From the Department of Immunology, The Scripps Research
Institute, La Jolla, California 92037 and the R. W. Johnson Pharmaceutical Research Institute, San
Diego, California 92121
Received for publication, November 28, 2000, and in revised form, January 24, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation of the mammalian mitogen-activated
protein kinase known as BMK1 is required for growth
factor-induced cell proliferation. To understand the mechanism by which
BMK1 mediates this cellular response, this kinase was used as bait in a
yeast two-hybrid-based library screening. Here, we report the
identification of serum and glucocorticoid-inducible kinase (SGK) as a
cellular protein that physically interacts with BMK1. During growth
factor-induced cell stimulation, BMK1 activates SGK by phosphorylation
at serine 78. This BMK1-mediated phosphorylation event is necessary for the activation of SGK and, more importantly, for cell proliferation induced by growth factors.
Genetic and biochemical studies have identified the
mitogen-activated protein
(MAP)1 kinases as central
intracellular molecules that deliver signals from activated cell
surface receptors to downstream regulatory proteins. These MAP kinases
have been conserved in all eukaryotes, ranging from yeast to mammals,
and have a universal role in controlling cell growth through the
regulation of cell cycle progression (1-6). The rate of cell cycle
progression is tightly regulated by both growth factors and
stress-related stimuli, and MAP kinases deliver and integrate both
types of these extracellular signals to the cell cycle machinery by
modulating the phosphorylation state of intracellular proteins. The MAP
kinases ERK1/2, JNK1, and p38 control cell cycle progression by
regulating either the expression or the activity of key molecules
required for G1 to S phase transition. We have previously
demonstrated that BMK1/ERK5, the newest member of the MAP kinase family
(7-11), is required for growth factor-induced cell proliferation and
cell cycle progression (10). Although we have established that the
activity of BMK1 is required for the growth factor-mediated entry of
cells into the S phase of the cell cycle (10), the downstream
effector(s) of this process have not yet been reported.
To investigate the mechanism by which BMK1 mediates the entry of cells
into S phase, this kinase was used as bait in a yeast two-hybrid
screening of a cDNA library. Here we report the identification of
serum- and glucocorticoid-inducible protein kinase (SGK) as a molecule
that physically interacts with BMK1. SGK is a serine/threonine protein
kinase with significant sequence homology throughout its catalytic
domain with protein kinase B, ribosomal protein S6 kinase, cAMP-dependent protein kinase, and members of the protein
kinase C family (12). A variety of stimuli, including glucocorticoids, hydrogen peroxide, hyperosmotic stress, serum, and insulin-like growth
factor, have been shown to induce both the cellular expression and
kinase activity of SGK (12-16). Similar to BMK1, the activity of SGK
is closely linked to the G1/S transition of the cell cycle (17). Here, we show that BMK1 activates SGK as a result of growth factor-induced cellular activation through the phosphorylation of
serine 78. Moreover, we demonstrate that the BMK1-mediated phosphorylation of SGK is critical for growth factor-induced entry of
cells into the S phase of the cell cycle.
Two-hybrid Screening--
The NdeI to
BamHI fragment encoding full-length BMK1(AEF) was fused in
frame with the GAL4 DNA binding domain of pGBKT7
(CLONTECH) to create BD/BMK1(AEF). A human
epithelial carcinoma cDNA library was separately fused with the
GAL4 activation domain (AD) of the vector pGAD-GH
(CLONTECH). The yeast strain PJ69-2A was
co-transformed with this library along with BD/BMK1(AEF), and
transformed yeast were screened for their ability to grow on plates
lacking histidine and adenine according to the supplier's protocols.
Expression Vectors and Recombinant Adenovirus--
A full-length
cDNA encoding human SGK was obtained from The Integrated Molecular
Analysis of Genome and Their Expression (IMAGE) Consortium
(clone ID 42669) and was cloned, in frame with the GST sequence, into
the mammalian expression vector pEBG (18). All point mutations in the
SGK gene were generated by polymerase chain reaction-based
mutagenesis as described previously (8, 11). Recombinant BMK1
and MEK5(D) were produced using expression vectors encoding human BMK1
and rat MEK5(D) as described in Ref. 8. To construct recombinant
adenovirus, SGK(S78A) was cloned into the
XhoI/XbaI site of the vector pShuttle-CMV
(Quantum). Recombinant adenoviral particles were generated as described
in protocols provided by the supplier.
Coupled in Vitro Kinase Assay--
HEK 293T cells were
co-transfected with expression vectors encoding FLAG-BMK1 and MEK5(D),
a dominant active form of the upstream kinase for BMK1 (8). Activated
FLAG-BMK1 protein was purified from HEK 293T cell extracts using
anti-FLAG (M2) antibody gel (Sigma) and eluted in kinase buffer
containing flag peptide (1 mg/ml). Wild type and mutant GST-SGK
proteins were similarly expressed in HEK 293T cells and affinity
purified from cell extracts using glutathione-Sepharose beads. Purified
GST-SGK proteins were dephosphorylated by incubation with 10 ng of
protein phosphatase 2A (Calbiochem) in 50 µl of 20 mM
Tris-HCl pH 7.5, 0.1% 2-mercaptoethanol, 0.1 mM EGTA, 1 mg/ml bovine serum albumin for 30 min at 30 °C. The reactions were
stopped by addition of 1 µM microcystin-LR (Calbiochem). The dephosphorylated GST-SGK protein was then mixed with eluted FLAG-BMK1 in the presence of 10 mM MgCl2 and
500 µM ATP. After incubation for 30 min at 30 °C, the
glutathione-Sepharose beads were collected by centrifugation. Kinase
assays were performed in a 50-µl reaction volume containing 20 mM Tris, pH 7.5, 10 mM MgCl2, and
[ Mapping of BMK1-catalyzed Phosphorylating Site in SGK--
Wild
type or S78A SGK proteins was incubated for 20 min at 30 °C with
protein phosphatase 2A (Calbiochem) and followed by the addition of
microcystin-LR to 1 µM to inactivate protein phosphatase 2A. These SGK proteins were then incubated with activated
GST-BMK1 and [ Cell Cycle Analysis--
MCF10A cells were starved for 48 h
in unsupplemented DMEM/F-12 medium. Some cells were exposed to
growth factors by the addition of DMEM/F-12 medium containing
10% fetal calf serum, 10 ng/ml EGF, 5 µg/ml insulin, and 0.5 µg/ml
hyrocortisone. After 15 h, the percentage of cells in S phase was
measured in triplicate by adding 10 µM bromodeoxyuridine
(Roche Molecular Biochemicals) directly to the culture medium
for 45 min. The cells were then harvested, stained with both propidium
iodide and anti-bromodeoxyuridine fluorescein
isothiocyanate-conjugated antibody (Roche Molecular Biochemicals), and
the fraction of cells in S phase was determined by flow cytometry as
described previously (10).
BMK1, a recently identified mammalian MAP kinase, is activated by
diverse stimuli, including hydrogen peroxide, hyperosmotic stress, and
serum factors such as EGF (8, 10, 19). The growth factor-induced
activation of BMK1 proceeds through a MAP kinase signaling cascade
involving the upstream kinases MEKK3 and MEK5 (11, 20). To identify
downstream targets of BMK1 activity, a dominant negative form of BMK1,
in which the catalytic TEY site has been mutated to AEF (8), was used
as bait in a yeast two hybrid screen. A total of 2 × 107 transformants were screened from a human epithelial
carcinoma cDNA library, and 83 positive clones were selected for
sequence analysis based on their potential interaction with BMK1. Using the BLAST algorithm and the nucleotide data base at the National Library of Medicine, two clones were found to encode carboxyl-terminal portions of the molecule SGK (Fig.
1A). As shown, the growth of transformed yeast in media lacking histidine and adenine is
completely dependent on the presence of sequences from both SGK and
BMK1(AEF) in this GAL4 system. One of the clones encodes only the
carboxyl-terminal 30 amino acids of SGK, indicating that this small
portion of the kinase is sufficient for its interaction with BMK1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (1000-2000 cpm/pmol) and 1 mM
crosstide (GRPRTSSFAEG) as substrate. After incubating for 30 min at 30 °C, the kinase reaction was spotted on Whatman
phosphocellulose paper, precipitated with 75 mM phosphoric
acid, and counted in a liquid scintillation counter.
-32P]ATP (2000 cpm/pmol) for 30 min at
30 °C as described in the previous section. The reactions were
stopped after 30 min by the addition of SDS and 2-mercaptoethanol to a
final concentration of 1% (w/v) and 1% (v/v), respectively, followed
by heating at 95 °C for 5 min. These samples were then incubated for
1 h at 30 °C with 2% (v/v) 4-vinylpyridine, separated by 10%
SDS-polyacrylamide gel electrophoresis, and transferred to
nitrocellulose to isolate 32P-labeled SGK proteins.
Phosphopeptide and Phosphoamino acid analysis of these
32P-SGK proteins was performed as described previously (8,
9). Trypsin was used to digest these 32P-SGK proteins in
all the phosphopeptide mapping experiments performed herein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 1.
SGK physically interacts with BMK1.
A, the yeast strain PJ69-2A was co-transformed with vectors
encoding the indicated GAL4 DNA binding domain (BD) fusion
protein along with the indicated GAL4 activating domain (AD)
fusion protein. Transformed yeast were cultured on plates containing
the indicated selective media. Two clones obtained from the yeast
two-hybrid screen, AD/SGK N221 and AD/SGK
N401, encode a fusion
between the GAL4 activation domain and portions of SGK with
amino-terminal deletions up to amino acids 221 and 401, respectively.
B, diagram of GST fusion constructs of various SGK deletion
mutants. The cross-hatched area represents the catalytic
domain of SGK. C, HEK 293T cells were co-transfected
with a vector encoding FLAG-tagged BMK1(AEF) along with the indicated
GST-SGK constructs. After transfection, GST affinity-purified cell
lysates were analyzed for co-purification of BMK1 by Western blotting
using anti-FLAG antibody as described under "Materials and Methods"
(top panel). Western blot analysis of cell lysates using
antibodies against FLAG (middle panel) or GST (bottom
panel) confirm the expression of FLAG-BMK1(AEF) and the GST-SGK
proteins.
To verify that BMK1 and SGK interact, GST-tagged portions of SGK were expressed in HEK 293T cells along with FLAG-tagged BMK1(AEF). After transfection, cell lysates were prepared, and glutathione-Sepharose was used to affinity purify GST-tagged SGK proteins. BMK1 and SGK co-purified in this system, and this interaction was dependent upon the presence of amino acid residues 401-431 of SGK (Fig. 1C). These results support the findings from the yeast two-hybrid screen and confirm that the COOH-terminal 30 amino acids of SGK are sufficient for its physical interaction with BMK1 in mammalian cells.
To identify putative site(s) in SGK that are phosphorylated by BMK1, we
individually mutated the five consensus MAP kinase phosphorylation
sites (Ser/Thr followed by Pro) in SGK to alanine at positions
Ser74, Ser78, Thr260,
Ser369, and Ser401. All of the mutants,
except SGK(S78A), were substrates for BMK1 in an in vitro
protein kinase assay, indicating that Ser78 is the site in
SGK that is phosphorylated by BMK1 (Fig.
2A). In agreement with this,
tryptic phosphopeptide mapping of wild type SGK protein treated
in vitro with BMK1 revealed one phosphorylated peptide that
only contained phosphoserine upon subsequent phosphoamino acid analysis
(Fig. 2, B and C). In contrast, this radioactive tryptic peptide was not detected when the mutant SGK(S78A) was used as
a BMK1 substrate. These data demonstrate that BMK1 phosphorylates serine 78 of SGK in vitro.
|
We next examined the effect of BMK1-mediated phosphorylation on the kinase activity of SGK. Although the downstream cellular targets of SGK are currently unknown, a peptide substrate of RAC protein kinase, known as crosstide, has been shown to be a substrate for SGK (21). To assess the activity of the SGK mutants in response to activated BMK1, we used crosstide in a coupled in vitro protein kinase assay. The activity of all of the SGK mutants was up-regulated 5-fold by BMK1 with the exception of SGK(S78A) whose activity was unaffected (Fig. 2D). These results show that SGK is activated by BMK1-induced phosphorylation at Ser78.
As EGF has been shown to be a potent stimulus for BMK1 (10), we
evaluated the cellular phosphorylation of SGK as a result of EGF
stimulation. To this end, HeLa cells expressing GST-tagged SGK were
labeled in vivo with [32P]orthophosphate and
treated with EGF. One EGF-inducible phosphopeptide was detected by
tryptic peptide mapping (Fig.
3A). To confirm that the
phosphorylation of SGK obtained in vivo and in
vitro is identical, we mixed equal radiolabeled counts of SGK from
these two sources followed by phosphopeptide mapping. The identical migration of the induced phosphopeptide in the mixture confirmed that
SGK is phosphorylated at the same site both in vivo and
in vitro. This EGF-induced phosphopeptide was absent when
SGK(S78A) was expressed in this experiment, confirming that the
in vivo phosphorylation site of SGK is indeed
Ser78 (Fig. 3A). In agreement with this,
subsequent phosphoamino acid analysis revealed that the EGF-inducible
phosphopeptide of wild type SGK obtained in vivo contained
only phosphoserine (Fig. 3B).
|
The above findings prompted us to evaluate SGK kinase activity as a result of EGF-mediated cell stimulation. EGF induced the kinase activity of wild type SGK about 3-fold but had no effect on the activity of SGK(S78A) (Fig. 3C). These results indicate that phosphorylation of Ser78 is necessary for growth factor-induced activation of SGK. To confirm that the observed cellular activation of SGK is mediated by BMK1, we tested the effects of dominant negative forms of the MAP kinases BMK1 and p38, BMK1(AEF) and p38(APF), on EGF-induced SGK activation. BMK1(AEF), but not p38(APF), completely abrogated EGF-induced cellular activation of SGK (Fig. 3D). Together, these results confirm a critical role for BMK1 in EGF-induced cellular activation of SGK.
We have shown previously that BMK1 activity is required for growth
factor-mediated cell proliferation and progression into the S phase of
the cell cycle (10). Therefore, we hypothesized that SGK, as a
downstream substrate of BMK1, is involved in mediating this cellular
function of BMK1. To test this, MCF10A cells were infected with
recombinant adenovirus expressing SGK(S78A), starved in growth
factor-deficient medium for 48 h, stimulated with growth factors,
and assayed for cell cycle progression 15 h later. The addition of
growth factors to both mock and control virus-infected cells caused the
fraction of cells in S phase to increase from about 7 to 40% (Fig.
4A). In contrast, expression of SGK(S78A) prevented cells from entering S phase following the addition of growth
factors. These results demonstrate that the BMK1-induced activation of
SGK is required for mammalian cells to enter the S phase of the cell
cycle in response to growth factors.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies in our laboratory have established that BMK1 is required for growth factor-induced cell proliferation and cell cycle progression (10). Herein, we identify SGK as a direct downstream substrate of BMK1 and show that upon EGF stimulation BMK1 activates SGK through phosphorylation of Ser78. Moreover, we demonstrate that Ser78 of SGK is required for growth factor-induced cell cycle progression, a finding that strongly suggests that BMK1-mediated regulation of SGK activity is critical for this cellular response. Interestingly, others have shown that SGK actively shuttles in synchrony with the cell cycle between the cytoplasm (in G1 phase) and the nucleus (in S and G2/M phase) of the cell (17). Similarly, we have found that upon activation, BMK1 actively accumulates in the nucleus of the cell (8). Thus, it is possible that these kinases are co-localized during cell cycle progression through the physical association of BMK1 with the carboxyl-terminal 30 amino acids of SGK.
Members of the 3-phosphoinositide-dependent kinase (PDK)
family have been shown to phosphorylate residues within the activation loop of the conserved catalytic domain of protein kinase B, ribosomal protein S6 kinase, and protein kinase C (22-24). Given that the catalytic domain of SGK is homologous to that of these PDK targets (12), SGK was suspected to be a target of PDK phosphorylation. Indeed,
two recent studies have reported that the PDKs phosphorylate SGK at
Thr256, which resides in the activation loop of the
catalytic domain of this kinase (21, 25). However, in these studies
mutagenesis of Thr256 did not abrogate the total cellular
phosphorylation of SGK, suggesting the existence of other
phosphorylation sites within this kinase (25). Herein, we show that SGK
is a downstream target of the MAP kinase family member BMK1. In
contrast to PDK, changing Thr256 to alanine does not alter
the BMK1-induced activation of SGK (Fig. 2D). Conversely, we
have shown that changing Ser78 to alanine abrogates the
phosphorylation of SGK by BMK1 (Fig. 2D). We have found that
the S78A mutation has no effect on PDK-mediated phosphorylation of SGK
and have confirmed that the T256A mutation abrogates this event (data
not shown). Together, these results demonstrate that PDK1 and BMK1 can
independently regulate SGK activity through the phosphorylation of two
entirely different sites. It is noteworthy that although
Ser78 is located outside the catalytic domain of SGK, there
are other examples of kinase activation as a result of phosphorylation
outside the catalytic region (26, 27).
In conclusion, SGK appears to be a point of convergence for at least
two different signaling pathways. Given the fact that SGK activity is
induced by a wide variety of cellular stimuli, it is not surprising
that this kinase is regulated by more than one upstream pathway. In
this regard, the PDK- and BMK1-mediated activation of SGK need not be
mutually exclusive and may in fact act together to coordinate the
appropriate activation of SGK under certain physiologic conditions.
Until natural cellular substrates for SGK are identified, the mechanism
by which the BMK1-induced activation of SGK mediates cell cycle
progression in response to growth factors will remain an important area
of future investigation.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant CA79871 from the National Institutes of Health and a grant from the American Heart Association. This publication was made possible by funds received from the Cancer Research Fund, under Interagency Agreement 97-12013 (University of California Contract 98-00924V) with the Department of Health Services, Cancer Research Program. Mention of trade name, proprietary product, or specific equipment does not constitute a guaranty or warranty by the Department of Health Services nor does it imply approval to the exclusion of other products. The views expressed herein represent those of the authors and do not necessarily represent the position of the State of California, Department of Health Services.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. E-mail: jdlee@scripps.edu.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.C000838200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated protein kinase; BMK1, big mitogen-activated protein kinase 1; MEK, MAP kinase kinase/ERK kinase; MKK, MAP kinase kinase; MEKK, MAP kinase kinase kinase/ERK kinase kinase; JNK, c-Jun NH2-terminal kinase; EGF, epidermal growth factor; SGK, serum and glucocorticoid-inducible kinase; AD, activation domain; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; PDK, 3-phosphoinositide-dependent kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Roovers, K., and Assoian, R. K. (2000) Bioessays 22, 818-826[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Wilkinson, M. G.,
and Millar, J. B.
(2000)
FASEB J.
14,
2147-2157 |
3. | Fiddes, R. J., Janes, P. W., Sivertsen, S. P., Sutherland, R. L., Musgrove, E. A., and Daly, R. J. (1998) Oncogene 16, 2803-2813[CrossRef][Medline] [Order article via Infotrieve] |
4. | Pelech, S. L., and Charest, D. L. (1995) Prog. Cell Cycle Res. 1, 33-52[Medline] [Order article via Infotrieve] |
5. | Shiozaki, K., and Russell, P. (1995) Nature 378, 739-743[CrossRef][Medline] [Order article via Infotrieve] |
6. | Shibuya, E. K., Boulton, T. G., Cobb, M. H., and Ruderman, J. V. (1992) EMBO J. 11, 3963-3975[Abstract] |
7. | Lee, J.-D., Ulevitch, R. J., and Han, J. (1995) Biochem. Biophys. Res. Commun. 213, 715-724[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Kato, Y.,
Kravchenko, V. V.,
Tapping, R. I.,
Han, J.,
Ulevitch, R. J.,
and Lee, J.-D.
(1997)
EMBO J.
16,
7054-7066 |
9. |
Kato, Y.,
Zhao, M.,
Morikawa, A.,
Sugiyama, T.,
Chakravoritty, D.,
Koide, N.,
Yoshida, T.,
Tapping, R. I.,
Yang, Y.,
Yokochi, T.,
and Lee, J.-D.
(2000)
J. Biol. Chem.
275,
18534-18540 |
10. | Kato, Y., Tapping, R. I., Huang, S., Watson, M. H., Ulevitch, R. J., and Lee, J. D. (1998) Nature 395, 713-716[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Chao, T. H.,
Hayashi, M.,
Tapping, R. I.,
Kato, Y.,
and Lee, J. D.
(1999)
J. Biol. Chem.
274,
36035-36038 |
12. | Webster, M. K., Goya, L., Ge, Y., Maiyar, A. C., and Firestone, G. L. (1993) Mol. Cell. Biol. 13, 2031-2040[Abstract] |
13. |
Webster, M. K.,
Goya, L.,
and Firestone, G. L.
(1993)
J. Biol. Chem.
268,
11482-11485 |
14. |
Waldegger, S.,
Barth, P.,
Raber, G.,
and Lang, F.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
4440-4445 |
15. | Kobayashi, T., Deak, M., Morrice, N., and Cohen, P. (1999) Biochem. J. 344, 189-197[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Bell, L. M.,
Leong, M. L.,
Kim, B.,
Wang, E.,
Park, J.,
Hemmings, B. A.,
and Firestone, G. L.
(2000)
J. Biol. Chem.
275,
25262-25272 |
17. |
Buse, P.,
Tran, S. H.,
Luther, E.,
Phu, P. T.,
Aponte, G. W.,
and Firestone, G. L.
(1999)
J. Biol. Chem.
274,
7253-7263 |
18. | Sanchez, I., Hughes, R. T., Mayer, B. J., Yee, K., Woodgett, J. R., Avruch, J., Kyriakis, J. M., and Zon, L. I. (1994) Nature 372, 794-798[Medline] [Order article via Infotrieve] |
19. |
Abe, J.-I.,
Kushuhara, M.,
Ulevitch, R. J.,
Berk, B. C.,
and Lee, J.-D.
(1996)
J. Biol. Chem.
271,
16586-16590 |
20. |
Zhou, G.,
Bao, Z. Q.,
and Dixon, J. E.
(1995)
J. Biol. Chem.
270,
12665-12669 |
21. | Kobayashi, T., and Cohen, P. (1999) Biochem. J. 339, 319-328[CrossRef][Medline] [Order article via Infotrieve] |
22. | Chou, M. M., Hou, W., Johnson, J., Graham, L. K., Lee, M. H., Chen, C. S., Newton, A. C., Schaffhausen, B. S., and Toker, A. (1998) Curr. Biol. 8, 1069-1077[Medline] [Order article via Infotrieve] |
23. | Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B., and Cohen, P. (1997) Curr. Biol. 7, 261-269[Medline] [Order article via Infotrieve] |
24. | Alessi, D. R., Kozlowski, M. T., Weng, Q. P., Morrice, N., and Avruch, J. (1998) Curr. Biol. 8, 69-81[Medline] [Order article via Infotrieve] |
25. |
Park, J.,
Leong, M. L.,
Buse, P.,
Maiyar, A. C.,
Firestone, G. L.,
and Hemmings, B. A.
(1999)
EMBO J.
18,
3024-3033 |
26. | Stokoe, D., Campbell, D. G., Nakielny, S., Hidada, H., Leevers, S. J., Marshall, C., and Cohen, P. (1992) EMBO J. 11, 3985-3994[Abstract] |
27. | Brushia, R. J., and Walsh, D. A. (1999) Front Biosci. 4, D618-D641[Medline] [Order article via Infotrieve] |