Sin1 but not PDK1 is required for PKCα and Akt TM phosphorylation. (A) Phosphorylation of the TM and HM in PKCα and Akt, but not PKCδ and S6K, is eliminated in Sin1−/− MEF. Sin1+/+ or −/− MEF cells were cultured in normal medium (medium), serum-free medium for 12 h (starved) or restimulated with serum for 30 min (serum). Immunoblottings were performed using indicated antibodies. (B) Reintroduction of Sin1 in Sin1−/− MEF restores phosphorylation. Sin1−/− MEFs were infected with either empty vector or HA-Sin1-containing retroviruses. The cells were treated with or without 400 nM insulin for 30 min. (C) PDK1 is required for PKCα HM but not TM phosphorylation. Immunoblotting of lysates from PDK1+/+ and −/− ES cells was performed for PKCα and Akt phosphorylation. The cells were serum-starved for 3 h and then stimulated with or without 100 nM IGF for 30 min in the presence or absence of pretreatment with 100 nM wortmannin (W) for 30 min.

Effect of Rictor and Sin1 deletion on PKC isoforms. (A) Protein levels of PKCα and ɛ but not PKCδ and λ are decreased in Rictor or Sin1−/− MEFs. Expression levels of endogenous PKCs were monitored by immunoblotting. (B) Expression levels of transfected PKC isoforms in Rictor+/+ and −/− MEFs. The Rictor+/+ and −/− MEFs were transfected with HA–PKC isoforms as indicated. Expression levels and mobility of the transfected PKCs were monitored by HA antibody.

PKCα is not directly phosphorylated by mTORC2 in vitro but associated with Rictor. (A–C) Immunoprecipitated mTORC2 fails to phosphorylate PKCα in vitro. Endogenous proteins from HeLa cells were immunoprecipitated with control, mTOR or Rictor antibody. The immunoprecipitates were used to phosphorylate GST–PKCα (CT) purified from E. coli and His–Akt (FL: full-length) from baculovirus (A), GST–Akt (CT) from E. coli (B) or GST–Akt and GST–PKCα from HeLa cells (C), which had mTOR knockdown and rapamycin treatment. FL and CT denotes full-length and C-terminal fragment (a.a. 125–480 for Akt and a.a. 322–672 for PKCα), respectively. ^**Denotes the non-specific signal detected by phospho-Akt HM antibody. (D) Rictor preferentially interacts with hypophosphorylated PKCα. HEK293 cells were transfected with HA–PKCα and Myc–Rictor, Myc–Sin1 or empty vector as indicated. Cell lysates were immunoprecipitated with Myc antibody, and then co-immunoprecipitated HA–PKCα was determined by immunoblotting. The transfected PKCα migrates as doublet. The slow-migrating PKCα (phosphorylated form) and fast-migrating PKCα (hypophosphorylated form) are denoted by two asterisks and a dash, respectively. The faster migrating HA–PKCα was co-immunoprecipitated with Myc–Rictor (SE denotes short exposure). Longer exposure (LE) shows that a small amount of PKCα was co-immunoprecipitated with Myc–Sin1. Phosphorylation of the co-immunoprecipitated HA–PKCα was determined with indicated phospho-PKCα antibodies (right panels).

PKCα functions are compromised in Rictor and Sin1−/− cells. (A) Phosphorylation of PKC substrates induced by thymeleatoxin (TX) is diminished in Rictor−/− MEFs. Rictor+/+ and −/− MEFs were treated with 100 nM thymeleatoxin (an activator for cPKC and nPKC). The presence of 1 μM bisindolylmaleimide I (BIM, pan PKC inhibitor) is indicated. Total cell lysates were prepared and immunoblotting was performed with indicated antibodies. (B) PMA-induced MARCKS phosphorylation is compromised in Rictor−/− cells. Cells were treated with 400 nM PMA. Immunoblottings were performed with indicated antibodies. (C) Effect of PMA on PKCα translocation in Rictor+/+ and −/− MEFs. Cells were treated with 400 nM PMA for 30 min. Subcellular fractionation was performed. PKCα protein was detected by immunoblotting. (D) Mutation of TM and HM in PKCα abolishes kinase activity. HeLa cells were transfected with PKCα WT, kinase inactive (K368R) and TM and HM mutants (T638A/S657A). HA–PKCα was immunoprecipitated and kinase activity was measured using a synthetic peptide as a substrate and radioactive ATP. (E) PKCα expressed in Sin1−/− MEFs is inactive. Sin1+/+ and −/− MEFs were transfected with various concentrations of HA–PKCα. HA–PKCα was immunoprecipitated and kinase activity was measured similar to Figure 7D.

Rictor is required for TM and HM phosphorylation of PKCα and Akt. (A) Deletion of Rictor gene decreases PKCα protein levels and phosphorylation. E10.5 embryos were lysed and used for immunoblot analysis. Immunoblotting was performed for PKCα and S6K phosphorylation states and mTOR complex components in lysates prepared from individual Rictor +/+, +/− and −/− embryos. (B) Phosphorylation of PKCα TM is eliminated in Rictor−/− embryos. Endogenous PKCα proteins were immunoprecipitated and a similar protein level was loaded. Phosphorylation of A-loop, TM and HM was determined by immunoblotting. (C) A-loop, TM and HM phosphorylation of transfected PKCα. HA–PKCα plasmid was transfected into Rictor+/+ or −/− MEF and HA–PKCα protein was immunoprecipitated and probed for phosphorylation. (D) Deletion of Rictor gene eliminates Akt TM and HM phosphorylation. E10.5 embryos were analysed for Akt phosphorylation.

Relationship among different PKCα phosphorylation sites. (A) Kinase domain is not required for PKCα TM phosphorylation. HEK293T cells were transfected with indicated GST–PKCα full length or C-terminal fragment (CT, a.a. 603–672). Cell lysates were probed with antibody for GST, TM and HM as indicated. (B) Phosphorylation status of PKCα mutants. Transfected HA-tagged PKCα (in HEK293T) were immunoprecipitated with HA-antibody. Phosphorylation was determined by immunoblotting with indicated antibodies. K368R and T497A denote PKCα kinase inactive and A-loop mutant, respectively. (C) Relationship between PKCα TM and HM phosphorylation. HEK293T cells were transfected with PKCα wild type (WT), TM mutant (T638A) and HM mutant (S657A). Phosphorylation of each PKCα construct was determined.

mTORC2 integrity and mTOR kinase activity are required for PKCα and Akt TM phosphorylation. (A) Prolonged treatment of rapamycin decreases both TM and HM phosphorylation in PKCα and Akt. HepG2 cells were treated with 100 nM rapamycin for indicated times. Total cell lysates were probed with PKC and Akt antibodies as indicated. Rictor or mTOR immunoprecipitates were analysed by immunoblotting with antibodies against TORC2 components. (B) The PI3K/mTOR inhibitor P103 inhibits phosphorylation of transfected PKCα and Akt. HEK293T cells were transfected with GST–PKCα and GST–Akt in the presence or absence of 10 μM PI-103. Immunoblottings were performed using indicated antibodies. (C) mTOR and Rictor are required for maintaining both phosphorylation and protein level of PKCα. HeLa cells stably expressing control, mTOR shRNA or Rictor shRNA were transfected with control, mTOR or Rictor siRNA oligos, respectively. (D) Knockdown of mTOR combined with prolonged rapamycin treatment abolishes TM phosphorylation. HeLa cells with mTOR knockdown using shRNA-containing lentivirus were pretreated with 100 nM rapamycin for 24 h. mTOR siRNA oligos together with GST–PKCα, GST–Akt, and siRNA-resistant Myc-mTOR were transfected as indicated under rapamycin treatment. (E) Kinase activity of mTOR is required for PKCα TM phosphorylation. Experiments were similar to Figure 5D except that the siRNA-resistant kinase dead (KD) mTOR was included. Protein levels and phosphorylation of both the transfected GST–PKCα and the endogenous PKCα were determined.

TM phosphorylation protects PKCα from proteasome-dependent degradation. (A) The residual PKCα in Sin−/− MEF cells is stable. Cells were treated with 100 μg/ml cycloheximide (CHX) for the indicated time. PKCα protein levels were determined. (B) Inhibition of Hsp90 destabilizes PKCα and PKCɛ in Rictor−/− but not in +/+ MEFs. Cells were treated with 20 μM radicicol, an Hsp90 inhibitor, for the indicated time, and PKC protein levels were monitored by immunoblotting. (C) Mutation of the TM phosphorylation site destabilizes PKCα in the presence of Hsp90 inhibition. HEK293 cells transfected with HA–PKCα wild type, TM mutant (T638A) or HM mutant (S657A) were treated with 20 μM radicicol for the indicated time. The protein levels of HA–PKCα were monitored by immunoblotting. (D) Inhibition of proteasome accumulates insoluble PKCα in the Rictor−/− but not +/+ cells. Cells were treated with 10 μM MG132 for 12 h, lysed in NP-40 buffer and then fractionated into soluble (S) and insoluble fractions (P). (E) Mutation of the PKCα TM phosphorylation site enhances ubiquitination. HEK293 cells were transfected with HA–PKCα wild type (WT), TM mutant (T638A), TM and compensation site mutant (T631A/T638A), HM mutant (S657A) or TM and HM mutant (T638A/S657A) together with FLAG–Ub. The transfected cells were treated with or without 10 μM MG132 for 12 h as indicated. Soluble fractions were immunoprecipitated with anti-HA, and ubiquitinated PKCα was determined by immunoblotting using anti-FLAG antibody. (F) The TM mutant PKCα is more prone to be in the insoluble fraction. HEK293 cells were transfected with HA–PKCα WT and TM mutants and treated with 10 μM MG132 for 12 h as described in Figure 6D. The levels of HA–PKCα in both soluble and insoluble fractions were determined by immunoblotting using anti-HA antibody. P and S denote insoluble pellet and soluble fractions, respectively. (G) PKCα TM mutant, but not the wild type, accumulates in aggresome when proteasome is inhibited. HeLa cells transfected with wild-type or mutant HA–PKCα (T631A/T638A) were treated with 5 μM MG132 for 24 h. Double immunofluorescent staining was performed using anti-HA antibody for HA–PKCα (red colour) and anti-Ub antibody for conjugated ubiquitin (green colour).