Phosphorylation sites in members of the protein kinase A (PKA), PKG, and PKC kinase subfamily are conserved. Thus, the PKB kinase PDK1 may be responsible for the phosphorylation of PKC isotypes. PDK1 phosphorylated the activation loop sites of PKCzeta and PKCdelta in vitro and in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner in vivo in human embryonic kidney (293) cells. All members of the PKC family tested formed complexes with PDK1. PDK1-dependent phosphorylation of PKCdelta in vitro was stimulated by combined PKC and PDK1 activators. The activation loop phosphorylation of PKCdelta in response to serum stimulation of cells was PI 3-kinase-dependent and was enhanced by PDK1 coexpression.

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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.

PKCzeta is identified as a component of the upstream kinase responsible for the phosphorylation of the PKCdelta hydrophobic site in vitro and in vivo. PKCzeta can therefore control the phosphorylation of this PKCdelta site, antagonizing a rapamycin-sensitive pathway.

Phosphorylation of protein kinase C (PKC) provides an amplitude control that operates in conjunction with allosteric effectors. Under many conditions, PKC isotypes appear to be highly phosphorylated; however, the cellular inputs that maintain these phosphorylations are not characterized. In the present work, it is shown that there is a differential phosphorylation of PKCdelta in adherent versus suspension cultures of transfected HEK-293 cells. It is established that integrin activation is sufficient to trigger PKCdelta phosphorylation and that this signals through phosphoinositide 3-kinase (PI3-kinase) to stimulate the phosphorylation of two sites, T505 and S662. The loss of signal input to PKCdelta in suspension culture is dependent on the tumour suppressor gene PTEN, which encodes a bi-functional phosphotyrosine/phosphoinositide 3-phosphate phosphatase. In the PTEN(-/-) UM-UC-3 bladder carcinoma cell line grown in suspension, transfected PKCdelta no longer accumulates in a dephospho-form on serum removal. By contrast, in a UM-UC-3-derivative cell line stably expressing PTEN, PKCdelta does become dephosphorylated under these conditions. Employing the PTEN Gly(129)-->Glu mutant, which is selectively defective in lipid phosphatase activity, it was established that it is the lipid phosphatase activity that controls PKCdelta phosphorylation. The evidence indicates that PKCdelta phosphorylation and its latent activity are maintained in serum-deprived adherent cultures through integrin-matrix interactions. This control acts through a pathway involving a lipid product of PI3-kinase in a manner that can be suppressed by PTEN.