Mechanism of phorbol ester activation of calcium-activated, phospholipid-dependent protein kinase

Tumor-promoting phorbol esters activated a calcium-activated, phospholipid-dependent protein kinase by direct compJexation with this protein kinase and phospholipid in the presence of divalent cations and this complexation was iden-tical to association of kinase/receptor activity with cell mem-branes. Treatment of isolated mouse spleen lymphocytes with phorbol ester tumor promoters resulted in a rapid shift in the subcelhilar localization of both the phorbol ester recep-tor and a calcium-activated, phospholipid-dependent protein kinase activity. Both activities shifted from almost entirely soluble to largely membrane-associated, which is consistent with a single protein possessing both activities. Activation of partially purified kinase/receptor activity by phorbol ester or calcium alone or in combination occurred in parallel to the formation of a complex between the kinase/receptor and phos-pholipid. Magnesium also was important both for complex formation and for activation of the protein kinase. Although phorbol ester did not appear to affect the affinity of the kinase/receptor for phospholipid, it did increase the extent of formation of a stable complex between the receptor and the phospholipid. These observations support the hypothesis that the cell membrane is the locus of action of both the phor-bol esters and the calcium-activated, phospholipid-dependent protein kinase activity