Phosphatidylinositol-3-kinase regulates PKCθ activity in cytotoxic T cells

Protein kinase C (PKC) θ plays a crucial role in T cell activation. We, therefore, examined the regulation of PKCθ activity in cytotoxic T lymphocytes (CTL). We demonstrated that PMA did not stimulate PKCθ activation and phospholipase C inhibition did not block anti-CD3-stimulated PKCθ activation in a CTL clone. This suggests that diacylglycerol is neither sufficient nor required for PKCθ activation. Furthermore, PKCθ was only activated in a CTL clone stimulated with plate-bound anti-CD3 but not soluble anti-CD3. However, PMA or cross-linked anti-CD3 stimulated phosphorylation of PKCθ as measured by a migratory shift, suggesting that phosphorylation was not sufficient for activity. Phosphatidylinositol 3-kinase activity was required for anti-CD3, but not PMA, stimulated phosphorylation and for immobilized anti-CD3-triggered PKCθ activity. A substantial fraction of PKCθ was constitutively membrane associated and PMA or CD3 stimulation did not significantly increase membrane association. Our data indicate that phosphorylation of PKCθ is not a suitable surrogate measurement for PKCθ activity and that additional, yet to be defined steps, are required for the regulation of PKCθ enzymatic activity in CTL.