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Phosphorylation of PKC activation loop plays an important role in receptor‐mediated translocation of PKC

Protein kinase C (PKC) is translocated to various cellular regions in a subtype and stimulation‐dependent manner. Thereafter, the activated PKC phosphorylates its substrate and causes subsequent cellular responses (PKC targeting). The 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1) has an essential role in the maturation of PKC by phosphorylating a threonine residue in the PKC activation loop. To elucidate the role of PDK1 in PKC targeting, we expressed mutant γ‐ or δ‐PKC fused with GFP (γ‐ or δ‐PKC‐ALM (activation loop mutant)‐GFP), whose threonine residue in the activation loop was replaced with alanine, and compared their P2Y receptor‐mediated translocation with wild‐type PKC‐GFP in CHO cells. ATP (1 mm) induced the transient translocation of wild‐type γ‐ or δ‐PKC‐GFP from cytoplasm to plasma membrane and following retranslocation from membrane to the cytoplasm. γ‐ or δ‐PKC‐ALM‐GFP was also translocated to plasma membrane, which was, however, retained at the membrane for a longer period than wild type. Similar results were observed in kinase‐negative PKC mutants, indicating that the phosphorylation by PDK1 affects the retranslocation step of PKC by regulating the kinase activity. The simultaneous monitoring of [Ca2+]i and diacylglycerol (DG) levels with the translocation of PKC demonstrated that PKC‐ALM induced the prolonged accumulation of DG, resulting in the prolonged retention of PKC‐ALM at the plasma membrane. It is possible that PKC‐ALM with decreased kinase activity could delay the conversion of DG at the plasma membrane. Our present study suggests that the activation loop phosphorylation plays an important role in receptor‐mediated PKC targeting.