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 (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).
Barnes
J A
JA
Department of Pharmacology and Therapeutics, University of Calgary, Faculty of Medicine, Alta., Canada.
Michiel
D
D
Hollenberg
M D
MD
D017302:Annexins; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002451:Cell Compartmentation; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D010766:Phosphorylation; D011257:Pregnancy Proteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology
D017302:Annexins; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D010710:Phosphates; D010718:Phosphatidylserines; D010743:Phospholipids; D011257:Pregnancy Proteins; D012964:Sodium; D011493:Protein Kinase C; D002118:Calcium