Regulatory Autophosphorylation Sites on Protein Kinase C-δ at Threonine-141 and Threonine-295

Protein kinase C-delta (PKC delta) is a Ser/Thr kinase that regulates a wide range of cellular responses. This study identifies novel in vitro PKC delta autophosphorylation sites at Thr(141) adjacent to the pseudosubstrate domain, Thr(218) in the C1A-C1B interdomain, Ser(295), Ser(302), and Ser(304) in the hinge region, and Ser(503) adjacent to Thr(505) in the activation loop. Cell-based studies show that Thr(141) and Thr(295) also are phosphorylated in vivo and that Thr(141) phosphorylation regulates the kinetics of PKC delta downregulation in COS7 cells. In vitro studies implicate Thr(141) and Thr(295) autophosphorylation as modifications that regulate PKC delta activity. A T141D substitution markedly increases basal lipid-independent PKC delta activity; the PKC delta-T141D mutant is only slightly further stimulated in vitro by PMA treatment, suggesting that Thr(141) phosphorylation relieves autoinhibitory constraints that limit PKC delta activity. Mutagenesis studies also indicate that a phosphorylation at Thr(295) contributes to the control of PKC delta substrate specificity. We previously demonstrated that PKC delta phosphorylates the myofilament protein cardiac troponin I (cTnI) at Ser(23)/Ser(24) when it is allosterically activated by lipid cofactors and that the Thr(505)/Tyr(311)-phosphorylated form of PKC delta (that is present in assays with Src) acquires as additional activity toward cTnI-Thr(144). Studies reported herein show that a T505A substitution reduces PKC delta-Thr(295) autophosphorylation and that a T295A substitution leads to a defect in Src-dependent PKC delta-Tyr(311) phosphorylation and PKC delta-dependent cTnI-Thr(144) phosphorylation. These results implicate PKC delta-Thr(295) autophosphorylation as a lipid-dependent modification that links PKC delta-Thr(505) phosphorylation to Si-c-dependent regulation of PKC delta catalytic function. Collectively, these studies identify novel regulatory autophosphorylations on PKC delta that serve as markers and regulators of PKC delta activity.