[PubMed] [Google Scholar] 77

[PubMed] [Google Scholar] 77. NIHMS1708431-supplement-Figure_S9.pdf (47M) GUID:?2907090E-AEAF-47F3-9DA5-0787B41C6FD5 Figure S10: Fig. S10. TOR-Interaction Motif Mutations Affect Kinase Conformations NIHMS1708431-supplement-Figure_S10.pdf (2.9M) GUID:?B462C992-00D7-41E6-AAE6-E4472151F6A4 Number S11: Fig. S11. Regulatory Domains Attenuate PKC Dimerization NIHMS1708431-supplement-Figure_S11.pdf (4.1M) GUID:?A3148E5D-356B-46B3-AA33-CC7F3C02CC8B Supplementary Text. NIHMS1708431-supplement-Supplementary_Text.docx (38M) GUID:?C8660DD7-65B6-49BB-AB57-9268FA1A677E Abstract The kinase complex mTORC2 is definitely widely approved as controlling phosphorylation of Ginsenoside F2 the hydrophobic motif, a key regulatory switch in the C-terminal tail of protein kinase C Ginsenoside F2 (PKC), Akt, and additional AGC kinases. Yet the biochemical mechanism by which it controls this site and whether mTOR is the direct hydrophobic motif kinase remain controversial. Here we determine a distinct mTOR-mediated phosphorylation site we term the TOR-Interaction Motif (TIM; F-x3-F-pT), which settings hydrophobic motif phosphorylation and activity of PKC and Akt. The TIM is definitely invariant in all mTOR-dependent Ginsenoside F2 kinases, is evolutionarily conserved, and co-evolved with mTORC2 parts. Mutation of this motif only in Akt1 (Thr443) or together with the change motif in PKCII (Thr634/Thr641) abolishes cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylates the PKC TIM by PDK1 phosphorylation of the activation loop (6, 32). Third, genuine PKC selectively dephosphorylated in the hydrophobic motif re-autophosphorylates at this site by an intramolecular mechanism (6). Additionally, we have previously reported that hydrophobic motif phosphorylation of Akt, but not kinase-inactive Akt, is definitely induced in the absence of mTORC2 by any transmission that disengages the PH website of Akt from its kinase website, as happens upon binding to the plasma membrane (33). These results suggest that phosphate incorporation in the hydrophobic motif is definitely governed by a kinase-intrinsic mechanism. The related AGC kinases SGK, PKN, and RSK also depend upon mTORC2, (34-37), which is definitely evolutionarily conserved in candida (38). Hydrophobic motif phosphorylation of the AGC kinase member S6K depends, instead, upon mTORC1 (39-41). Contributing to the puzzle of understanding mTORC2 rules, three novel PKCs (, , and ) are mTOR-independent and don’t require mTOR kinase activity for phosphorylation (13). Therefore, in the absence of a mechanism for hydrophobic motif phosphorylation, the function of mTORC2 in regulating AGC kinases remains unclear. Here we identify a distinct AGC kinase phosphorylation site controlled by mTORC2, which we term the TOR-Interaction Motif (TIM), that settings the activation of PKC and Akt by facilitating PDK1 phosphorylation of the activation loop. We find that phosphorylation of the PKC and Akt hydrophobic motif is definitely governed by autophosphorylation in an mTORC2- and PDK1-dependent manner. We detect phosphorylation of the TGFB2 PKCII TOR-interaction motif (Thr634) by mass spectrometry in mouse mind and display that mTORC2 phosphorylates Ginsenoside F2 the TIM site +PKC) compared with WT MEFs (+PKC) (Fig. 1H). Reconstitution of Sin1 KO MEFs with Sin1 (+PKC+Sin1) slowed the translocation rate to that observed in WT MEFs. The enhanced rate of PKC translocation in mTORC2-deficient cells was attributable to an revealed C1A domain: impairing ligand binding of the domain by intro of a W58A mutation (53) reduced the pace of PKCII translocation in Rictor KO MEFs (+PKC-W58A) (Fig. S1F). Furthermore, PKC with phosphomimetic Glu substitutions in the change and hydrophobic motif phospho-acceptor sites (T641E/S660E) translocated rapidly in Sin1 KO MEFs (+PKC-EE), indicative of the unprimed conformation of PKC. This suggests that mTORC2 regulates PKC conformation by a mechanism self-employed of phosphorylation in the C-tail change motif and hydrophobic motif Ginsenoside F2 sites (Fig. 1H). This quick translocation was not a result of the phosphomimetics not permitting the primed, autoinhibited conformation because this same construct translocated slowly in WT MEFs, indicating normal autoinhibition. Therefore, mTORC2 promotes the autoinhibited and primed conformation of PKC by a mechanism that is self-employed of phosphorylation of the known C-tail sites. We next tackled whether mTOR activity regulates the initial phosphorylation of newly-synthesized PKC or the steady-state phosphorylation of adult PKC. The stoichiometry of PKC phosphorylation can be assessed by monitoring the electrophoretic mobility shift that accompanies phosphorylation of the two C-terminal sites (7) :the slower mobility species (asterisk, top band) is definitely phosphorylated at both the change motif and hydrophobic motif sites and the faster mobility varieties (dash, lower band) is definitely unphosphorylated. Using electrophoretic mobility to assess the phosphorylation state of PKC, we observed that Torin treatment of WT MEFs overexpressing PKCII resulted in a relatively sluggish build up of unphosphorylated PKCII (faster electrophoretic mobility). This sluggish rate of appearance of unphosphorylated PKC contrasted with the rapid loss of phosphate within the mTORC1-regulated S6K1.