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J Biol Chem, Vol. 274, Issue 14, 9133-9136, April 2, 1999

COMMUNICATION
A Human Protein Kinase Bgamma with Regulatory Phosphorylation Sites in the Activation Loop and in the C-terminal Hydrophobic Domain*

Daniela Brodbeck, Peter Cron, and Brian A. HemmingsDagger

From the Friedrich Miescher-Institut, P. O. Box 2543, 4002 Basel, Switzerland

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

We have cloned human protein kinase Bgamma (PKBgamma ) and found that it contains two regulatory phosphorylation sites, Thr305 and Ser472, which correspond to Thr308 and Ser473 of PKBalpha . Thus it differs significantly from the previously published rat PKBgamma . We have also isolated a similar clone from a mouse cDNA library. In human tissues, PKBgamma is widely expressed as two transcripts. A mutational analysis of the two regulatory sites of human PKBgamma showed that phosphorylation of both sites, occurring in a phosphoinositide 3-kinase-dependent manner, is required for full activity. Our results suggest that the two phosphorylation sites act in concert to produce full activation of PKBgamma , similar to PKBalpha . This contrasts with rat PKBgamma , which is thought to be regulated by 3-phosphoinositide-dependent protein kinase 1 alone.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Three members of the protein kinase B (PKB)1 subfamily of second-messenger regulated serine/threonine protein kinases have been identified and termed alpha , beta , and gamma  (1-4). The isoforms are homologous, particularly in regions encoding the N-terminal pleckstrin homology (PH) and the catalytic domains. PKBs are activated by phosphorylation events occurring in response to phosphoinositide 3-kinase (PI3K) signaling (5-8). PI3K phosphorylates membrane inositol phospholipids, generating the second messengers phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (reviewed in Ref. 9), which have been shown to bind to the PH domain of PKB (10, 11). The current model of PKB activation proposes recruitment of the enzyme to the membrane by 3'-phosphorylated phosphoinositides, where phosphorylation of the regulatory sites of PKB by the upstream kinases occurs (12-14).

Phosphorylation of PKBalpha /beta occurs on two regulatory sites, Thr308/309 in the activation loop in the catalytic domain and Ser473/474 in the C-terminal domain (15, 16). The upstream kinase, which phosphorylates PKBalpha at the activation loop site Thr308, has been cloned and termed 3-phosphoinositide-dependent protein kinase 1 (PDK1; Refs. 17-20). PDK1 phosphorylates not only PKBalpha , but also equivalent sites in the p70 ribosomal S6 kinase (21, 22), protein kinase A (23), and protein kinase C (24). The upstream kinase phosphorylating the second regulatory site of PKBalpha /beta , Ser473/474, has not been identified yet, but a recent report implies a role for the integrin-linked kinase (ILK-1), a serine/threonine protein kinase (25).

Only a few studies have been reported on the third member of the PKB family, PKBgamma , and these all involved a clone originating from a rat brain cDNA library (4, 26). A major feature distinguishing rat PKBgamma from the otherwise very similar alpha  and beta  isoforms is the C terminus, which is truncated by 23 amino acids and lacks Ser473/474, one of the two phosphorylation sites essential for activation of PKBalpha and beta  (15, 16). Consequently, it has been suggested that rat PKBgamma activation depends solely on the upstream kinase PDK1. We now report the cloning and characterization of human PKBgamma . This isoform differs significantly from the rat enzyme in that it contains a C-terminal domain similar to PKBalpha /beta , with a putative second regulatory phosphorylation site at Ser472.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cloning of Human HA-PKBgamma and Mutant Isoforms-- A 525-bp PCR product corresponding to nucleotides 815-1340 of the rat PKBgamma sequence (4) was amplified from mouse brain cDNA and used to screen several different human cDNA libraries. Twelve overlapping clones were isolated and assembled to a cDNA encoding amino acids 16-479 of human PKBgamma . This cDNA was repaired by PCR-mediated addition of a hemagglutinin (HA) tag and the missing N-terminal amino acids deduced from the rat PKBgamma sequence and ligated as a KpnI/XbaI fragment into the pCMV5 eucaryotic expression vector (27). Subsequently, the 5' end of PKBgamma was amplified by 5'-rapid amplification of cDNA ends from human brain cDNA. A mouse brain cDNA library screened with the same probe yielded 13 overlapping clones which could be assembled into a cDNA encoding the entire reading frame of mouse PKBgamma . Mutations in HA-PKBgamma (HA-PKBgamma T305A and HA-PKBgamma T305D) were done by Quikchange (Stratagene) or with mutagenizing 3' primers (HA-PKBgamma S472A and HA-PKBgamma S472D). HA-Delta PHPKBgamma was obtained by PCR with a primer encoding the HA-tag and amino acids 119-126. All PCR-cloned constructs were verified by DNA sequencing.

Northern Blot Analysis-- Human adult and fetal multiple tissue Northern blots (CLONTECH) were hybridized with a 825-bp fragment encoding amino acids 110-384 of PKBgamma according to the manufacturer's instructions.

Cell Culture, Immunoprecipitation, in Vitro Kinase Assays, and Immunoblot Analysis-- Human embryonic kidney (HEK) 293 cells were maintained and transfected by a modified calcium phosphate method as described previously (15, 28). Stimulation was for 5 min with 0.2 mM pervanadate (7) or for 15 min with 500 nM insulin (Boehringer Mannheim). Pretreatment with the PI3K inhibitor wortmannin (200 nM; gift of Dr. Markus Thelen, Theodor Kocher Institute, Bern, Switzerland) was for 15 min. Cells were extracted and HA-PKBgamma activity determined exactly as described in Ref. 28. Western blot analysis was performed as described before (15) and developed with the polyclonal phospho-specific Ser473 antibody (1:1000, New England Biolabs), an alkaline phosphatase (AP)-coupled goat-anti mouse IgG secondary antibody (1:2000, Sigma), and alkaline phosphatase color development reagents (Boehringer Mannheim).

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Screening of several human cDNA libraries led to the isolation of 12 clones encoding partial and overlapping sections of the open reading frame of human PKBgamma , and the cDNA was assembled as described under "Experimental Procedures." The 479-residue amino acid sequence of human PKBgamma is presented in Fig. 1, as an alignment with human PKBalpha , PKBbeta , mouse PKBgamma (see below), and with the C-terminal domain of rat PKBgamma . Human PKBgamma is 83% identical to PKBalpha , 78% identical to PKBbeta , and 99% identical to rat PKBgamma (two changes in 451 amino acids and a different C terminus), indicating that we have isolated the authentic human PKBgamma isoform and not a PKBalpha or PKBbeta variant. Moreover, we cloned a similar PKBgamma from a mouse brain cDNA library, demonstrating that this isoform is not restricted to one species. Human and mouse PKBgamma were found to be more than 99% identical (2 amino acid changes in 479). The major characteristic distinguishing human and mouse PKBgamma from the rat isoform is the presence of a C-terminal domain similar to PKBalpha and beta , containing a second putative regulatory phosphorylation site at Ser472 (marked with an asterisk in Fig. 1). To ascertain whether the human PKBgamma cDNA corresponded to the major mRNA species, we performed 3'-rapid amplification of cDNA ends and found that all 15 clones sequenced, which spanned the C terminus of the protein contained the Ser472 domain. However, human and rat PKBgamma diverge in amino acid sequence precisely at a site where an exon boundary has been mapped for the mouse PKBalpha gene (29). Thus, it is possible that the published rat cDNA sequence constitutes a minor splice variant of PKBgamma or a partially processed mRNA.


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  		Fig. 1.   Sequence alignment of human PKBalpha , PKBbeta , PKBgamma , mouse PKBgamma , and the C terminus of rat PKBgamma . Shown are the amino acid sequences of human PKBalpha (1), PKBbeta (3), and PKBgamma , of mouse PKBgamma , and the C-terminal domain of rat PKBgamma (4). The numbering refers to PKBalpha . The regulatory phosphorylation sites (Thr308 and Ser473 in PKBalpha ) are indicated with an asterisk.

To assess the tissue distribution of transcripts encoding PKBgamma , we used an isoform-specific radiolabeled cDNA fragment to probe two human multiple tissue Northern blots. Two equally expressed transcripts of 8.5 and 6.5 kilobases were detected in all tissues tested, with highest levels found in adult brain, lung, and kidney and very low levels in heart and liver (Fig. 2A). Two transcripts of similar size were detected in fetal tissues (Fig. 2B), with high levels found in heart, brain, and liver, but none in the kidney. This observation, and the size of the transcripts, which are much larger than the 3.2-3.4-kilobases transcripts of PKBalpha /beta ,2 indicated the presence of long untranslated regions, and thus the possibility of developmental regulation of expression or post-transcriptional modifications affecting mRNA stability.


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  		Fig. 2.   Northern blot analysis of PKBgamma expression in human tissues. A, adult and B, fetal multiple tissue Northern blots were probed for expression of PKBgamma with a [alpha -32P]dATP-random-prime-labeled probe derived from a 825-bp fragment of the human PKBgamma cDNA spanning amino acids 110-384 and exposed for 3 days at -70 °C. RNA molecular weight markers (in kilobases) are indicated.

In contrast to the rat enzyme, human PKBgamma contains two predicted regulatory phosphorylation sites, Thr305 in the activation loop, and Ser472 in the C-terminal domain, as does PKBalpha /beta . To determine the importance of these two residues, we mutated them to alanine which cannot be phosphorylated, or to aspartate to mimic the phosphorylated state, and assayed HA-PKBgamma kinase activity following transient transfection and stimulation with the insulin mimetic compound pervanadate (Fig. 3). The results presented here show that wild type HA-PKBgamma , which had a low basal activity, could be stimulated 67-fold by pervanadate treatment; furthermore, mutation of Thr305 to alanine completely ablated activation. No activity above basal levels was observed for HA-PKBgamma T305A, and the same was true for the double mutant HA-PKBgamma T305A,S472A. On the other hand, mutation of the C-terminal regulatory site (HA-PKBgamma S472A) reduced but did not abolish activation by pervanadate (10-fold). We also tested the effects of aspartate mutations, since in the case of PKBalpha , a double aspartate mutant was constitutively active (15). However, PKBgamma was not active above basal levels upon mutation of the activation loop site to aspartate (HA-PKBgamma T305D and HA-PKBgamma T305D,S472D) and, furthermore, could not be stimulated by pervanadate treatment. Thus the aspartic acid moiety could not substitute for the phosphorylated threonine residue. Again, mutation of Ser472 to aspartate (HA-PKBgamma S472D) resulted in a protein that could still be activated by pervanadate (35-fold), albeit to a lesser extent than the wild type. These results establish that the phosphorylation site Thr305 in the activation loop is absolutely necessary for activation of PKBgamma , with conformational constraints around the active site apparently so stringent that substitution by a negatively charged residue is not tolerated.


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  		Fig. 3.   Mutational analysis of the two regulatory phosphorylation sites of human HA-PKBgamma . HEK-293 cells were transfected with constructs encoding HA-PKBgamma , HA-PKBgamma T305A, HA-PKBgamma T305A,S472A, HA-PKBgamma S472A, HA-PKBgamma T305D, HA-PKBgamma T305D,S472D, or HA-PKBgamma S472D. After serum starvation for 18 h, cells were stimulated with 0.2-mM pervanadate for 5 min and kinase activity determined as described in Ref. 28. Specific activity (pmol/min/mg of protein) is the mean (±S.D.) of two experiments assayed in duplicate.

To determine the role of the C-terminal Ser472 in regulation of human PKBgamma , we tested whether activation of HA-PKBgamma , HA-PKBgamma S472A, and HA-PKBgamma S472D by insulin was sensitive to inhibition of PI3K. Fig. 4A depicts the results of a representative experiment, which show that insulin activated HA-PKBgamma , HA-PKBgamma S472D, and, to a lesser extent, HA-PKBgamma S472A. This stimulation was dependent on the activity of PI3K, since pretreatment of transfected cells with the PI3K inhibitor wortmannin inhibited activation by insulin. Furthermore, we subjected the immunoprecipitated proteins to Western blot analysis with an antibody generated specifically against the phosphorylated Ser473 peptide of PKBalpha (Fig. 4A, inset). The antibody cross-reacted with HA-PKBgamma only upon stimulation with insulin, and phosphorylation of Ser472 was prevented by wortmannin. Since Ser472 was mutated in HA-PKBgamma S472A and HA-PKBgamma S472D and could not be phosphorylated, we concluded that the wortmannin-sensitive, insulin-stimulated activity of these proteins was entirely due to phosphorylation at Thr305, dependent on the presence of 3-phosphorylated phospholipids.


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  		Fig. 4.   HA-PKBgamma activation is dependent on PI3K. HEK-293 cells were transfected with constructs encoding HA-PKBgamma , HA-PKBgamma S472A, or HA-PKBgamma S472D (A) or HA-Delta PHPKBgamma , HA-Delta PHPKBgamma S472A, or HA-Delta PHPKBgamma S472D (B) and serum-starved for 18 h. Cells were treated with the solvent Me2SO or 200 nM wortmannin for 15 min, stimulated with 500 nM insulin in the presence of Me2SO or wortmannin for 15 min, and kinase activity determined as described in Ref. 28. Specific activity (pmol/min/mg of protein) is the mean (±average deviation) of duplicate immunoprecipitates from a representative experiment. The experiment was repeated twice, with similar results. Insets: HA-PKBgamma (A) and HA-Delta PHPKBgamma (B) were immunoprecipitated from 50-µg aliquots of cell extracts from the same experiment and subjected to Western blot analysis with a phospho-specific Ser473 antibody. Molecular mass markers (in kDa) are indicated.

In this analysis, we also included PKBgamma constructs lacking the N-terminal PH domain. In the basal state, this domain is thought to restrict access to the phosphorylation site in the activation loop, thus leaving Thr305 more accessible to phosphorylation by upstream kinases when it is removed. The proteins lacking the PH domain now presented a different picture (Fig. 4B): HA-Delta PHPKBgamma was maximally activated under basal conditions and could not be stimulated further by insulin treatment. This contrasts with results obtained for Delta PHPKBalpha , shown to be activated by insulin (6). Furthermore, pretreatment of the cells with wortmannin led to a reduction in activity of HA-Delta PHPKBgamma , indicating that it was still a target for PI3K-dependent phosphorylation. HA-Delta PHPKBgamma S472A activity was comparable with that of wortmannin-treated HA-Delta PHPKBgamma and was not responsive to insulin or wortmannin. In contrast, HA-Delta PHPKBgamma S472D was again fully active in the absence of stimulation but, unlike HA-Delta PHPKBgamma , was not inhibited by wortmannin. The Western blot signals with the phospho-specific Ser473 antibody correlated with the activities observed (Fig. 4B, inset); HA-Delta PHPKBgamma was strongly phosphorylated in extracts of unstimulated and insulin-stimulated cells, but pretreatment with wortmannin reduced the signal. We found previously that transiently transfected PDK1, the upstream kinase phosphorylating Thr308 in PKBalpha , is active in serum-starved HEK-293 cells.3 The present results seem to indicate that removal of the PH domain causes a conformational change of PKBgamma favorable to phosphorylation at Thr305, so as to make it independent of PI3K activity. Basal activity of PI3K in unstimulated cells allowed phosphorylation of HA-Delta PHPKBgamma by the Ser473 kinase, resulting in full activation. Conversely, inhibiting this basal PI3K activity by wortmannin treatment reduced HA-Delta PHPKBgamma activity, probably due to the rapid action of phosphatases on phosphorylated Ser472. Thus, HA-Delta PHPKBgamma is a model for studying phosphorylation of Ser472, the second regulatory site of human PKBgamma , almost independent of Thr305.

In summary, we report the cloning and characterization of human PKBgamma , a PKB isoform distinct from its rat counterpart in having two regulatory phosphorylation sites, Thr305 and Ser472, both of which are required for full activation of the protein. Our results suggest markedly similar regulation mechanisms for PKBgamma and the alpha  and beta  isoforms, with both upstream kinases phosphorylating the regulatory sites being sensitive to PI3K-derived signals. Furthermore, we found a high abundance of PKBgamma mRNAs encoding the C-terminal hydrophobic domain and have isolated a similar mouse PKBgamma , showing that this isoform is not restricted to humans. Taken together, we conclude that the truncated rat PKBgamma used in all studies so far (26) probably constitutes a minor splice variant of endogenous PKBgamma protein. The crucial question now emerging is that of the specific roles of the three different PKB isoforms.

    ACKNOWLEDGEMENTS

We thank Drs. M. Andjelkovic, T. Millward, and P. King for comments on the manuscript; P. Mueller for oligonucleotide synthesis; and Dr. H. Angliker for DNA sequencing.

    FOOTNOTES

* 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF124141 and AF124142.

Dagger To whom correspondence should be addressed: Friedrich Miescher-Institut, P. O. Box 2543, CH-4002 Basel, Switzerland. Tel.: 41-61-697-40-46; Fax: 41-61-697-39-76; E-mail: hemmings{at}fmi.ch.

2 B. A. Hemmings, unpublished results.

3 M. Andjelkovic and D. Brodbeck, unpublished results.

    ABBREVIATIONS

The abbreviations used are: PKB, protein kinase B; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PDK1, 3-phosphoinositide-dependent protein kinase 1; HA, hemagglutinin; HEK, human embryonic kidney; bp, base pair; PCR, polymerase chain reaction.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.


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Tumor cell sensitization to apoptotic stimuli by selective inhibition of specific Akt/PKB family members
Mol. Cancer Ther., February 1, 2005; 4(2): 271 - 279.
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 Cancer Res.Home page
J. M. Stahl, A. Sharma, M. Cheung, M. Zimmerman, J. Q. Cheng, M. W. Bosenberg, M. Kester, L. Sandirasegarane, and G. P. Robertson
Deregulated Akt3 Activity Promotes Development of Malignant Melanoma
Cancer Res., October 1, 2004; 64(19): 7002 - 7010.
[Abstract] [Full Text] [PDF]

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 ReproductionHome page
Q Qiu, M Yang, B K Tsang, and A Gruslin
EGF-induced trophoblast secretion of MMP-9 and TIMP-1 involves activation of both PI3K and MAPK signalling pathways
Reproduction, September 1, 2004; 128(3): 355 - 363.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
M. Mogi, J. Yang, J.-F. Lambert, G. A. Colvin, I. Shiojima, C. Skurk, R. Summer, A. Fine, P. J. Quesenberry, and K. Walsh
Akt Signaling Regulates Side Population Cell Phenotype via Bcrp1 Translocation
J. Biol. Chem., October 3, 2003; 278(40): 39068 - 39075.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Endocrinol. Metab.Home page
C. E. McCurdy, R. T. Davidson, and G. D. Cartee
Brief calorie restriction increases Akt2 phosphorylation in insulin-stimulated rat skeletal muscle
Am J Physiol Endocrinol Metab, October 1, 2003; 285(4): E693 - E700.
[Abstract] [Full Text] [PDF]

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 Genes Dev.Home page
X.-d. Peng, P.-Z. Xu, M.-L. Chen, A. Hahn-Windgassen, J. Skeen, J. Jacobs, D. Sundararajan, W. S. Chen, S. E. Crawford, K. G. Coleman, et al.
Dwarfism, impaired skin development, skeletal muscle atrophy, delayed bone development, and impeded adipogenesis in mice lacking Akt1 and Akt2
Genes & Dev., June 1, 2003; 17(11): 1352 - 1365.
[Abstract] [Full Text] [PDF]

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 Circ. Res.Home page
I. Shiojima and K. Walsh
Role of Akt Signaling in Vascular Homeostasis and Angiogenesis
Circ. Res., June 28, 2002; 90(12): 1243 - 1250.
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 J. Biol. Chem.Home page
M. K. Dahle, L. M. Gronning, A. Cederberg, H. K. Blomhoff, N. Miura, S. Enerback, K. A. Tasken, and K. Tasken
Mechanisms of FOXC2- and FOXD1-mediated Regulation of the RIalpha Subunit of cAMP-dependent Protein Kinase Include Release of Transcriptional Repression and Activation by Protein Kinase Balpha and cAMP
J. Biol. Chem., June 14, 2002; 277(25): 22902 - 22908.
[Abstract] [Full Text] [PDF]

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 Clin. Cancer Res.Home page
S. R. Chinni and F. H. Sarkar
Akt Inactivation Is a Key Event in Indole-3-carbinol-induced Apoptosis in PC-3 Cells
Clin. Cancer Res., April 1, 2002; 8(4): 1228 - 1236.
[Abstract] [Full Text] [PDF]

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 Mol. Cell. Biol.Home page
G. Kunstle, J. Laine, G. Pierron, S.-i. Kagami, H. Nakajima, F. Hoh, C. Roumestand, M.-H. Stern, and M. Noguchi
Identification of Akt Association and Oligomerization Domains of the Akt Kinase Coactivator TCL1
Mol. Cell. Biol., March 1, 2002; 22(5): 1513 - 1525.
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 Mol. Cell. Biol.Home page
T. Jahn, P. Seipel, S. Urschel, C. Peschel, and J. Duyster
Role for the Adaptor Protein Grb10 in the Activation of Akt
Mol. Cell. Biol., February 15, 2002; 22(4): 979 - 991.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
J. Laine, G. Kunstle, T. Obata, and M. Noguchi
Differential Regulation of Akt Kinase Isoforms by the Members of the TCL1 Oncogene Family
J. Biol. Chem., January 25, 2002; 277(5): 3743 - 3751.
[Abstract] [Full Text] [PDF]

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 Clin. Cancer Res.Home page
M. J. Zinda, M. A. Johnson, J. D. Paul, C. Horn, B. W. Konicek, Z. H. Lu, G. Sandusky, J. E. Thomas, B. L. Neubauer, M. T. Lai, et al.
AKT-1, -2, and -3 are Expressed in Both Normal and Tumor Tissues of the Lung, Breast, Prostate, and Colon
Clin. Cancer Res., August 1, 2001; 7(8): 2475 - 2479.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
S. L. Pogue, T. Kurosaki, J. Bolen, and R. Herbst
B Cell Antigen Receptor-Induced Activation of Akt Promotes B Cell Survival and Is Dependent on Syk Kinase
J. Immunol., August 1, 2000; 165(3): 1300 - 1306.
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 Hum Mol GenetHome page
F. D. Ciccarelli, A. Acciarito, and S. Alberti
Large and diverse numbers of human diseases with HIKE mutations
Hum. Mol. Genet., April 1, 2000; 9(6): 1001 - 1007.
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 J. Biol. Chem.Home page
A. Toker and A. C. Newton
Akt/Protein Kinase B Is Regulated by Autophosphorylation at the Hypothetical PDK-2 Site
J. Biol. Chem., March 17, 2000; 275(12): 8271 - 8274.
[Abstract] [Full Text] [PDF]

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 Genes Dev.Home page
S. R. Datta, A. Brunet, and M. E. Greenberg
Cellular survival: a play in three Akts
Genes & Dev., November 15, 1999; 13(22): 2905 - 2927.
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 J. Biol. Chem.Home page
K. Nakatani, D. A. Thompson, A. Barthel, H. Sakaue, W. Liu, R. J. Weigel, and R. A. Roth
Up-regulation of Akt3 in Estrogen Receptor-deficient Breast Cancers and Androgen-independent Prostate Cancer Lines
J. Biol. Chem., July 30, 1999; 274(31): 21528 - 21532.
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 J. Biol. Chem.Home page
M. Takata, W. Ogawa, T. Kitamura, Y. Hino, S. Kuroda, K. Kotani, A. Klip, A.-C. Gingras, N. Sonenberg, and M. Kasuga
Requirement for Akt (Protein Kinase B) in Insulin-induced Activation of Glycogen Synthase and Phosphorylation of 4E-BP1 (PHAS-1)
J. Biol. Chem., July 16, 1999; 274(29): 20611 - 20618.
[Abstract] [Full Text] [PDF]