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Mammalian TOR controls one of two kinase pathways acting upon nPKCdelta and nPKCepsilon
D Parekh1, W Ziegler, K Yonezawa
1Imperial Cancer Research Fund, Protein Phosphorylation Lab, 44 Lincoln's Inn Fields, London WC2A 3PX.
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 nPKCdelta 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 nPKCdelta 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 nPKCdelta. It is established that nPKCepsilon behaves in a manner similar to nPKCdelta with respect to phosphorylation at its C-terminal hydrophobic site, Ser-729. The results define the regulatory inputs to nPKCdelta and nPKCepsilon and establish these PKC isotypes downstream of mTOR and on an amino acid sensing pathway. The multiple signals integrated in PKC are discussed.
Insights
Novel protein kinase C (PKC) phosphorylation involves two independent pathways for nPKCdelta, regulated by mammalian target of rapamycin (mTOR) and amino acid availability, impacting enzyme activity.
Area of Science:
- Cellular signaling
- Molecular biology
- Enzymology
Background:
- Protein kinase C (PKC) isotypes have conserved phosphorylation sites crucial for their function.
- Novel PKC (nPKC) phosphorylation pathways require investigation to understand their regulatory mechanisms.
Purpose of the Study:
- To investigate the phosphorylation requirements and pathways for novel (nPKC) isotypes, specifically nPKCdelta and nPKCepsilon.
- To elucidate the role of mammalian target of rapamycin (mTOR) and amino acid sensing in nPKC regulation.
Main Methods:
- Investigated phosphorylation sites (Thr-505, Ser-662, Ser-643) in nPKCdelta using biochemical assays.
- Utilized co-expression of wild-type and rapamycin-resistant mTOR mutants to study pathway regulation.
- Examined the effect of amino acid deprivation on nPKCdelta phosphorylation.
Main Results:
- nPKCdelta exhibits two independent kinase pathways for activation loop (Thr-505) and hydrophobic motif (Ser-662) phosphorylation, with Thr-505 protecting Ser-662 from dephosphorylation.
- Phosphorylation of Thr-505 and Ser-662, requiring C1 domain activation, increases nPKCdelta activity up to 80-fold.
- Ser-662 phosphorylation is regulated by mTOR, and its phosphorylation is inhibited by amino acid deprivation, establishing nPKCdelta and nPKCepsilon downstream of mTOR.
Conclusions:
- nPKCdelta and nPKCepsilon are regulated by distinct phosphorylation events controlled by mTOR and amino acid availability.
- These findings define key regulatory inputs for nPKC isotypes, integrating multiple signaling pathways.
- PKC isotypes are positioned downstream of mTOR within an amino acid sensing pathway.