Protein Kinase B/Akt Is Activated by Polyomavirus Middle-T Antigen Via a Phosphatidylinositol 3-Kinase-Dependent Mechanism
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Oncogene (1998) 16, 903 ± 907 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 Protein kinase B/Akt is activated by polyomavirus middle-T antigen via a phosphatidylinositol 3-kinase-dependent mechanism R Meili, P Cron, BA Hemmings and K Ballmer-Hofer1 Friedrich Miescher Institute, PO Box 2543, CH-4002 Basel, Switzerland The middle tumor antigen (middle-T) of mouse poly- threonine protein phosphatase 2A (PP2A) (Pallas et al., omavirus is responsible for the transforming potential of 1990; Walter et al., 1990), Src family tyrosine kinases this virus. Middle-T has been shown to interact with a (Courtneidge and Smith, 1983 and reviewed in Kiefer variety of cellular proteins known to mediate mitogenic et al., 1994), the adaptor protein SHC which binds to signaling, like phosphatase-2A, Src family kinases, phosphotyrosine 250 of middle-T via its phosphotyr- phosphatidylinositol 3-kinase (PI 3-kinase), the adapter osine binding (PTB) domain (Dilworth et al., 1994; protein SHC, phospholipase Cg-1 and 14-3-3 family Campbell et al., 1994), PI 3-kinase (Whitman et al., proteins. Association with SHC and PI 3-kinase, 1985; Talmage et al., 1989; Courtneidge et al., 1989) respectively, stimulates two independent signaling path- and phospholipase Cg-1 (Su et al., 1995). When ways that are indispensible for viral oncogenicity. SHC associated with middle-T, SHC gets phosphorylated activates the Ras/MAPK pathway via Grb2/SOS at tyrosine residues. This renders this protein resulting in changes in early gene expression. The competent to initiate downstream signaling through downstream targets of PI 3-kinase are less well studied Grb2 and SOS resulting in increased GTP loading of but seem to impinge on serum response factor (SRF) Ras. Activated Ras propagates and presumably which is also involved in regulating early gene expres- ampli®es incoming signals and initiates the MAP sion. Recently, the protein kinase B/Akt (PKB/Akt) has kinase cascade (Robinson and Cobb, 1997). Associa- been identi®ed as a target of PI 3-kinase in receptor tion of PI 3-kinase with tyrosine kinase receptors or tyrosine kinase signaling. Here we show that PKB/Akt is middle-T leads to activation of this enzyme, resulting a target of wild type middle-T, but not of mutants unable in increased production of D3 phosphatidylinositides. to activate PI 3-kinase. These data were con®rmed using These lipids have been implicated in the regulation of a inhibitors of PI 3-kinase as well as dominant-negative plethora of cellular phenomena such as cell growth, alleles of the catalytic subunit of this lipid kinase. In rearrangement of cytoskeletal structures, cellular addition, mutants of PKB/Akt lacking a pleckstrin motility, receptor tracking, cell survival and chemo- homology domain and therefore unable to bind to D3 taxis (reviewed in Toker and Cantley, 1997). Recent phospatidylinositides were not activated by middle-T. work shows that these lipids undergo speci®c interac- Taken together these data suggest that middle-T tions with target proteins having SH2- (Rameh et al., activates PKB/Akt in a PI 3-kinase-dependent manner. 1995) or PH-domains (Klarlund et al., 1997; Franke et Furthermore, direct association with D3 phosphatidyli- al., 1997). Protein kinase B/Akt (PKB/Akt), also nositides seems to be essential for activation of PKB/ known as RAC-PK, is one of these targets and is Akt. activated in response to growth stimulation (Franke et al., 1995). The phospholipid products of PI 3-kinase Keywords: middle-T; PI 3-kinase; protein kinase B/Akt; bind to the PH-domain of PKB/Akt and recruit this polyomavirus protein to the plasma membrane. This leads to activation upon phosphorylation by a not yet identi®ed cellular kinase, presumably also located at cellular membranes (Alessi et al., 1996). It has also Introduction been shown that PI 3-kinase is required for full activation of the Ras/MAPK pathway by middle-T as Mouse polyomavirus induces a broad spectrum of well as for signaling to serum response factor (Urich et tumors in mice and transforms cells in culture al., 1997). Mutants of middle-T still capable of (Treisman et al., 1981; Rassoulzadegan et al., 1982). activating PI 3-kinase, but lacking a binding site for The viral proteins mediating this function are the SHC, trigger stimulation of p70S6-kinase (Dahl et al., tumor antigens, in particular the middle-T antigen. 1996) or p21Rac-mediated transcriptional regulation Oncogenic transformation by polyomavirus has been (Urich et al., 1997). used as a paradigm for the study of mitogenic signaling in mammalian cells since this protein activates many of the cellular pathways also targeted by tyrosine kinase Results growth factor receptors. Signaling by middle-T requires interactions with cellular proteins such as the serine/ Middle-T activates PKB/Akt To determine the eect of middle-T on PKB/Akt Correspondence: K Ballmer-Hofer activity, a HA-tagged version of this kinase was 1Current address: Institute for Medical Radiobiology, CH-5232 Villigen-PSI, Switzerland transiently expressed together with middle-T in Received 7 May 1997; revised 29 September 1997; accepted 29 NIH3T3 cells. Kinase activity was measured in September 1997 immunecomplex assays using histone 2B as substrate. PKB/Akt activation by middle-T RMeiliet al 904 In the absence of serum, wild type middle-T activated reverted middle-T- as well as PDGF-induced PKB/ PKB/Akt about 20-fold (Figure 1a, compare lanes 1 Akt activation (Figure 3, compare lane 2 with 5 and 3 and 2) whereas in the presence of serum activation was with 6). Similarly, coexpression of an enzymatically only twofold (Figure 1a, compare lanes 6 and 7) inactive mutant of the catalytic p110 subunit of PI 3- indicating that PKB/Akt is maintained at high kinase blocked PKB/Akt activation by middle-T enzymatic activity by serum growth factors. (Figure 3, lane 9). A constitutively active form of the p110 subunit, on the other hand, slightly increased PKB/Akt activity (Figure 3, lane 8). Taken together PI 3-kinase but not SHC is required for PKB/Akt these data indicate that middle-T-stimulated activation activation by middle-T of PI 3-kinase results in activation of PKB/Akt. Mutants of middle-T were used to further de®ne the requirements for PKB/Akt activation. The mutant The PH-domain of PKB/Akt is required for activation Y315F, which does not bind p85 and therefore cannot by middle-T activate PI 3-kinase, was unable to activate PKB/Akt (Figure 1a, lanes 5 and 9 and 1b, bar 4). The mutant To investigate whether the phosphoinositides produced Y250F, unable to associate with SHC, activated PKB/ upon activation of PI 3-kinase by middle-T are Akt almost to the level of wild type middle-T (Figure required for PKB/Akt activation, we studied the 1a, lanes 4 and 8 and 1b, bar 3). The fact that this activity of a mutant of PKB/Akt, DPH-PKB/Akt, mutant was slightly less potent at activating PKB/Akt lacking a pleckstrin homology domain and therefore might be explained by the observation that it binds less unable to bind D3 phosphoinositides (Franke et al., p85 as shown earlier (Dilworth et al., 1994) (Figure 2, 1997; Alessi et al., 1996). This mutant was not compare lanes 2 and 3). Finally, the mutant dl1015 activated by middle-T (Figure 4, lanes 3 and 4). The which binds p85 (Figure 2, lane 4), but fails to activate fact that DPH-PKB/Akt was almost fully activated by PI 3-kinase in vivo (Ling et al., 1992), showed that the nonspeci®c tyrosine phosphatase inhibitor vanadyl interaction of PI 3-kinase with middle-T per se is not hydroperoxide as previously described (Andjelkovic et sucient for PKB/Akt activation (Figure 1b, bar 5). al., 1996), con®rms that the catalytic center of the Additional experiments using the PI 3-kinase kinase domain was unaected by the deletion (Figure inhibitor LY294002 were performed to demonstrate 4, lane 5). From these data we conclude that middle-T that lipid kinase activity is essential for PKB/Akt signaling to PKB/Akt is mediated by binding of D3 activation. The data show that LY294002 rapidly phosphoinositides to the PH-domain of PKB/Akt. a 0.1% CS 14h 10% CS b Relative activation of HA PKB — mT wt mT Y250F mT Y315F mT dl1015 — 20% CS mT wt mT Y250F mT Y315F HA PKB H2B Figure 1 Activation of PKB/Akt by middle-T. (a) Cells were transfected as described and serum deprived (lanes 1 ± 5) for 14 h. Cells in lane 2 were treated with 20% calf serum for 30 min prior to cell lysis. Cells were lysed and HA-tagged PKB/Akt was immunoprecipitated with the monoclonal antibody 12CA5. The immunoprecipitates were assayed for kinase activity using histone 2B as substrate and analysed by SDS ± PAGE (bottom panel). To determine the amount of kinase in the immunoprecipitates, a Western blot was decorated with the anti-HA- tag antibody (top panel). The bar graph at the bottom shows relative kinase activities of one representative experiment. (b) Shows the mean relative activation of PKB in serum-deprived NIH3T3 cells by various middle-T mutants calculated from ®ve separate experiments (except for dl1015: n=2). Activation by wild type middle-T was taken as 100% PKB/Akt activation by middle-T RMeiliet al 905 Anti-middle-T immunoprecipitates Blot HA PKB + mT wt + mT Y250F + mT dl1015 + mT Y315F HA PKB + mT wt + mT Y250F + mT dl1015 + mT Y315F a p85 p85 a mT Src mT p43 a p36 p36 1 2 3 4 5 1 2 3 4 5 Westernblot in vitro kinase Figure 2 Analysis of middle-T associated proteins.