Simultaneous phosphorylation of three 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 (Vmax values = 0.3–0.5 nmol phosphate incorporated/min), with calpactin-II and calpactin-p70 exhibiting lower apparent Km values (40 and 30 nM, respectively) than did calpactin-I (Km, 200 nM). Because of the higher Vmax/Km 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 (≥ 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 (≤ 0.25 mM).Key words: calpactins, kinase C, lipocortins, phosphorylation.