [Abstract] [Full Text] [PDF]
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R. K. Bommakanti, S. Vinayak, and W. F. Simonds Dual Regulation of Akt/Protein Kinase B by Heterotrimeric G Protein Subunits J. Biol. Chem., December 1, 2000; 275(49): 38870 - 38876. [Abstract] [Full Text] [PDF]
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J. C. Stam, W. J. C. Geerts, H. H. Versteeg, A. J. Verkleij, and P. M. P. v. B. en Henegouwen The v-Crk Oncogene Enhances Cell Survival and Induces Activation of Protein Kinase B/Akt J. Biol. Chem., June 29, 2001; 276(27): 25176 - 25183. [Abstract] [Full Text] [PDF]
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J. F. Kuemmerle and K. S. Murthy Coupling of the Insulin-like Growth Factor-I Receptor Tyrosine Kinase to Gi2 in Human Intestinal Smooth Muscle. Gbeta gamma -DEPENDENT MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION AND GROWTH J. Biol. Chem., March 2, 2001; 276(10): 7187 - 7194. [Abstract] [Full Text] [PDF]
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S. Gelkop, Y. Babichev, and N. Isakov T Cell Activation Induces Direct Binding of the Crk Adapter Protein to the Regulatory Subunit of Phosphatidylinositol 3-Kinase (p85) via a Complex Mechanism Involving the Cbl Protein J. Biol. Chem., September 21, 2001; 276(39): 36174 - 36182. [Abstract] [Full Text] [PDF]
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K. Kurokawa, N. Mochizuki, Y. Ohba, H. Mizuno, A. Miyawaki, and M. Matsuda A Pair of Fluorescent Resonance Energy Transfer-based Probes for Tyrosine Phosphorylation of the CrkII Adaptor Protein in Vivo J. Biol. Chem., August 10, 2001; 276(33): 31305 - 31310. [Abstract] [Full Text] [PDF]
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