Interaction between Src and a C-terminal Proline-rich Motif of
Akt Is Required for Akt Activation*
Tianyun
Jiang and
Yun
Qiu
From the Department of Pharmacology and Experimental Therapeutics,
University of Maryland School of Medicine,
Baltimore, Maryland 21201
Received for publication, December 9, 2002
 |
ABSTRACT |
Activation of Akt by growth factors is a
multistep process. Here, we provide evidence that tyrosine
kinase Src is directly associated with Akt through the interaction
between its SH3 domain and a conserved proline-rich motif
(PXXP) in the C-terminal regulatory region of Akt.
Substitution of the proline residues Pro-424 and Pro-427 by alanines
results in loss of Akt activity and phosphorylation induced by the
epidermal growth factor (EGF), possibly because these mutations disrupt
the interaction between Akt and the SH3 domain of Src. This possibility
is corroborated by our observation that the Akt mutant lacking these
two prolines fails to bind to Src both in vivo and in
vitro. We also showed that phosphorylation of Tyr-315 in Akt
induced by Src or EGF is dependent on the integrity of this
proline-rich motif. Furthermore, the Akt mutant lacking this proline
motif fails to block the transcription activity of Forkhead in 293 cells and poorly stimulates the proliferation of Madin-Darby canine
kidney cells. Taken together, our data suggest that the
interaction between the SH3 domain of Src family kinases and the
proline-rich motif in the C-terminal regulatory region of Akt is
required for tyrosine phosphorylation of Akt and its subsequent
activation. It is noteworthy that this PXXP motif is conserved throughout several members of AGC kinase family, implying that association of this motif with the SH3 domain of an upstream regulator may represent a general mechanism applicable to these kinases
as well.
 |
INTRODUCTION |
Serine/threonine kinase Akt/protein kinase B (PKB) is a key
regulator in a variety of cellular processes including apoptosis, proliferation, intermediary metabolism, and response to inflammatory agents (1-3). Given its critical role in physiological response as
well as in malignant transformation, the mechanisms underlying Akt
activation are of great interest. Akt belongs to the AGC protein kinase
family. Structural analysis showed that Akt contains an N-terminal
PH1 domain, a central
catalytic domain, and a C-terminal regulatory region. The intact PH
domain is essential for activation of Akt. Upon growth factor
stimulation, binding of the PH domain to the secondary messenger, PIP3,
mediates translocation of Akt to the plasma membrane and the
conformational change, which renders the kinase accessible to its
upstream kinases such as PDK1 and Src (4-8). As a conserved feature
shared by members of AGC protein kinase family, full activation of Akt
requires phosphorylation at Thr-308 in the activation loop and Ser-473
in the C-terminal regulatory domain (2, 9, 10). In addition,
phosphorylation of Akt at several tyrosine residues is also involved in
the regulation of Akt activation (7, 8). Furthermore, protein-protein
interaction between signaling molecules and Akt, especially at its
C-terminal regulatory region, plays important roles in regulation of
its activity. The turn motif of the AGC family kinase is reported to provide a binding site for HSP70, which may stabilize the kinase and
allow it to be re-activated (11). The C-terminal modulator protein
(CTMP), a recently identified negative regulator of Akt, is also
associated with Akt at the C-terminal region encompassing amino acids
411-480 in a phosphorylation-dependent manner (12). These
observations suggest an essential role for the C-terminal regulatory
region in mediating interaction of Akt with key regulatory molecules
during its activation process. It is noteworthy that there are several
proline-rich "patches" in the C-terminal region that may
potentially serve as protein-protein interaction site(s). However,
their functional implications still remain elusive.
In this report, we show that a conserved SH3-domain binding motif
(PXXP), located in the C-terminal regulatory region of Akt, is involved in the interaction between Akt and its upstream tyrosine kinase Src and is essential for Akt activation in response to growth
factors. Mutation of this motif in Akt dramatically diminished its
interaction with Src, which seems to be required for tyrosine phosphorylation of Akt and its subsequent activation by other upstream
kinases such as PDK1. Our data further demonstrated the important role
of the C-terminal regulatory domain and the direct involvement of Src
family kinases in the regulation of Akt activity.
 |
MATERIALS AND METHODS |
cDNA Constructs and Antibodies--
Mammalian expression
constructs of HA-tagged Akt wt and Akt K179M were kindly gifts from Dr.
Philip Tsichlis. Full-length Akt cDNA was inserted in pGEX3X for
generating the GST·Akt fusion protein. P424A/P427A mutants
were generated by a PCR based method using a QuikChange kit
(Stratagene). The mouse monoclonal antibody against the HA epitope used
for immunoprecipitation and immunoblotting was from Berkeley Antibody.
Phospho-specific antibodies against Akt pT308, pS473, and pY315 were
from Cell Signaling Technology, Inc. Anti-phosphotyrosine PY99 was from
Santa Cruz Biotechnology.
Cell Culture and Transfection--
COS1 cells (from ATCC) were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum. The cells were transfected with FuGENE 6 (Roche Molecular Biochemicals) following the manufacturer's
instruction. 1 µg of Akt wt or mutants together with 1 µg of vector
or Src were used. At 24 h post-transfection, the medium was
removed, and the cells were serum starved for 24 h. The cells were
then left untreated or treated with 100 ng/ml of EGF for 10 min.
Transient Transfection Reporter Assay--
Human embryonic
kidney 293 cells grown in 12-well plates were transfected with
3XIRS-luc (kindly provide by Dr. K.-L. Guan's lab) (13), FLAG-tagged
FKHR, and HA-tagged Akt or its mutants. pRL-TK, a control reporter, was
cotransfected as an internal control. The total amount of DNA was kept
consistent using empty vector. Cells were lysed 24 h later, and
Dual luciferase assays were performed according to the manufacturer's
instruction (Promega, Madison, WI).
Cell Proliferation Assay--
MDCK cells were transfected with
vector, HA-tagged myristoylated (myr) Akt wt, and myr Akt P424A/P427A,
respectively. 24 h later, the transfected cells were seeded on
96-well plates and incubated in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum for 6 h to allow the
cells to attach. The cells were then maintained in serum-free medium
for another 12 h. The proliferation rate was measured using WST-1
reagent (Roche Molecular Biochemicals). An aliquot of cells was lysed
and followed by Western blotting with anti-HA to monitor the
transfection efficiency.
Immunoprecipatation, Western Blot, and in Vitro Kinase
Assay--
The transfected cells were washed twice with ice cold
phosphate-buffered saline and then lysed with lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1% TritonX-100, 2.5 mM sodium
pyrophosphate, 1 mM Na3VO4, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride) at 4 °C for 30 min. The cell lysates
were centrifuged to remove cell debris before incubation with anti-HA
at 4 °C for 1 h. The immunocomplexes were collected with
protein G-Sepharose beads. The immunoprecipitates for Western blot were
resuspended in SDS sample buffer and resolved by SDS-polyacrylamide gel
electrophoresis. Immunoblotting was performed as described previously
(7, 14). The immunoprecipitates for in vitro kinase assay
were washed twice with kinase buffer (20 mM Tris, pH 7.4, 10 mM MgCl2, and 2 mM
dithiothreitol) and then incubated at room temperature with kinase
buffer containing 3 µg of H2B, 2 µM ATP, and 10 µCi
of [
-32P]ATP. The reaction was terminated by adding 30 µl of 2× SDS sample buffer and boiling for 10 min. The reaction
mixtures were separated by 12% SDS-polyacrylamide gel electrophoresis.
The gel was dried, and the phosphorylation of H2B as indicated by
-32P incorporation was visualized by autoradiography
(15).
In Vitro Binding Assay--
The expression and purification of
GST-fusion proteins containing wild type Akt or Akt P424A/P427A were
described previously (7, 14, 16). The bacteria were harvested and
resuspended in PBS containing protease inhibitors and lysed by
sonication. After centrifugation (13,000 × g for 10 min), the fusion proteins were collected by incubating the supernatants
with glutathione-Sepharose 4B beads (Amersham Biosciences) at 4 °C
for 1 h with continuous shaking. The beads were washed three times
to remove the unbound proteins. The GST-fusion proteins attached to
glutathione-Sepharose 4B beads were mixed with the lysates of COS1
cells transfected with c-Src for 1 h at 4 °C by rotating. The
beads were washed three times, and the bound proteins were
immunoblotted with anti-Src antibody.
 |
RESULTS |
Alignments of the C-terminal regulatory region of Akt kinases from
various organisms revealed an evolutionarily conserved SH3
domain-binding motif, PXXP, encompassing the residues
424-427 (Fig. 1A). To test
whether this proline-rich motif is of functional importance, we
substituted both proline residues (Pro-424 and Pro-427) with alanines.
As shown in Fig. 1B, the P424A/P427A mutant failed to
respond to EGF stimulation, as evidenced by its lack of phosphorylation
at Thr-308 and Ser-473 as well as in vitro kinase activity
using H2B as a substrate. These data suggest that the integrity of this
proline-rich motif is essential for Akt activity induced by growth
factors. We further examined the effects of the mutation of these two
proline residues on Akt activation induced by Src, which contains an
SH3 domain and can potentially interact with the PXXP motif
in Akt. Fig. 1C shows that mutation of Pro-424 and Pro-427
abolished the activation of Akt induced by a constitutively active
Src527F. These results prompted us to test whether Src is physically
associated with Akt upon growth factor stimulation. As shown in Fig.
2A, we were able to detect the
co-precipitation of endogenous Akt with Src in human prostate cancer
cell PC3 cells after 5 min of treatment with EGF. This interaction
seemed to be transient and peaked at 15 min of treatment and decayed
rapidly at 30 min. Similar results were also obtained in COS1 cells. To
further confirm this interaction, c-Src and Akt were co-expressed in
COS1 cells, and immunoprecipitations were performed using anti-Src
antibody followed by Western blotting with an anti-Akt antibody. As
shown in Fig. 2B, in the presence of serum wild-type Akt was
efficiently co-precipitated with Src, but the mutant Akt was not,
suggesting that Akt is associated with Src and that this interaction
depends on the PXXP motif. These results are further
supported by the following GST pull-down experiments. GST·Akt or its
mutant, P424A/P427A, was immobilized to glutathione beads and then
mixed with the cell extracts from COS1 cells overexpressing c-Src. In
Fig. 2C, we showed that c-Src was only pulled down by the
wild-type Akt but not the P424A/P427A mutant. These data suggest that
the PXXP motif could serve as a binding site for the SH3
domain of Src, and such an interaction seems to be required for Akt
activation by Src as shown above. The interaction between Akt and the
Src SH3 domain appeared to be specific, because no detectable
association of Akt with the SH3 domain of p130Cas was observed (data
not shown).

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Fig. 1.
The proline-rich motif is required for Akt
activation. a, alignments of amino acid sequence of the
C-terminal regulatory region of Akt from different species. The
evolutionary conserved PXXP motifs are highlighted. The
numbers of amino acids are denoted based on mouse Akt1. b,
the role of proline-rich motif in EGF-induced Akt activation. COS1
cells were transiently transfected with the indicated constructs.
At 24 h post-transfection, the cells were serum starved for
24 h and then left untreated or treated with 100 ng/ml EGF for 10 min. Immunoprecipitations (IP) were performed as described
under "Materials and Methods." The immunoprecipitates were subject
to in vitro kinase (IVK) assays or immunoblotting
(IB) with phospho-specific antibodies against Akt. The
amount of Akt in each immunoprecipitate was monitored by anti-HA
antibody. c, a proline-rich motif is necessary for the
activation of Akt by Src. COS1 cells were cotransfected with the
indicated constructs of Akt and Src527F. At 24 h
post-transfection, the cells were serum starved for 24 h.
Immunoprecipitation, in vitro kinase assays, and
immunoblotting were performed as in panel
b.
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Fig. 2.
Interaction between the proline-rich motif of
Akt and the SH3 domain of Src. a,
co-immunoprecipitation of endogenous Akt with Src upon EGF-treatment.
PC3 and COS1 cells were serum starved overnight and followed by
treatment with EGF for the indicated time. The cells were then lysed,
and immunoprecipitations (IP) were performed with anti-Src
antibody followed by immunoblotting (IB) with anti-Akt. The
membrane was then stripped and reprobed with anti-Src antibody. The
immunoblotting of total cell lysates (TCL) with anti-Akt was
used to monitor the level of Akt in each lysate. C, the
control cells without EGF treatment. b, the proline-rich
motif is required for the association of Akt with Src in
vivo. COS1 cells were co-transfected with the indicated plasmids
for 24 h. The cell lysates were subjected to immunoprecipitation
with anti-Src antibody followed by immunoblotting with anti-Akt
antibody. c, proline-rich motif is required for the
interaction of Akt and src in vitro. COS1 cells were
transfected with c-Src. The cell lysates were incubated with GST·Akt
wt or GST·Akt P424A/P427A bound to glutathione-Sepharose 4B beads at
4 °C for 1 h. The beads were washed intensively, and the bound
proteins were detected by immunoblotting with an anti-Src
antibody.
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Our previous study showed that tyrosine phosphorylation of Tyr-315 and
Tyr-326 by Src plays an important role in the regulation of Akt
activity (7). We would like to know whether the interaction between Src
and Akt could be a prerequisite step for phosphorylating Akt by Src. As
shown in Fig. 3A, the mutation
of Pro-424 and Pro-427 dramatically diminished the level of tyrosine
phosphorylation of Akt induced by Src. These results are further
confirmed by using an antibody specific for phosphorylated Tyr-315.
Similar results were also obtained when the cells were stimulated with EGF (Fig. 3B). To understand the relationship between
tyrosine phosphorylation and Ser/Thr phosphorylation of Akt
during its activation, we also examined the tyrosine phosphorylation of
Akt derivatives containing two major Ser/Thr phosphorylation site mutations. As shown in Fig 2C, phosphorylation of Tyr-315
was readily detected in wild-type Akt, Akt T308A, and Akt S473A in EGF-treated or Src527F-contransfected cells. This suggests that phosphorylation of Tyr-315 may be prior to and independent of the
phosphorylation of Thr-308 and Ser-473. Taken together, these observations allow us to conclude that the interaction between the
PXXP motif of Akt with the SH3 domain of Src family kinases is required for tyrosine phosphorylation of Akt and its subsequent activation by other upstream kinases.

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Fig. 3.
The proline-rich motif is required for
tyrosine phosphorylation of Akt. a, mutation of the
proline-rich motif diminishes tyrosine phosphorylation of Akt.
HA-tagged wild type Akt or Akt P424A/P427A were cotransfected with
Src527F into COS1 cells. The cells were serum starved, and
immunoprecipitations were performed with anti-HA antibody. Tyrosine
phosphorylation (pY) and phosphorylation of Tyr-315
(pY315) were determined by immunoblotting (IB)
with anti-phosphotyrosine 99 ( pY) and
anti-phosphotyrosine 315 ( pY315). b, effects
of the mutation of the proline-rich motif on tyrosine phosphorylation
induced by EGF. COS1 cells were transfected with the indicated Akt
constructs and serum starved for 24 h before treated with EGF (100 ng/ml). Tyrosine phosphorylation and phosphorylation of Tyr-315 were
determined by immunoblotting as in panel a.
c, effects of the mutations of Thr-308 (T308) and
Ser-473 (S473) on tyrosine phosphorylation of 315 (pY315) of Akt. HA-tagged Akt and its mutants were
transfected into COS1 cells and followed with treatment as described in
panels a and b. Phosphorylation status
of Akt was determined by phosphor-specific antibodies against Tyr-315
( pY315), Thr-308 ( pT308), or Ser-473 ( pS473).
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To investigate whether this proline-rich motif in Akt is required for
its biological function, we first examined the effects of the mutation
of the proline-rich motif on the transcriptional activity of Forkhead,
which is negatively regulated by Akt (13). Fig.
4A shows that co-transfection
of wild-type Akt with FKHR into 293 cells dramatically inhibited FKHR
activity as evidenced by a marked reduction of the luciferase activity
under the control of a promoter containing three copies of the FKHR
recognition motif IRS. The mutant Akt P424A/P427/A virtually lost its
inhibitory effect on FKHR, which is consistent with its lack of kinase
activity in the in vitro kinase assays. As another
functional assay, we also examined the effects of the mutation of this
proline-rich motif on cell proliferation. As shown in Fig.
4B, overexpression of constitutively active myr Akt in MDCK
cells resulted in 2-fold increase in cell proliferation, whereas its
derivative containing P424A/P427A mutations had very little effect. Our
data suggest that the integrity of this proline-rich motif is required
for its biological functions.

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Fig. 4.
The proline-rich motif is required for the
biological functions of Akt. a, mutation of the
proline-rich motif attenuated the inhibitory effects of Akt on FKHR
transcription activity. 293 cells were transfected with 3XIRS-luc,
FLAG-tagged FKHR, pRL-TK, and HA-tagged Akt or its mutants. Luciferase
activity was determined as described under "Materials and Methods."
The results were expressed as mean ± S.D. of three independent
experiments. The expression of Akt or FKHR was monitored by
immunoblotting with anti-HA or anti-FLAG antibodies. b, the
effects of proline-rich motif on cell proliferation. MDCK cells were
transfected with vector, myr Akt, and its mutant. 24 h later, the
cells were seeded on 96-well plates, and the proliferation rate was
assayed using WST-1 reagent. The data are given as mean ± S.D. of
three independent experiments. Error bar
represent the S.D. *, p < 0.01, compared with the
vector control; **, p < 0.01, compared with myr Akt. A
fraction of these cells were lysed and subjected to immunoblotting with
anti-HA to monitor the expression level of Akt.
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 |
DISCUSSION |
Protein-protein interactions, which control the organization of
the complex network of molecules involved in signal transduction, are
mediated via interactions of various structure domains and their
recognition motifs. In this study, we demonstrated, for the first time,
that a PXXP motif, which is evolutionarily conserved in Akt
kinases among organisms from Caenorhabditis
elegans to human, plays a crucial role in regulating Akt
activity. This PXXP motif is located in the C-terminal
regulatory region of Akt. When it is mutated, Akt is no longer
activated by EGF. Because phosphorylation of Thr-308 and Ser-473 is
necessary for Akt activation, we then examined the effects of the
mutation of the PXXP element on the phosphorylation state of
these two critical sites. As expected, EGF failed to induce
phosphorylation of either site. According to the current model of Akt
activation, translocation of Akt from cytosol to the plasma membrane is
required for its phosphorylation by upstream kinases. The loss of
response of the mutant to growth factors may result from incapability
of translocation to the plasma membrane, because the interaction
between proline-rich motif and SH3 domain has been implicated in plasma
membrane localization of some proteins (17-19). However, we found that
the mutant Akt P424A/P427A can still be efficiently recruited to the
plasma membrane as the wild-type (data not shown). These data suggested
that this PXXP element regulates Akt activation through
mechanisms other than affecting its recruitment to plasma membrane.
Recently, we and others have reported that, in addition to
phosphorylation of Thr-308 and Ser-473, tyrosine phosphorylation is
also implicated in the regulation of Akt activation (7, 8). In our
previous study, we provided evidence showing that Src directly
regulates Akt activity by phosphorylating Tyr-315 and Tyr-326 in the
activation loop of Akt. Because Src contains an SH3 domain, it is
likely that Src could potentially interact with the PXXP
motif in Akt. In this report, we showed that endogenous Akt is
associated with Src in response to EGF. Such interaction was confirmed
by both in vivo and in vitro binding assays. As
expected, the mutation of the PXXP motif markedly reduced
the amount of Src associated with Akt. This indicated that binding of
the PXXP motif on Akt and the SH3 domain of Src plays a
major role in mediating their interaction. We then demonstrated that
this interaction could be a prerequisite for the Src family kinases to
phosphorylate the two critical tyrosine residues in the activation loop
of Akt, because replacement of the two proline residues with alanines dramatically blocked the tyrosine phosphorylation of Akt. Moreover, the
phosphorylation of Tyr-315 was also significantly reduced. Furthermore,
we showed that that EGF- or Src527F-induced phosphorylation of Tyr-315
is not affected in Akt T308A or Akt S473A, suggesting that tyrosine
phosphorylation of Akt may occur prior to serine/threonine phosphorylation, at least in COS1 cells. The interaction between Akt
and Src is functionally important because the mutant Akt P424A/P427A failed to inhibit FKHR transcriptional activity and can hardly stimulate cell proliferation. Our data are consistent with the model in
which Akt translocates to the plasma membrane through the binding of
its PH domain to the second messenger PIP3 generated by
phosphatidylinositol 3-kinase, where Akt meets with membrane-bound Src
via the interaction between its PXXP motif in the C-terminal regulatory region and the SH3 domain of Src. This allows Src family kinase(s) to phosphorylate Akt at Tyr-315 and Tyr-326, which is followed by phosphorylation of Akt at Thr-308 and Ser-473 by other upstream kinases such as PDK1. Our results further support the notion
that Src family tyrosine kinases can directly regulate Akt activity in
concert with phosphatidylinositol 3-kinase and PDK1. Most intriguingly,
this PXXP motif is conserved throughout several members of
the AGC kinase family including protein kinase A, protein kinase C, and
S6K, suggesting that the association of this motif with its recognition
domain in the corresponding upstream regulator may be a general
mechanism applicable to these kinases.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Tung Chan, Philip Tsichlis, and
Kun-Liang Guan for providing the constructs used in this study.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant CA85380 and Department of Defense Grant PC020169 (to Y. Q.).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.
To whom correspondence should be addressed: Dept. of Pharmacology
and Experimental Therapeutics, University of Maryland School of
Medicine, Baltimore, MD 21201. Tel.: 410-706-4535; Fax: 410-706-0032; E-mail: yqiu@som.umaryland.edu.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M212525200
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ABBREVIATIONS |
The abbreviations used are:
PH, pleckstrin
homology;
SH3, Src homology 3;
HA, hemagglutinin A;
wt, wild-type;
GST, glutathione S-transferase;
EGF, epidermal growth factor;
MDCK, Madin-Darby canine kidney;
PIP3, phosphatidylinositol(3,4,5)P3;
FKHR, Forkhead homologue in
rhabdomyosarcoma;
myr, myristoylated.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.