Mammalian TOR Controls One of Two Kinase Pathways Acting upon nPKCδ and nPKCε

Abstract

There are three conserved phosphorylation sites in protein kinase C (PKC) isotypes that have been termed priming sites and play an important role in PKC function. The requirements and pathways involved in novel (nPKC) phosphorylation have been investigated here. The evidence presented for nPKC␦ shows that there are two independent kinase pathways that act upon the activation loop (Thr-505) and a C-terminal hydrophobic site (Ser-662) and that the phosphorylation of the Ser-662 site is protected from dephosphorylation by the Thr-505 phosphorylation. Both phosphorylations require C1 domain-dependent allosteric activation of PKC. The third site (Ser-643) appears to be an autophosphorylation site. The serum-dependent phosphorylation of the Thr-505 and Ser-662 sites increases nPKC␦ activity up to 80-fold. Phosphorylation at the Ser-662 site is independently controlled by a pathway involving mammalian TOR (mTOR) because the rapamycin-induced block of its phosphorylation is overcome by co-expression of a rapamycin-resistant mutant of mTOR. Consistent with this role of mTOR, amino acid deprivation selectively inhibits the serum-induced phosphorylation of the Ser-662 site in nPKC␦. It is established that nPKC⑀ behaves in a manner similar to nPKC␦ with respect to phosphorylation at its C-terminal hydrophobic site, Ser-729. The results define the regulatory inputs to nPKC␦ and nPKC⑀ and establish these PKC isotypes downstream of mTOR and on an amino acid sensing pathway. The multiple signals integrated in PKC are discussed.

T Cell Effector Function and Anergy Avoidance Are Quantitatively Linked to Cell Division

“…PKC is normally coupled to the IL-2R through PI 3 K, as PKC is activated by PDK1-dependent phosphorylation (40), and a role for PKC in IL-2-mediated signal transduction has been defined using the IL-2-dependent cell line CTLL (59,60). Interestingly, full activation of the PKC-␦ isoform requires the activity of mTOR/FRAP (61), which is in turn dependent on PKB/Akt (62). These two distinct PI 3 K-dependent pathways leading to PKC activation, which can be bypassed using phorbol esters, represent a set of biochemical events that could potentially be defective in the undivided pool.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…This implies that other signalling pathways or autophosphorylation may regulate these isoforms at this site. Indeed, the TM of PKC has been reported to be an autophosphorylation site [54,66]. While deletion of mTORC2-specific components in MEFs appears to have no effect on TM phosphorylation or expression of PKC , this pathway regulates TM phosphorylation of PKC in T lymphocytes [61].…”

“…This theme developed because it was demonstrated that PKCs (predominantly cPKCs) are phosphorylated at these sites shortly after synthesis [83,42] and are often highly phosphorylated at these sites in many types of cells grown in culture, even under quiescent conditions [26,66,84]. In the absence of phosphorylation at one or more of these sites, catalytic activation of these isoforms is impaired or enzyme stability is compromised.…”

“…Interestingly, while the levels of expression of PKC are unaffected following deletion of mTORC2 components in MEFs [59,60], phosphorylation of PKC (and PKC * ) at the HM motif is sensitive to rapamycin treatment in human embryonic kidney cells, which therefore implicates the mTORC1 complex in phosphorylation of these isoforms [73,66]. It is clear therefore that mTORC1, mTORC2 and possibly other pathways impact on the HM of different…”

mTOR Signaling in Lymphangioleiomyomatosis

“…PDK1 can also directly phosphorylate the activation loop of other AGC kinases that undergo phosphorylation by mTOR at their hydrophobic motifs (e.g., PKCa, PKCd, S6K). 38,39,[76][77][78] Therefore, the PI3K=PDK1 pathway can activate AGC kinases independently of TSC2 or Rheb. The concurrent phosphorylation of AGC kinases by PDK1 and mTOR may be required for their maximal activation or stability.…”