Cleavage Alters the Molecular Determinants of Protein Kinase C-δ Catalytic Activity

Protein kinase C-δ (PKCδ) is an allosterically activated enzyme that acts much like other PKC isoforms to transduce growth factor-dependent signaling responses. However, PKCδ is unique in that activation loop (Thr⁵⁰⁷) phosphorylation is not required for catalytic activity. Since PKCδ can be proteolytically cleaved by caspase-3 during apoptosis, the prevailing assumption has been that the kinase domain fragment (δKD) freed from autoinhibitory constraints imposed by the regulatory domain is catalytically competent and that Thr⁵⁰⁷ phosphorylation is not required for δKD activity. This study provides a counternarrative showing that δKD activity is regulated through Thr⁵⁰⁷ phosphorylation. We show that Thr⁵⁰⁷-phosphorylated δKD is catalytically active and not phosphorylated at Ser³⁵⁹ in its ATP-positioning G-loop. In contrast, a δKD fragment that is not phosphorylated at Thr⁵⁰⁷ (which accumulates in doxorubicin-treated cardiomyocytes) displays decreased C-terminal tail priming-site phosphorylation, increased G-loop Ser³⁵⁹ phosphorylation, and defective kinase activity. δKD is not a substrate for Src, but Src phosphorylates δKD-T507A at Tyr³³⁴ (in the newly exposed δKD N terminus), and this (or an S359A substitution) rescues δKD-T507A catalytic activity. These results expose a unique role for δKD-Thr⁵⁰⁷ phosphorylation (that does not apply to full-length PKCδ) in structurally organizing diverse elements within the enzyme that critically regulate catalytic activity.