SIMULTANEOUS PHOSPHORYLATION OF 3 HUMAN CALPACTINS BY KINASE-C

We evaluated the concurrent phosphorylation of reconstituted mixtures of three purified human placental calpactins (or lipocortins) by purified bovine brain protein kinase C (PKC). Calpactin-I (p36 or lipocortin-II), calpactin-II (p38 or lipocortin-I), and a 70-kilodalton calpactin-related protein, calpactin-p70, when present together as substrates for PKC, all demonstrated comparable kinetic parameters (V(max) values = 0.3-0.5 nmol phosphate incorporated/min), with calpactin-II and calpactin-p70 exhibiting lower apparent K(m) values (40 and 30 nM, respectively) than did calpactin-I (K(m), 200 nM). Because of the higher V(max)/K(m) ratios for calpactin-II and calpactin-70 (12.5 and 10.0, respectively) compared with the ratio for calpactin-I (2.0), our data suggest that, intracellularly, where all three calpactins might be co-localized, the higher molecular mass calpactins could be preferred substrates for PKC. Nonetheless, the requirement for relatively high calcium concentrations (greater-than-or-equal-to 0.5 mM) suggests that PKC-mediated phosphorylation of the calpactins may take place only in restricted intracellular compartments, wherein calcium concentrations might transiently reach levels much higher than those that are normally found intracellularly (less-than-or-equal-to 0.25 mM).