Rapamycin inhibition of baculovirus recombinant (BVr) ribosomal protein S6 kinase (S6K1) is mediated by an event other than phosphorylation
© Beigh et al; licensee BioMed Central Ltd. 2012
Received: 31 December 2011
Accepted: 1 March 2012
Published: 1 March 2012
Ribosomal protein S6 kinase 1(S6K1) is an evolutionary conserved kinase that is activated in response to growth factors and viral stimuli to influence cellular growth and proliferation. This downstream effector of target of rapamycin (TOR) signaling cascade is known to be directly activated by TOR- kinase mediated hydrophobic motif (HM) phosphorylation at Threonine 412 (T412). Selective loss of this phosphorylation by inactivation of TOR kinase or activation/recruitment of a phosphatase has accordingly been implicated in mediating inhibition by rapamycin.
We present evidence that baculovirus driven expression of S6K1 in insect cells (Sf9) fails to activate the enzyme and instead renders it modestly active representing 4-6 folds less activity than its fully active mammalian counterpart. Contrary to the contention that viral infection activates TOR signaling pathway, we report that BVr enzyme fails to exhibit putative TOR dependent phosphorylation at the HM and the resultant phosphorylation at the activation loop (AL) of the enzyme, correlating with the level of activity observed. Surprisingly, the BVr enzyme continued to exhibit sensitivity to rapamycin that remained unaffected by mutations compromised for TOR phosphorylation (T412A) or deletions compromised for TOR binding (ΔNH 2-46/ΔCT104).
These data together with the ability of the BVr enzyme to resist inactivation by phosphatases indicate that inhibition by rapamycin is not mediated by any phosphorylation event in general and TOR dependent phosphorylation in particular.
KeywordsS6 kinase Baculovirus Rapamycin mTOR Threonine 412 and Threonine 252
S6K1 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates 40S ribosomal protein S6, and coordinates cellular growth and proliferation. Multiple independent phosphorylations have been proposed to account for complete activation of the enzyme in response to growth factor stimulation . A battery of protein kinases coordinate to accomplish the activation of the enzyme through a series of phosphorylation events that culminate in phosphorylating Threonine 412 (T412) at HM and Threonine 252 (T252) at the AL [2–7]. The dynamics of these critical phosphorylations, in particular the one at the HM therefore, dictates the activation state of the enzyme. Accordingly selective loss of this TOR kinase dependent phosphorylation has been implicated in mediating the inhibitory effects of rapamycin, through direct inactivation of TOR kinase, or through activation/recruitment of a phosphatase [8–11]. In addition to Insulin and other growth factor stimulation, S6K1 has also been reported to get activated in response to viral infection, such that baculovirus mediated expression of the enzyme in insect cells activates the enzyme by phosphorylation at similar sites as identified in the enzyme from regulated cells . Since it stands established that insect S6 kinase, behaves similar to that of its mammalian counterpart, it is conceivable that the activation state of the enzyme and its inhibition by rapamycin would be no different than the one established for mammalian systems [13, 14]. Furthermore, since the stimulus due to viral infection can at no point be disengaged in Sf9 cells, the state of S6K1 activation could be deemed as constitutive and therefore, ideal to investigate the dynamics of activating phosphorylations in presence of rapamycin. Herein we provide evidence that activity or rapamycin sensitivity of Baculovirus recombinant enzyme (BVr) is not dependent on any posttranslational phosphorylation events in general and TOR- mediated phosphorylation in particular.
In the absence of the activating phosphorylations and lesser activity associated with the BVr-enzyme, it can be concluded that the viral infection per se does not activate the enzyme but instead appears to lock it in a state of activity comparable to a rather amplified basal (serum starved) state of the mammalian cells, in complete disagreement with the conclusion drawn by some investigators . The stimulus required to bring about activating phosphorylations may therefore, be either absent in insect cells or inactivated due to viral infection, undermining the existence of kinases responsible to bring about both AL and HM phosphorylations [13, 23]. It is therefore, obvious to contemplate that TOR signaling pathway that supposedly mediates both activation and rapamycin inhibition otherwise established in the insect cell system [13, 24], fails to phosphorylate and activate the enzyme yet continues to mediate inhibition by rapamycin. Therefore, it seems that while the process of activation remains unaccomplished, viral infection preserves the events responsible for mediating rapamycin inhibition. In other words the activation and sensitivity to rapamycin appear to be two independent events, in complete contravention to prevalent hypotheses. Baculoviral infection in a way serves to provide a better system where the basal state, i.e. the form of the enzyme without phosphorylations at the HM and AL, is completely disengaged from the activation state otherwise quite difficult to achieve in a mammalian system. Since, it would be strange to contemplate a unique mechanism of rapamycin action for BVr-enzyme it is possible to contemplate the loss of activating phosphorylations in mammalian cells otherwise construed as mechanistic may in effect be only the consequence of rapamycin inhibition. It is however, imperative to validate the contention for better understanding of the process.
We greatly appreciate the kind gift of HA-S6Kα1, (ΔNH2-46/ΔCT104) S6K α1 from Dr. Joseph Avruch Mass. General Hospital, Harvard Medical School, Boston-MA. Junior Research fellowships in favor of MAB (09/251/0025) and MS (20-6/2009) from Council of Scientific and Industrial Research (CSIR) and University Grants Commission (UGC) respectively are great fully acknowledged. Infrastructural Grant under FIST program and Special Assistance grant under SAP program, from DST, India and UGC, India is duly acknowledged.
- Fenton TR, Gout IT: Functions and regulation of the 70 kDa ribosomal S6 kinases. Int J Biochem Cell Biol. 2011, 43: 47-59. 10.1016/j.biocel.2010.09.018.View ArticlePubMedGoogle Scholar
- Weng QP, Andrabi K, Klippel A, Kozlowski MT, Williams LT, Avruch J: Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation. Proc Natl Acad Sci USA. 1995, 92: 5744-5748. 10.1073/pnas.92.12.5744.PubMed CentralView ArticlePubMedGoogle Scholar
- Mukhopadhyay NK, Price DJ, Kyriakis JM, Pelech S, Sanghera J, Avruch J: An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase. J Biol Chem. 1992, 267: 3325-3335.PubMedGoogle Scholar
- Frodin M, Antal TL, Dummler BA, Jensen CJ, Deak M, Gammeltoft S, Biondi RM: A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation. EMBO J. 2002, 21: 5396-5407. 10.1093/emboj/cdf551.PubMed CentralView ArticlePubMedGoogle Scholar
- Ferrari S, Bannwarth W, Morley SJ, Totty NF, Thomas G: Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs. Proc Natl Acad Sci USA. 1992, 89: 7282-7286. 10.1073/pnas.89.15.7282.PubMed CentralView ArticlePubMedGoogle Scholar
- Dennis PB, Pullen N, Pearson RB, Kozma SC, Thomas G: Phosphorylation sites in the autoinhibitory domain participate in p70(s6k) activation loop phosphorylation. J Biol Chem. 1998, 273: 14845-14852. 10.1074/jbc.273.24.14845.View ArticlePubMedGoogle Scholar
- Weng QP, Andrabi K, Kozlowski MT, Grove JR, Avruch J: Multiple independent inputs are required for activation of the p70 S6 kinase. Mol Cell Biol. 1995, 15: 2333-2340.PubMed CentralView ArticlePubMedGoogle Scholar
- Saitoh M, Pullen N, Brennan P, Cantrell D, Dennis PB, Thomas G: Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site. J Biol Chem. 2002, 277: 20104-20112. 10.1074/jbc.M201745200.View ArticlePubMedGoogle Scholar
- Price DJ, Grove JR, Calvo V, Avruch J, Bierer BE: Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science. 1992, 257: 973-977. 10.1126/science.1380182.View ArticlePubMedGoogle Scholar
- Peterson RT, Desai BN, Hardwick JS, Schreiber SL: Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein. Proc Natl Acad Sci USA. 1999, 96: 4438-4442. 10.1073/pnas.96.8.4438.PubMed CentralView ArticlePubMedGoogle Scholar
- Isotani S, Hara K, Tokunaga C, Inoue H, Avruch J, Yonezawa K: Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro. J Biol Chem. 1999, 274: 34493-34498. 10.1074/jbc.274.48.34493.View ArticlePubMedGoogle Scholar
- Kozma SC, McGlynn E, Siegmann M, Reinhard C, Ferrari S, Thomas G: Active baculovirus recombinant p70s6k and p85s6k produced as a function of the infectious response. J Biol Chem. 1993, 268: 7134-7138.PubMedGoogle Scholar
- Stewart MJ, Berry CO, Zilberman F, Thomas G, Kozma SC: The Drosophila p70s6k homolog exhibits conserved regulatory elements and rapamycin sensitivity. Proc Natl Acad Sci USA. 1996, 93: 10791-10796. 10.1073/pnas.93.20.10791.PubMed CentralView ArticlePubMedGoogle Scholar
- Watson KL, Chou MM, Blenis J, Gelbart WM, Erikson RL: A Drosophila gene structurally and functionally homologous to the mammalian 70-kDa s6 kinase gene. Proc Natl Acad Sci USA. 1996, 93: 13694-13698. 10.1073/pnas.93.24.13694.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim DH, Sarbassov DD, Ali SM, Latek RR, Guntur KV, Erdjument-Bromage H, Tempst P, Sabatini DM: GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol Cell. 2003, 11: 895-904. 10.1016/S1097-2765(03)00114-X.View ArticlePubMedGoogle Scholar
- Pullen N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, Hemmings BA, Thomas G: Phosphorylation and activation of p70s6k by PDK1. Science. 1998, 279: 707-710. 10.1126/science.279.5351.707.View ArticlePubMedGoogle Scholar
- Alessi DR, Kozlowski MT, Weng QP, Morrice N, Avruch J: 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr Biol. 1998, 8: 69-81. 10.1016/S0960-9822(98)70037-5.View ArticlePubMedGoogle Scholar
- Keshwani MM, von Daake S, Newton AC, Harris TK, Taylor SS: Hydrophobic Motif Phosphorylation Is Not Required for Activation Loop Phosphorylation of p70 Ribosomal Protein S6 Kinase 1 (S6K1). J Biol Chem. 2011, 286: 23552-23558. 10.1074/jbc.M111.258004.PubMed CentralView ArticlePubMedGoogle Scholar
- Nojima H, Tokunaga C, Eguchi S, Oshiro N, Hidayat S, Yoshino K, Hara K, Tanaka N, Avruch J, Yonezawa K: The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. J Biol Chem. 2003, 278: 15461-15464. 10.1074/jbc.C200665200.View ArticlePubMedGoogle Scholar
- Schalm SS, Tee AR, Blenis J: Characterization of a conserved C-terminal motif (RSPRR) in ribosomal protein S6 kinase 1 required for its mammalian target of rapamycin-dependent regulation. J Biol Chem. 2005, 280: 11101-11106. 10.1074/jbc.M413995200.View ArticlePubMedGoogle Scholar
- Schalm SS, Blenis J: Identification of a conserved motif required for mTOR signaling. Curr Biol. 2002, 12: 632-639. 10.1016/S0960-9822(02)00762-5.View ArticlePubMedGoogle Scholar
- Mahalingam M, Templeton DJ: Constitutive activation of S6 kinase by deletion of amino-terminal autoinhibitory and rapamycin sensitivity domains. Mol Cell Biol. 1996, 16: 405-413.PubMed CentralView ArticlePubMedGoogle Scholar
- Montagne J, Stewart MJ, Stocker H, Hafen E, Kozma SC, Thomas G: Drosophila S6 kinase: a regulator of cell size. Science. 1999, 285: 2126-2129. 10.1126/science.285.5436.2126.View ArticlePubMedGoogle Scholar
- Oldham S, Montagne J, Radimerski T, Thomas G, Hafen E: Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 2000, 14: 2689-2694. 10.1101/gad.845700.PubMed CentralView ArticlePubMedGoogle Scholar
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