Abstract
Following the induction of apoptosis in mammalian cells, protein kinase C zeta (PKC zeta) is processed between the regulatory and catalytic domains by caspases, which increases its kinase activity. The catalytic domain fragments of PKC isoforms are considered to be constitutively active, because they lack the autoinhibitory amino-terminal regulatory domain, which includes a pseudosubstrate segment that plugs the active site. Phosphorylation of the activation loop at Thr(410) is known to be sufficient to activate the kinase function of full-length PKC zeta, apparently by inducing a conformational change, which displaces the amino-terminal pseudosubstrate segment from the active site. Amino acid substitutions for Thr(410) of the catalytic domain of PKC zeta (CAT zeta) essentially abolished the kinase function of ectopically expressed CAT zeta in mammalian cells. Similarly, substitution of Ala for a Phe of the docking motif for phosphoinositide-dependent kinase-1 prevented activation loop phosphorylation and abolished the kinase activity of CAT zeta. Treatment of purified CAT zeta with the catalytic subunit of protein phosphatase 1 decreased activation loop phosphorylation and kinase activity. Recombinant CAT zeta from bacteria lacked detectable kinase activity. Phosphoinositide-dependent kinase-1 phosphorylated the activation loop and activated recombinant CAT zeta from bacteria. Treatment of HeLa cells with fetal bovine serum markedly increased the phosphothreonine 410 content of CAT zeta and stimulated its kinase activity. These findings indicate that the catalytic domain of PKC zeta is intrinsically inactive and dependent on the transphosphorylation of the activation loop.
Highlights
Following the induction of apoptosis in mammalian cells, protein kinase C (PKC ) is processed between the regulatory and catalytic domains by caspases, which increases its kinase activity
Treatment of HeLa cells with fetal bovine serum markedly increased the phosphothreonine 410 content of CAT and stimulated its kinase activity. These findings indicate that the catalytic domain of PKC is intrinsically inactive and dependent on the transphosphorylation of the activation loop
We found that CAT , the free catalytic domain of PKC depends on phosphorylation of Thr410 for activity
Summary
Following the induction of apoptosis in mammalian cells, protein kinase C (PKC ) is processed between the regulatory and catalytic domains by caspases, which increases its kinase activity. Treatment of HeLa cells with fetal bovine serum markedly increased the phosphothreonine 410 content of CAT and stimulated its kinase activity These findings indicate that the catalytic domain of PKC is intrinsically inactive and dependent on the transphosphorylation of the activation loop. An acidic residue (Glu500 of PKC ␦) that immediately precedes the PDK-1 substrate motif is critical for the inherent catalytic activity of bacterial expressed nontransphosphorylated PKC ␦ and partially fulfills the role of activation loop phosphorylation of other. That the free catalytic domain of PKC is inherently inactive and dependent on transphosphorylation by PDK-1 for activation, in contrast to the kinase domain of PKC ␦
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