From the Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel and the § Department of Oncology, Novartis Pharma AG, CH-4057 Basel, Switzerland
Received for publication, April 6, 2001, and in revised form, May 9, 2001
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ABSTRACT |
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Full activation of protein kinase B (PKB, also
called Akt) requires phosphorylation on two regulatory sites, Thr-308
in the activation loop and Ser-473 in the hydrophobic C-terminal
regulatory domain (numbering for PKB Protein kinase B (PKB)1
is activated by growth factors in a phosphoinositide 3-kinase
(PI3K)-dependent manner, through translocation to the
plasma membrane and phosphorylation on two regulatory sites, Thr-308 in
the activation loop in the kinase domain and Ser-473 in the hydrophobic
C-terminal regulatory domain (1). Phosphorylation on both sites are
required for full activation of PKB; however, the contribution of each
site toward PKB activation is not equal. Thus, whereas phosphorylation
on Thr-308 alone is able to increase PKB activity, phosphorylation on
Ser-473 alone does not significantly stimulate the kinase (1, 2).
Although the upstream kinase responsible for phosphorylation of Thr-308
has been identified as 3'-phosphoinositide-dependent
kinase-1 (PDK1), the identity of the Ser-473 kinase has yet to be
determined (3, 4).
Mitogen-activated protein kinase-activated protein kinase-2 was
the first kinase shown to phosphorylate PKB To further characterize the upstream kinase(s) involved in the
activation of PKB, we have adopted a pharmacological approach by
screening for protein kinase inhibitors that differentially inhibit
either Thr-308 or Ser-473 phosphorylation. We found that staurosporine,
a broad-specificity protein kinase inhibitor, attenuated PKB activation
specifically through inhibition of PDK1, with an IC50 of
~0.22 µM in vitro. Staurosporine has been
widely used as an inducer of apoptosis; however, the cellular target(s)
of its proapoptotic action are not known. Our data suggest that at least part of the apoptotic effects of staurosporine is due to inhibition of PKB signaling. In contrast to Thr-308 phosphorylation, insulin-stimulated phosphorylation of the Ser-473 site was not reduced
by staurosporine treatment (up to 1 µM). Taken together, our results suggest that phosphorylation of PKB on Ser-473 does not
occur by autophosphorylation but rather through the action of an
upstream kinase that is resistant to staurosporine and distinct from
PDK1.
Expression Constructs and Transfection of Cells--
Culture and
transfection of HEK293 cells and the expression constructs used in this
study have been described previously (1, 10-12).
Recombinant Proteins--
Expression and infection of insect
Sf9 cells have been described previously for PKB and PKC (13,
14). Human PDK1-glutathione S-transferase fusion protein
(GST-PDK1) was cloned in a modified pFastBac vector (Life Technologies,
Inc.) and prepared as described previously (13).
Immunoprecipitation, Immunoblotting, and in Vitro Kinase
Assays--
Cell lysis, immunoprecipitation, immunoblotting, and PKB
assay using Crosstide peptide (GRPRTSSAEG) were performed as described previously (11). Phospho-specific PKB antibodies were purchased from
Cell Signaling Technologies. In addition, we also produced and purified
an anti-phospho-Ser-473 PKB antibody using the peptide Arg-Pro-His-Phe-Pro-Gln-Phe-Ser(PO3H2)-Tyr-Ser-Ala-Ser
(15). Assays for recombinant PKC We have previously reported the characterization of a PKB To extend our observations, the effect of a panel of protein kinase
inhibitors was compared with staurosporine (Fig.
1). Staurosporine obtained from Alexis or
Novartis (CGP 39360) inhibited TPA-stimulated Thr-308 phosphorylation
and activation of C1-PKB/Akt-1). Although
3'-phosphoinositide-dependent protein kinase 1 (PDK1) has now been identified as the Thr-308 kinase, the mechanism of
the Ser-473 phosphorylation remains controversial. As a step to further
characterize the Ser-473 kinase, we examined the effects of a range of
protein kinase inhibitors on the activation and phosphorylation of PKB.
We found that staurosporine, a broad-specificity kinase inhibitor and
inducer of cell apoptosis, attenuated PKB activation exclusively
through the inhibition of Thr-308 phosphorylation, with Ser-473
phosphorylation unaffected. The increase in Thr-308 phosphorylation
because of overexpression of PDK1 was also inhibited by staurosporine.
We further show that staurosporine (CGP 39360) potently inhibited PDK1
activity in vitro with an IC50 of ~0.22 µM. These data indicate that agonist-induced
phosphorylation of Ser-473 of PKB is independent of PDK1 or PKB
activity and occurs through a distinct Ser-473 kinase that is not
inhibited by staurosporine. Moreover, our results suggest that
inhibition of PKB signaling is involved in the proapoptotic action of staurosporine.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
on Ser-473 in vitro (1). However, mitogen-activated protein kinase-activated protein kinase-2 is unlikely to be the physiological Ser-473 kinase as it is activated downstream of p38 mitogen-activated protein kinase
in response to stress, in a PI3K-independent manner, and inhibition of
p38 mitogen-activated protein kinase by SB 203580 did not interfere
with activation of PKB (1). Integrin-linked kinase (ILK) was also shown
to phosphorylate Ser-473 on PKB
in vitro, and
overexpression of a kinase-inactive ILK inhibited Ser-473 phosphorylation (5). However, certain kinase-inactive ILK mutants also
induced Ser-473 phosphorylation, suggesting that ILK is unlikely to be
the direct Ser-473 kinase in vivo (6). Two further candidate Ser-473 kinases have been proposed recently, PDK1 (7) and PKB itself
(8). PDK1, in the presence of a peptide resembling the phosphorylated
Ser-473 region of PKB, is able to phosphorylate Ser-473, in addition to
Thr-308 of PKB
(7). However, PDK1 is clearly not the in
vivo Ser-473 kinase, as PDK1-null embryonic stem cells are
impaired in Thr-308 but not Ser-473 phosphorylation (9).
Autophosphorylation was originally ruled out, because kinase-inactive
PKB
undergoes insulin-like growth factor-1 (IGF-1)-induced phosphorylation at both Thr-308 and Ser-473 when overexpressed in human
embryonic kidney (HEK) 293 cells (1). In contrast to these
observations, Toker and Newton (8) recently demonstrated that IGF-1
stimulated phosphorylation of kinase-inactive PKB
on Thr-308 but not
on Ser-473 when overexpressed in the same cells and that PKB
is able
to autophosphorylate on Ser-473 in vitro (8). Thus, it seems
possible that agonist-induced Ser-473 phosphorylation may be mediated
by PKB itself.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and PKC
have been described
previously (14). Recombinant GST-PDK1 was similarly assayed, using 0.1 mg/ml casein (Sigma) as substrate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
construct in which the pleckstrin homology (PH) domain was replaced by
the C1 domain of PKC (C1-PKB
-
PH) (11). C1-PKB
-
PH
translocated to the membrane upon stimulation with the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA) and was activated
and phosphorylated on both Thr-308 and Ser-473 (11). TPA-stimulated
activation of C1-PKB
-
PH was inhibited by PI3K inhibitors, as well
as the broad range protein kinase inhibitor, staurosporine (11).
Interestingly, whereas the former inhibited phosphorylation on both
sites, staurosporine treatment specifically attenuated phosphorylation
on Thr-308 without affecting Ser-473 phosphorylation (11).
-
PH, without affecting Ser-473
phosphorylation (Fig. 1B). CGP 39360 was more potent than
staurosporine (Alexis), requiring 0.1 and 1 µM to reduce
kinase activity and Thr-308 phosphorylation to basal levels, respectively (Fig. 1B). This difference may be because of
improved purity of the chemical produced by Novartis. The staurosporine derivative CGP 41251 also inhibited Thr-308 phosphorylation and activation of C1-PKB
-
PH but was much less potent than CGP 39360 (Fig. 1B). The inactive analog of CGP 41251, CGP 42700, had
no significant effect (Fig. 1B). Three other protein kinase
inhibitor compounds examined (CGP 25956, CGP 45910, and CGP 57148B) had an inhibitory effect only at concentrations above 10 µM,
where they reduced phosphorylation at both sites to below basal levels (Fig. 1C). Notably, an effect of CGP 57148B (STI571 or
Glivec) was only observed at 40 µM (Fig. 1C).
CGP 57148B is a potent inhibitor of Abl and PDGF receptor tyrosine
kinases that selectively inhibits the growth of Bcr/Abl-transformed
cells (16) and is now in clinical trials for treatment of chronic
myeloid leukemia. The effects of CGP 57148B were observed
concentrations <10 µM, and our results show that it does
not significantly affect the PDK1/PKB pathway at this concentration
(Fig. 1C).
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Fig. 1.
Effect of staurosporine and its derivatives
on TPA-stimulated activation of
C1-PKB -
PH.
HEK293 cells transiently transfected with HA-C1-PKB
-
PH were
treated with the indicated concentration of staurosporine
(Stauro) (Alexis), CGP 39360, CGP 41251, CGP 42700 (B), CGP 25956, CGP 45910, or CGP 57148B/STI571
(C) for 30 min prior to stimulation with TPA (200 ng/ml;
Life Technologies, Inc.) for 15 min. HA-C1-PKB
-
PH was
immunoprecipitated and assayed for kinase activity or analyzed by
immunoblotting with phospho-specific antibodies. Structures of the
compounds used are shown in A.
To extend our observations to wild type PKB, we examined the effect of
staurosporine on insulin-stimulated activation of HA-PKB expressed
in HEK293 cells. Staurosporine treatment inhibited insulin-stimulated HA-PKB
activation in a dose-dependent manner, with
complete inhibition observed at 1 µM (Fig.
2A). Similar to
C1-PKB
-
PH, this inhibitory effect of staurosporine on HA-PKB
activity correlated with an inhibition of Thr-308 phosphorylation (Fig.
2A). In contrast, phosphorylation on Ser-473 was slightly
enhanced with increasing concentrations of staurosporine (Fig.
2A). A similar inhibitory effect of staurosporine was
observed for insulin-stimulated Thr-308 phosphorylation of endogenous
PKB in HEK293 cells (Fig. 2B). Two other modes of PKB
activation were also examined, coexpression of PDK1 and constitutive
membrane targeting. In agreement with previous results (3),
coexpression of PDK1 with HA-PKB
resulted in a 3-fold increase in
basal PKB
activity, together with constitutive phosphorylation of
Thr-308 (Fig. 2C). Treatment with staurosporine reduced
Thr-308 phosphorylation and kinase activity (Fig. 2C). Interestingly, overexpression of PDK1 also induced an increase in
Ser-473 phosphorylation, reaching ~10% of the insulin-stimulated levels, that was reduced with staurosporine treatment, suggesting that
it occurs through a mechanism different from insulin-stimulated Ser-473
phosphorylation (Fig. 2C). Targeting of PKB to the plasma membrane using the Lck myristoylation/palmitylation signal (m/p-PKB
) results in the constitutive activation and phosphorylation of PKB (10).
In contrast to Thr-308 phosphorylation induced by insulin or
coexpression of PDK1, staurosporine did not reduce Thr-308
phosphorylation of m/p-PKB
(Fig. 2D). This observation indicates that dephosphorylation of PKB does not occur readily at the
plasma membrane and that the phosphorylation step is the target of
staurosporine.
|
Staurosporine is a competitive inhibitor that is thought to bind in the
ATP pocket of target protein kinases (17). The observed effect on
Thr-308 may occur via direct inhibition of PDK1, or staurosporine could
bind to PKB, thus blocking the access to the phosphorylation site in
the catalytic domain. To distinguish between these possibilities, we
determined the inhibitory profiles of the CGP inhibitor compounds using
recombinant GST-PDK1, GST-PKB, PKC
, and PKC
. CGP 39360 (staurosporine) was most potent against PKC
(IC50 < 3 nM) but also inhibited PDK1 (IC50 = 0.22 ± 0.09 µM), PKB
(IC50 = 0.83 ± 0.19 µM), and PKC
(IC50 = 1.03 ± 0.37 µM) at higher concentrations. CGP 41251 selectively
inhibited PKC
(IC50 = 0.04 ± 0.02 µM) and also inhibited PDK1 (IC50 = 1.72 ± 0.21 µM) but did not have significant effects on
PKB
or PKC
(up to 10 µM). These data suggest that
the target of staurosporine and its derivative CGP 41251 is PDK1 rather
than PKB. CGP 42700, CGP 25956, CGP 45910, and CGP 57148B did not
inhibit any of the four kinases tested.
To further examine the regulation of PKB activation by upstream
kinases, the effect of staurosporine on insulin-stimulated phosphorylation and activity of kinase-inactive (K179A) or
phosphorylation-site mutants (T308A and S473A) of PKB was examined
(Fig. 3). In agreement with previously
reported results (1), Thr-308 and Ser-473 phosphorylation occurred
independently of each other upon insulin stimulation, as observed in
the phosphorylation-site mutants (Fig. 3). In addition, the
kinase-inactive mutant (K179A) was phosphorylated on both Thr-308 and
Ser-473 upon insulin stimulation (Fig. 3). Staurosporine treatment
inhibited insulin-stimulated Thr-308 phosphorylation on wild type,
K179A, and S473A PKB
(Fig. 3), but its effect on Ser-473
phosphorylation of the different PKB
constructs was somewhat varied.
Staurosporine at 0.1 and 1 µM did not inhibit
insulin-stimulated Ser-473 phosphorylation of wild type and
T308A-PKB
but even enhanced their phosphorylation, which was more
readily observed in the T308A mutant (Fig. 3). Interestingly,
insulin-stimulated Ser-473 phosphorylation of kinase-inactive PKB
was inhibited by staurosporine at 1 µM but not at 0.1 µM (Fig. 3). As staurosporine is a broad specificity
kinase inhibitor, it is possible that complex effects are observed
because of inhibition of numerous kinases/pathways at higher doses.
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DISCUSSION |
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Phosphorylation at Thr-308 and Ser-473 is required for full
activation of PKB. Although the Thr-308 kinase has been identified (PDK1), the Ser-473 kinase remains elusive. Most recently, it has been
suggested that autophosphorylation may be the mechanism by which PKB is
phosphorylated on Ser-473 and that the previously reported
phosphorylation of kinase-deficient PKB at this site is due to the
activity of endogenous PKB (8). Our previous results with m/p-PKB
(10) and C1-PKB
-
PH (11) suggest that both upstream kinases are
present in a constitutively active form at the plasma membrane. As PKB
is present largely in the cytosol prior to stimulation, it seems
unlikely that PKB itself is the physiological Ser-473 kinase. More
directly, here we show that pretreatment with 1 µM
staurosporine abolished insulin-stimulated PKB activation of both
transiently expressed and endogenous PKB, without affecting Ser-473
phosphorylation (Fig. 2). In addition, using phospho-specific
antibodies, we have confirmed our previous results (1) that
kinase-inactive PKB
can be phosphorylated on both Thr-308 and
Ser-473 in response to insulin (Fig. 3). Taken together, these data do
not support the hypothesis that phosphorylation on Ser-473 occurs via
autophosphorylation or trans-phosphorylation. Rather, it confirms the
existence of a distinct Ser-473 kinase that is constitutively active at
the plasma membrane of quiescent cells.
One kinase that fulfills the above criteria is PDK1. Indeed, PDK1 has
the ability to phosphorylate PKB on Ser-473 in the presence of an
exogenous peptide that resembles phosphorylated Ser-473 (7). However,
PDK1 is not the physiological Ser-473 kinase, because PDK1-null
embryonic stem cells are not impaired in Ser-473 phosphorylation in
response to IGF-1 (9), and staurosporine inhibits PDK1 activity without
affecting insulin-stimulated Ser-473 phosphorylation (Fig. 2).
Interestingly, overexpression of PDK1 in HEK293 cells not only induced
constitutive phosphorylation of PKB at Thr-308 but also caused a slight
elevation of Ser-473 phosphorylation (Fig. 2C). In this
case, however, Ser-473 phosphorylation is dependent on PDK1 activity,
as it is attenuated by staurosporine treatment (Fig. 2C),
and overexpression of a kinase-inactive PDK1 mutant did not increase
basal Ser-473 phosphorylation in HEK293 cells (data not shown). Thus,
although PDK1 is not the physiological Ser-473 kinase, it likely plays
a role in Ser-473 phosphorylation, with the nature of this interaction
yet to be defined.
ILK has recently come to attention as a prime candidate kinase for Ser-473 phosphorylation (5). According to our observations, the Ser-473 kinase should be staurosporine-resistant. Unfortunately, we were unable to determine the effect of staurosporine on ILK as we have not been able to detect any significant kinase activity of overexpressed or endogenous ILK by autophosphorylation or on myelin basic protein (data not shown). Intriguingly, ILK possesses a hydrophobic motif similar to the Ser-473 site, and when this serine was mutated to an acidic residue to mimic phosphorylation, the ability of a kinase-inactive ILK to induce Ser-473 phosphorylation was rescued (6). It was recently shown that the hydrophobic phosphorylation site in p90 ribosomal S6 kinase acts as a docking site for the recruitment of PDK1 (18). Thus, it is possible that ILK mediates the colocalization of PKB with PDK1 and the Ser-473 kinase.
Staurosporine exhibits anti-proliferative properties on a wide range of mammalian cells, and its derivatives UCN-01, CGP 41251, Ro 31-8220, and PKC412 (19-22) are being examined as potential therapeutic agents for the treatment of cancer. Despite the common use of staurosporine as an inducer of apoptosis, the direct cellular target of staurosporine is not known. Our finding that staurosporine inhibits PDK1 activity raises the possibility that staurosporine and its derivatives may induce apoptosis by interfering with survival signaling mediated by PDK1. Indeed, it was recently shown that reduction of PDK1 expression by antisense oligonucleotides induced apoptosis (23). Apart from PKB, PDK1 also phosphorylates and activates other kinases that are involved in cell survival, including p70 ribosomal S6 kinase (12), p90 ribosomal S6 kinase (24), and serum- and glucocorticoid-inducible protein kinase (25). Thus the mechanisms of staurosporine-induced apoptosis needs to be readdressed in light of its effects on PDK1 activity.
In summary, we have demonstrated that staurosporine attenuates PKB
activation through direct inhibition of PDK1 activity, without
affecting insulin-stimulation of Ser-473 phosphorylation. These results
strongly suggest that insulin-stimulated phosphorylation on Ser-473 is
not dependent on the activity of PDK1 or PKB. Our data is consistent
with a model in which phosphorylation on Thr-308 and Ser-473 occurs via
two distinct upstream kinases that are constitutively active at the
plasma membrane, and of these, only the Ser-473 kinase is
staurosporine-resistant.
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FOOTNOTES |
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* This work was supported in part by the Swiss Cancer League (to M. M. H., M. A., and B. A. H.), and the Friedrich Miescher Institute is supported by the Novartis Research Foundation.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.
Present address: Dept. of Vascular and Metabolic Diseases, F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland.
¶ To whom correspondence should be addressed. Tel.: 41-61-697-40-46; Fax: 41-61-697-39-76.
Published, JBC Papers in Press, May 23, 2001, DOI 10.1074/jbc.C100174200
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ABBREVIATIONS |
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The abbreviations used are: PKB, protein kinase B; PI3K, phosphoinositide 3-kinase; PDK1, 3'-phosphoinositide-dependent protein kinase 1; ILK, integrin-linked kinase; IGF-1, insulin-like growth factor-1; HEK, human embryonic kidney; PKC, protein kinase C; GST, glutathione S-transferase; PH, pleckstrin homology; TPA, 12-O-tetradecanoylphorbol-13-acetate; HA, hemagglutinin; m/p, myristoylated/palmitylated.
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REFERENCES |
---|
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---|
1. | Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., and Hemmings, B. A. (1996) EMBO J. 15, 6541-6551[Abstract] |
2. | Bellacosa, A., Chan, T. O., Ahmed, N. N., Datta, K., Malstrom, S., Stokoe, D., McCormick, F., Feng, J., and Tsichlis, P. (1998) Oncogene 17, 313-325[CrossRef][Medline] [Order article via Infotrieve] |
3. | 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] |
4. |
Stokoe, D.,
Stephens, L. R.,
Copeland, T.,
Gaffney, P. R.,
Reese, C. B.,
Painter, G. F.,
Holmes, A. B.,
McCormick, F.,
and Hawkins, P. T.
(1997)
Science
277,
567-570 |
5. |
Delcommenne, M.,
Tan, C.,
Gray, V.,
Rue, L.,
Woodgett, J.,
and Dedhar, S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11211-11216 |
6. | Lynch, D. K., Ellis, C. A., Edwards, P. A., and Hiles, I. D. (1999) Oncogene 18, 8024-8032[CrossRef][Medline] [Order article via Infotrieve] |
7. | Balendran, A., Casamayor, A., Deak, M., Paterson, A., Gaffney, P., Currie, R., Downes, C. P., and Alessi, D. R. (1999) Curr. Biol. 9, 393-404[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Toker, A.,
and Newton, A. C.
(2000)
J. Biol. Chem.
275,
8271-8274 |
9. | Williams, M. R., Arthur, J. S., Balendran, A., van der Kaay, J., Poli, V., Cohen, P., and Alessi, D. R. (2000) Curr. Biol. 10, 439-448[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Andjelkovic, M.,
Alessi, D. R.,
Meier, R.,
Fernandez, A.,
Lamb, N. J.,
Frech, M.,
Cron, P.,
Cohen, P.,
Lucocq, J. M.,
and Hemmings, B. A.
(1997)
J. Biol. Chem.
272,
31515-31524 |
11. |
Andjelkovic, M.,
Maira, S. M.,
Cron, P.,
Parker, P. J.,
and Hemmings, B. A.
(1999)
Mol. Cell. Biol.
19,
5061-5072 |
12. |
Pullen, N.,
Dennis, P. B.,
Andjelkovic, M.,
Dufner, A.,
Kozma, S. C.,
Hemmings, B. A.,
and Thomas, G.
(1998)
Science
279,
707-710 |
13. | Fabbro, D., Batt, D., Rose, P., Schacher, B., Roberts, T. M., and Ferrari, S. (1999) Protein Expression Purif. 17, 83-88[CrossRef][Medline] [Order article via Infotrieve] |
14. | Geiges, D., Meyer, T., Marte, B., Vanek, M., Weissgerber, G., Stabel, S., Pfeilschifter, J., Fabbro, D., and Huwiler, A. (1997) Biochem. Pharmacol. 53, 865-875[CrossRef][Medline] [Order article via Infotrieve] |
15. | Hill, M. M., and Hemmings, B. A. (2001) Methods Enzymol. 345, in press |
16. | Druker, B. J., Tamura, S., Buchdunger, E., Ohno, S., Segal, G. M., Fanning, S., Zimmermann, J., and Lydon, N. B. (1996) Nat. Med. 2, 561-566[Medline] [Order article via Infotrieve] |
17. | Toledo, L. M., Lydon, N. B., and Elbaum, D. (1999) Curr. Med. Chem. 6, 775-805[Medline] [Order article via Infotrieve] |
18. |
Frodin, M.,
Jensen, C. J.,
Merienne, K.,
and Gammeltoft, S.
(2000)
EMBO J.
19,
2924-2934 |
19. | Shao, R. G., Shimizu, T., and Pommier, Y. (1997) Exp. Cell. Res. 234, 388-397[CrossRef][Medline] [Order article via Infotrieve] |
20. | Begemann, M., Kashimawo, S. A., Lunn, R. M., Delohery, T., Choi, Y. J., Kim, S., Heitjan, D. F., Santella, R. M., Schiff, P. B., Bruce, J. N., and Weinstein, I. B. (1998) Anticancer Res. 18, 3139-3152[Medline] [Order article via Infotrieve] |
21. | Begemann, M., Kashimawo, S. A., Heitjan, D. F., Schiff, P. B., Bruce, J. N., and Weinstein, I. B. (1998) Anticancer Res. 18, 2275-2282[Medline] [Order article via Infotrieve] |
22. | Fabbro, D., Ruetz, S., Bodis, S., Pruschy, M., Csermak, K., Man, A., Campochiaro, P., Wood, J., O'Reilly, T., and Meyer, T. (2000) Anticancer Drug Des. 15, 17-28[Medline] [Order article via Infotrieve] |
23. | Flynn, P., Wongdagger, M., Zavar, M., Dean, N. M., and Stokoe, D. (2000) Curr. Biol. 10, 1439-1442[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Jensen, C. J.,
Buch, M. B.,
Krag, T. O.,
Hemmings, B. A.,
Gammeltoft, S.,
and Frodin, M.
(1999)
J. Biol. Chem.
274,
27168-27176 |
25. |
Park, J.,
Leong, M. L.,
Buse, P.,
Maiyar, A. C.,
Firestone, G. L.,
and Hemmings, B. A.
(1999)
EMBO J.
18,
3024-3033 |