Phosphorylation Sites in the Autoinhibitory Domain Participate in p70s6k Activation Loop Phosphorylation
1998; Elsevier BV; Volume: 273; Issue: 24 Linguagem: Inglês
10.1074/jbc.273.24.14845
ISSN1083-351X
AutoresPatrick B. Dennis, Nick Pullen, Richard B. Pearson, Sara C. Kozma, George Thomas,
Tópico(s)Protein Degradation and Inhibitors
ResumoHere we have employed p70s6ktruncation and point mutants to elucidate the role played by the carboxyl-terminal autoinhibitory domain S/TP phosphorylation sites in kinase activation. Earlier studies showed that truncation of the p70s6k amino terminus severely impaired kinase activation but that this effect was reversed by deleting the carboxyl terminus, which in parallel led to deregulation of Thr229 phosphorylation in the activation loop (Dennis, P. B., Pullen, N., Kozma, S. C., and Thomas, G. (1996)Mol. Cell. Biol. 16, 6242–6251). In this study, substitution of acidic residues for the four autoinhibitory domainS/TP sites mimics the carboxyl-terminal deletion largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr229phosphorylation, suggesting the involvement of another regulatory element in the intact kinase. This element appears to be Thr389 phosphorylation, because substitution of an acidic residue at this position in the p70s6k variant containing the S/TP mutations leads to a large increase in basal Thr229 phosphorylation and kinase activity. In contrast, an alanine substitution at Thr389 blocks both responses. Consistent with these data, we show that a mutant harboring the acidicS/TP and Thr389 substitutions is an excellentin vitro substrate for the newly identified Thr229 kinase, phosphoinositide-dependent kinase-1 (Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S., Hemmings, B. A., and Thomas, G. (1998)Science 279, 707–710), whereas phosphoinositide-dependent kinase-1 poorly utilizes the two p70s6k variants that have only one set of mutations. These findings indicate that phosphorylation of the S/TP sites, in cooperation with Thr389 phosphorylation, controls Thr229 phosphorylation through an intrasteric mechanism. Here we have employed p70s6ktruncation and point mutants to elucidate the role played by the carboxyl-terminal autoinhibitory domain S/TP phosphorylation sites in kinase activation. Earlier studies showed that truncation of the p70s6k amino terminus severely impaired kinase activation but that this effect was reversed by deleting the carboxyl terminus, which in parallel led to deregulation of Thr229 phosphorylation in the activation loop (Dennis, P. B., Pullen, N., Kozma, S. C., and Thomas, G. (1996)Mol. Cell. Biol. 16, 6242–6251). In this study, substitution of acidic residues for the four autoinhibitory domainS/TP sites mimics the carboxyl-terminal deletion largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr229phosphorylation, suggesting the involvement of another regulatory element in the intact kinase. This element appears to be Thr389 phosphorylation, because substitution of an acidic residue at this position in the p70s6k variant containing the S/TP mutations leads to a large increase in basal Thr229 phosphorylation and kinase activity. In contrast, an alanine substitution at Thr389 blocks both responses. Consistent with these data, we show that a mutant harboring the acidicS/TP and Thr389 substitutions is an excellentin vitro substrate for the newly identified Thr229 kinase, phosphoinositide-dependent kinase-1 (Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S., Hemmings, B. A., and Thomas, G. (1998)Science 279, 707–710), whereas phosphoinositide-dependent kinase-1 poorly utilizes the two p70s6k variants that have only one set of mutations. These findings indicate that phosphorylation of the S/TP sites, in cooperation with Thr389 phosphorylation, controls Thr229 phosphorylation through an intrasteric mechanism. The concerted up-regulation of transcription and translation is required for a cell, responding to mitogenic stimuli, to grow and enter the cell cycle (1Brooks R.F. Cell. 1977; 12: 311-317Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 2Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar, 3Karin M. Hunter T. Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). Recent studies have implicated increased S6 phosphorylation in the selective translation of a subset of essential mRNAs containing an oligopyrimidine tract at their transcriptional start site (4Jefferies H.B.J. Thomas G. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control: Ribosomal Protein S6 Phosphorylation and Signal Transduction. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 389-409Google Scholar, 5Jefferies H.B.J. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 12: 3693-3704Crossref Scopus (806) Google Scholar). This event is regulated by p70s6k/p85s6k (6Pearson R.B. Thomas G. Meijer L. Guidet S. Tung H.Y.L. Progress in Cell Cycle Research. 1. Plenum Press, New York1995: 21-32Crossref Scopus (44) Google Scholar), two mitogen-stimulated protein kinase isoforms that rely on multiple phosphorylation as a principal mechanism for activation (7Pullen N. Thomas G. FEBS Lett. 1997; 410: 78-82Crossref PubMed Scopus (483) Google Scholar). The p85s6k isoform is expressed from the same transcript as p70s6k through an alternative translational initiation start site, 1Y. Chen, C. D. Hoemann, G. Thomas, and S. C. Kozma, submitted for publication. 1Y. Chen, C. D. Hoemann, G. Thomas, and S. C. Kozma, submitted for publication. adding a 23-amino acid extension at the amino terminus and constitutively targeting it to the nucleus (8Reinhard C. Fernandez A. Lamb N.J.C. Thomas G. EMBO J. 1994; 13: 1557-1565Crossref PubMed Scopus (179) Google Scholar). Little is known about the role of p85s6k in the nucleus; however, S6 has been shown to reside in both the nucleoplasm, in a free form, and in the nucleolus, in preribosomal particles, where it is also phosphorylated in response to mitogens (9Franco R. Rosenfeld M.G. J. Biol. Chem. 1990; 265: 4321-4325Abstract Full Text PDF PubMed Google Scholar). In studies conducted to date, regulation of the nuclear isoform parallels that of p70s6k, so a concomitant role for p85s6k in mitogenesis is predicted (10Reinhard C. Thomas G. Kozma S.C. Proc. Natl. Acad. Sci. USA. 1992; 89: 4052-4056Crossref PubMed Scopus (98) Google Scholar, 11Ming X.F. Burgering B.M. Th. Wennström S. Claesson-Welsh L. Heldin C.H. Bos J.L. Kozma S.C. Thomas G. Nature. 1994; 371: 426-429Crossref PubMed Scopus (204) Google Scholar), perhaps involving transcription or processing of RNA (8Reinhard C. Fernandez A. Lamb N.J.C. Thomas G. EMBO J. 1994; 13: 1557-1565Crossref PubMed Scopus (179) Google Scholar).The identification of intramolecular p70s6k regulatory elements, in the form of domains and phosphorylation sites, has increased our understanding of the mechanism by which the kinase autoregulates (7Pullen N. Thomas G. FEBS Lett. 1997; 410: 78-82Crossref PubMed Scopus (483) Google Scholar, 12Weng Q.-P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (210) Google Scholar, 13Dennis P.B. Pullen N. Kozma S.C. Thomas G. Mol. Cell. Biol. 1996; 16: 6242-6251Crossref PubMed Scopus (222) Google Scholar). To date, eight phosphorylation sites have been identified in the endogenous kinase (14Ferrari S. Bannwarth W. Morley S.J. Totty N.F. Thomas G. Proc. Natl. Acad. Sci. USA. 1992; 89: 7282-7285Crossref PubMed Scopus (131) Google Scholar, 15Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 21: 5279-5287Crossref Scopus (387) Google Scholar). In initial studies, Ser411, Ser418, Thr421, and Ser424, residing within a potential autoinhibitory domain at the carboxyl terminus of the kinase (16Price D.J. Mukhopadhyay N.K. Avruch J. J. Biol. Chem. 1991; 266: 16281-16284Abstract Full Text PDF PubMed Google Scholar, 17Flotow H. Thomas G. J. Biol. Chem. 1992; 267: 3074-3078Abstract Full Text PDF PubMed Google Scholar), were found to be principal sites of mitogen-induced phosphorylation (14Ferrari S. Bannwarth W. Morley S.J. Totty N.F. Thomas G. Proc. Natl. Acad. Sci. USA. 1992; 89: 7282-7285Crossref PubMed Scopus (131) Google Scholar). These sites are characterized by a proline in the +1 position and a hydrophobic residue in the −2 position. More recently, studies have led to the identification of Ser371 (18Moser B.A. Dennis P.B. Pullen N. Pearson R.B. Williamson N.A. Wettenhall E.H. Kozma S.C. Thomas G. Mol. Cell. Biol. 1997; 17: 5648-5655Crossref PubMed Scopus (86) Google Scholar) as a phosphorylation site that shares the same motif, and three additional sites, Thr229, Thr389, and Ser404, which are flanked in the +1 and −1 positions by bulky aromatic amino acids (15Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 21: 5279-5287Crossref Scopus (387) Google Scholar). The phosphorylation of these latter sites in response to mitogens is blocked by treatment of cells with the immunosuppressant rapamycin or the fungal metabolite wortmannin (15Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 21: 5279-5287Crossref Scopus (387) Google Scholar, 19Han J.-W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Based on mutation studies, Thr229, in the activation loop, as well as Ser371 and Thr389, in the linker region coupling the catalytic and autoinhibitory domains, appear to be critical for kinase activation (15Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 21: 5279-5287Crossref Scopus (387) Google Scholar, 18Moser B.A. Dennis P.B. Pullen N. Pearson R.B. Williamson N.A. Wettenhall E.H. Kozma S.C. Thomas G. Mol. Cell. Biol. 1997; 17: 5648-5655Crossref PubMed Scopus (86) Google Scholar). The activation loop phosphorylation site, Thr229 in p70s6k, is a common regulatory element found in many kinases (20Marshall C.J. Nature. 1994; 367: 686Crossref PubMed Scopus (7) Google Scholar, 21Hanks S.K. Hunter T. Hardie G. Hanks S.K. The Protein Kinase Facts Book. Protein-Serine Kinases: The Eukaryotic Protein Kinase Superfamily. Academic Press, London1995: 7-47Google Scholar). In parallel studies, we have shown that the phosphorylation of this site is regulated by the newly described phosphoinositide-dependent protein kinase, PDK1 2The abbreviations used are: PDK1, phosphoinositide-dependent kinase-1; FCS, fetal calf serum. 2The abbreviations used are: PDK1, phosphoinositide-dependent kinase-1; FCS, fetal calf serum. (22Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google Scholar). Thr229, Ser371, and Thr389, as well as the domains in which they reside, are strikingly conserved in most members of the AGC (protein kinases A, G, and C) family of Ser/Thr kinases (21Hanks S.K. Hunter T. Hardie G. Hanks S.K. The Protein Kinase Facts Book. Protein-Serine Kinases: The Eukaryotic Protein Kinase Superfamily. Academic Press, London1995: 7-47Google Scholar).In contrast to the domains containing Thr229, Ser371, and Thr389, the autoinhibitory domain, as well as the carboxyl terminus of p70s6k, are conspicuously absent in the other members of the AGC family of Ser/Thr kinases (21Hanks S.K. Hunter T. Hardie G. Hanks S.K. The Protein Kinase Facts Book. Protein-Serine Kinases: The Eukaryotic Protein Kinase Superfamily. Academic Press, London1995: 7-47Google Scholar). We have reported that mutation of the S/TP sites within the autoinhibitory domain, as well as Ser404, to alanines or acidic residues modulates kinase activity (15Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 21: 5279-5287Crossref Scopus (387) Google Scholar, 19Han J.-W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). However, others have claimed little to no effect of similar mutations on the activity of the kinase (23Mahalingam M. Templeton D.J. Mol. Cell. Biol. 1996; 16: 405-413Crossref PubMed Scopus (61) Google Scholar, 24Edelmann H.M.L. Kühne C. Petritsch C. Ballou L.M. J. Biol. Chem. 1996; 271: 963-971Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Indeed, the deletion of the p70s6k carboxyl terminus, containing the S/TP sites, has little effect on either basal or mitogen-induced kinase activity (12Weng Q.-P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (210) Google Scholar, 13Dennis P.B. Pullen N. Kozma S.C. Thomas G. Mol. Cell. Biol. 1996; 16: 6242-6251Crossref PubMed Scopus (222) Google Scholar, 25Cheatham L. Monfar M. Chou M.M. Blenis J. Proc. Natl. Acad. Sci. USA. 1995; 92: 11696-11700Crossref PubMed Scopus (114) Google Scholar), which has also been used to conclude that these sites are not involved in regulating kinase activation at either the G0/G1 or M/G1 transition state of the cell cycle (23Mahalingam M. Templeton D.J. Mol. Cell. Biol. 1996; 16: 405-413Crossref PubMed Scopus (61) Google Scholar, 24Edelmann H.M.L. Kühne C. Petritsch C. Ballou L.M. J. Biol. Chem. 1996; 271: 963-971Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Despite these observations, this domain is completely conserved in all the mammalian forms of p70s6kand is also present in the recently cloned Drosophila homolog, Dp70s6k (26Stewart M.J. Berry C.O.A. Zilberman F. Thomas G. Kozma S.C. Proc. Natl. Acad. Sci. USA. 1996; 93: 10791-10796Crossref PubMed Scopus (39) Google Scholar, 27Watson K.L. Chou M.M. Blenis J. Gelbart W.M. Erickson R.L. Proc. Natl. Acad. Sci. USA. 1996; 93: 13694-13698Crossref PubMed Scopus (28) Google Scholar). More notably, a p70s6k amino-terminal truncation, which blocks kinase activation (12Weng Q.-P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (210) Google Scholar, 13Dennis P.B. Pullen N. Kozma S.C. Thomas G. Mol. Cell. Biol. 1996; 16: 6242-6251Crossref PubMed Scopus (222) Google Scholar, 25Cheatham L. Monfar M. Chou M.M. Blenis J. Proc. Natl. Acad. Sci. USA. 1995; 92: 11696-11700Crossref PubMed Scopus (114) Google Scholar) and mitogen-induced Thr229 and Thr389 phosphorylation (13Dennis P.B. Pullen N. Kozma S.C. Thomas G. Mol. Cell. Biol. 1996; 16: 6242-6251Crossref PubMed Scopus (222) Google Scholar), is rescued by the same carboxyl-terminal deletion that removes the autoinhibitory domain (12Weng Q.-P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (210) Google Scholar,13Dennis P.B. Pullen N. Kozma S.C. Thomas G. Mol. Cell. Biol. 1996; 16: 6242-6251Crossref PubMed Scopus (222) Google Scholar, 25Cheatham L. Monfar M. Chou M.M. Blenis J. Proc. Natl. Acad. Sci. USA. 1995; 92: 11696-11700Crossref PubMed Scopus (114) Google Scholar). These observations suggest instead that the autoinhibitory domain, and possibly the phosphorylation sites residing within this domain, play a critical role in regulating p70s6k activity in the intact kinase through the modulation of Thr229 and Thr389 phosphorylation.In this study, we utilized phosphorylation site and truncation mutations to elucidate the role of the autoinhibitory domainS/TP sites in regulating mitogen-induced p70s6kactivation. Next, we examined the nature of this event as it relates to the ability of the S/TP sites, along with Thr389, to regulate Thr229 phosphorylationin vivo . Finally, by employing PDK1 in vitro , we have determined that the mechanism by which these carboxyl-terminal phosphorylation sites control Thr229 phosphorylationin vivo is synergistic and intrasteric.DISCUSSIONOf the eight known p70s6k phosphorylation sites, the first to be identified were the S/TP sites in the carboxyl-terminal autoinhibitory domain of the kinase (14Ferrari S. Bannwarth W. Morley S.J. Totty N.F. Thomas G. Proc. Natl. Acad. Sci. USA. 1992; 89: 7282-7285Crossref PubMed Scopus (131) Google Scholar). Although we reported that neutral and acidic mutations at these sites lower and raise basal kinase activity, respectively (15Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 21: 5279-5287Crossref Scopus (387) Google Scholar, 19Han J.-W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), others found little to no effect of similar mutations (23Mahalingam M. Templeton D.J. Mol. Cell. Biol. 1996; 16: 405-413Crossref PubMed Scopus (61) Google Scholar, 24Edelmann H.M.L. Kühne C. Petritsch C. Ballou L.M. J. Biol. Chem. 1996; 271: 963-971Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). This study emphasizes the importance of the autoinhibitory domain phosphorylation sites in p70s6k activation. First, acidic amino acid substitutions at these sites largely rescue the activity of an amino-terminal truncation mutant, and second, these sites cooperate with an acidic mutation at Thr389 to synergistically regulate phosphorylation of the activation loop site, Thr229. Although the p70s6kΔC104 mutant is still regulated by mitogens, indicating that other elements are involved in p70s6k activation, it does not exclude a role for the autoinhibitory domain S/TP in the activation of the intact kinase. Indeed, the synergistic effect conferred by theS/TP and Thr389 acidic mutations on Thr229 phosphorylation suggests a possible mechanism for the sensitive control of Thr229 phosphorylation, which is dependent on the stoichiometry of S/TP site phosphorylation in the autoinhibitory domain (see below). This is further supported by the observation that truncation of the carboxyl terminus largely deregulates Thr229 phosphorylation (Fig. 3).The ability of the acidic mutations of the S/TP sites to rescue p70s6kΔN54 activity was unexpected, because deletion of this domain did not significantly affect the serum-induced phosphorylation of these sites (13Dennis P.B. Pullen N. Kozma S.C. Thomas G. Mol. Cell. Biol. 1996; 16: 6242-6251Crossref PubMed Scopus (222) Google Scholar). However, this may be due to the degree of phosphorylation at the autoinhibitory domainS/TP sites in response to mitogen stimulation. Mitogen stimulation is hypothesized to first lead to an increase inS/TP site phosphorylation in the autoinhibitory domain, which functions together with the amino terminus to facilitate Thr389 phosphorylation (Fig.8). In the absence of the amino terminus, the level of mitogen-induced S/TP site phosphorylation may not be sufficient to promote a net increase in Thr389phosphorylation and subsequent Thr229 phosphorylation, attenuating kinase activation. However, substitution of an acidic amino acid at each of the S/TP sites would raise the overall of negative charge of this domain and overcome the effect of the amino-terminal truncation, triggering Thr389phosphorylation. In support of this model, phosphopeptide maps show that Thr389 phosphorylation is rescued when acidicS/TP site mutations are placed in the p70s6kΔN54background. 3P. B. Dennis and G. Thomas, unpublished results. Thus, Thr389 phosphorylation would act as an intermediary step between autoinhibitory S/TP and activation loop site phosphorylation, which would be the final step in mitogen-induced p70s6k activation (Fig. 8).Although a number of candidate autoinhibitory domain S/TP kinases have been suggested, including cyclin-dependent kinase-1 and the mitogen-activated kinases p42mapk/p44mapk (33Mukhopadhyay N.K. Price D.J. Kyriakis J.M. Pelech S.L. Sanghera J. Avruch J. J. Biol. Chem. 1992; 267: 3325-3335Abstract Full Text PDF PubMed Google Scholar), their requirement has not been substantiated to date. Indeed, utilization of interfering mutants of p21ras and p74raf, as well as deletion mutants of the platelet-derived growth factor receptor, demonstrated that p42mapk/p44mapk were not effectors of the p70s6k signaling pathway (11Ming X.F. Burgering B.M. Th. Wennström S. Claesson-Welsh L. Heldin C.H. Bos J.L. Kozma S.C. Thomas G. Nature. 1994; 371: 426-429Crossref PubMed Scopus (204) Google Scholar). It has been shown that over-expression of a kinase dead p70s6k blocks Thr229, Thr389, and Ser404phosphorylation as well as strongly suppressing Ser411 and Thr421 phosphorylation in the autoinhibitory domain (5Jefferies H.B.J. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 12: 3693-3704Crossref Scopus (806) Google Scholar,34Von Manteuffel S.R. Dennis P.B. Pullen N. Gingras A.-C. Sonenberg N. Thomas G. Mol. Cell. Biol. 1997; 17: 5426-5436Crossref PubMed Scopus (210) Google Scholar), a pattern of inhibition resembling that induced by rapamycin (15Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 21: 5279-5287Crossref Scopus (387) Google Scholar). Furthermore, it was found that over-expression of kinase dead or wild-type p70s6k blocks the same sites of phosphorylation in the suppressor of protein synthesis initiation factor 4E, eIF4E-binding protein-1, as does rapamycin (34Von Manteuffel S.R. Dennis P.B. Pullen N. Gingras A.-C. Sonenberg N. Thomas G. Mol. Cell. Biol. 1997; 17: 5426-5436Crossref PubMed Scopus (210) Google Scholar). These results suggest that overexpression of p70s6k might sequester a common upstream kinase that is also responsible for phosphorylating eIF4E-binding protein-1 (34Von Manteuffel S.R. Dennis P.B. Pullen N. Gingras A.-C. Sonenberg N. Thomas G. Mol. Cell. Biol. 1997; 17: 5426-5436Crossref PubMed Scopus (210) Google Scholar). Consistent with this hypothesis, the phosphorylation sites in eIF4E-binding protein-1 also displayS/TP motifs (35Fadden P. Haystead T.A.J. Lawrence Jr., J.C. J. Biol. Chem. 1997; 272: 10240-10247Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), and recently, it was shown that these sites are phosphorylated in vitro by the mammalian target of rapamycin (36Brunn G.J. Hudson C.C. Sekulic A. Williams J.M. Hosoi H. Houghton P.J. Lawrence Jr., J.C. Abraham R.T. Science. 1997; 277: 99-101Crossref PubMed Scopus (802) Google Scholar). However, it is unlikely that mammalian target of rapamycin is the S/TP kinase for p70s6k, because rapamycin has no effect on serum-induced S/TP phosphorylation in the amino-terminal truncation mutant, p70s6kΔN54. 4P. B. Dennis, N. Pullen, R. B. Pearson, S. C. Kozma, and G. Thomas, unpublished data. Interest in the identity of the S/TP kinase has been further increased by the recent observation that Pin1, the conserved mitotic peptidyl-prolyl isomerase, binds to p70s6k, apparently through phosphorylated Ser411 (37Yaffe M.B. Schutkowski M. Shen M. Zhou X.Z. Stukenberg P.T. Rahfeld J.-U. Xu J. Kuang J. Kirshner M.W. Fischer G. Cantley L.C. Lu K.P. Science. 1998; 278: 1957-1960Crossref Scopus (674) Google Scholar).The finding that in the carboxyl-terminal deletion mutant, p70s6k ΔC104, kinase activity and Thr389 phosphorylation are tightly regulated suggests the existence of an additional regulatory element in p70s6kthat is controlled by the phosphorylation of the S/TP and Thr389 sites. This element would function to modulate Thr229 phosphorylation and activity. An obvious candidate for such an element is the linker region, which couples the carboxyl and catalytic domains of p70s6k (Fig. 1). Previously, it was noted that many members of the AGC family of protein kinases (21Hanks S.K. Hunter T. Hardie G. Hanks S.K. The Protein Kinase Facts Book. Protein-Serine Kinases: The Eukaryotic Protein Kinase Superfamily. Academic Press, London1995: 7-47Google Scholar) contain a site homologous to Thr389, as well as the conserved motif surrounding this site (15Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 21: 5279-5287Crossref Scopus (387) Google Scholar). In a more recent study, it was also pointed out that this conservation extends through the entire linker region (18Moser B.A. Dennis P.B. Pullen N. Pearson R.B. Williamson N.A. Wettenhall E.H. Kozma S.C. Thomas G. Mol. Cell. Biol. 1997; 17: 5648-5655Crossref PubMed Scopus (86) Google Scholar). Within this region we identified a novel site, Ser371, the phosphorylation of which appears to be critical for kinase activation (18Moser B.A. Dennis P.B. Pullen N. Pearson R.B. Williamson N.A. Wettenhall E.H. Kozma S.C. Thomas G. Mol. Cell. Biol. 1997; 17: 5648-5655Crossref PubMed Scopus (86) Google Scholar). The site equivalent to Ser371has been identified as a major autophosphorylation site in protein kinase C βII (38Flint A.J. Paladini R.D. Koshland Jr., D.E. Science. 1990; 249: 408-411Crossref PubMed Scopus (108) Google Scholar, 39Tsutakawa S.E. Medzihradszky K.F. Flint A.J. Burlingame A.L. Koshland Jr., D.E. J. Biol. Chem. 1995; 270: 26807-26812Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and protein kinase C α (40Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar), Thr641 and Thr638, respectively. In both cases, there is a proline in the +1 position and a hydrophobic residue in the −2 position, as with Ser371 (18Moser B.A. Dennis P.B. Pullen N. Pearson R.B. Williamson N.A. Wettenhall E.H. Kozma S.C. Thomas G. Mol. Cell. Biol. 1997; 17: 5648-5655Crossref PubMed Scopus (86) Google Scholar). Furthermore, modeling studies with protein kinase C have suggested that the conserved linker region may interact with the amino terminus and that Thr641is juxtaposed to the active site allowing autophosphorylation by an intramolecular reaction (41Orr J.W. Newton A.C. J. Biol. Chem. 1994; 269: 8383-8387Abstract Full Text PDF PubMed Google Scholar, 42Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1466) Google Scholar, 43Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Although Ser371 is not an autophosphorylation site in p70s6k (18Moser B.A. Dennis P.B. Pullen N. Pearson R.B. Williamson N.A. Wettenhall E.H. Kozma S.C. Thomas G. Mol. Cell. Biol. 1997; 17: 5648-5655Crossref PubMed Scopus (86) Google Scholar), the modeling studies suggest that it could be strategically placed to modulate potential interactions between the amino terminus, the catalytic domain, and the autoinhibitory region. Indeed, mutation of this site to either an alanine or an aspartic acid blocked both Thr389phosphorylation and kinase activation, but surprisingly, it did not affect Thr229 phosphorylation (18Moser B.A. Dennis P.B. Pullen N. Pearson R.B. Williamson N.A. Wettenhall E.H. Kozma S.C. Thomas G. Mol. Cell. Biol. 1997; 17: 5648-5655Crossref PubMed Scopus (86) Google Scholar). This may indicate that mutations at Ser371 disrupt the normal function of the linker region in regulating Thr389 and Thr229phosphorylation.PDK1 has been identified as the common activation loop kinase for Thr229 phosphorylation in p70s6k (22Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google Scholar) and Thr308 in protein kinase B (32Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). In a manner similar to the p70s6kS/TP and Thr389phosphorylation sites, it has been shown that the access of PDK1 to the activation loop of protein kinase B is controlled by the binding of specific phosphatidylinositides to its PH domain (31Stokoe D. Stephens L.R. Copeland T. Gaffney P.R.J. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1045) Google Scholar, 32Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. 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