Selective phosphorylation of cationic polypeptide aggregated with phosphatidylserine/diacylglycerol/Ca2+/detergent mixed micelles by Ca(2+)-independent but not Ca(2+)-dependent protein kinase C isozymes.
Mixed micelles containing Nonidet P40 (NP-40) (829 microM or 4.8 mM), phosphatidylserine (PS) (14.5 or 8 mol%), and 1,2-diacylglycerol (DG) (0.5 or 1 mol%) when preincubated with protein kinase C (PKC) assay mixture containing cationic substrate and CaCl2 (400 microM) formed aggregates in a time-, temperature-, and substrate concentration-dependent manner with a t1/2 approximately 3-12 min (22 degrees C). Concomitant with the formation of these aggregates there was a substantial loss of substrate phosphorylation catalyzed by the Ca(2+)-dependent PKC alpha, beta, and gamma but not the Ca(2+)-independent PKC, delta and epsilon. All cationic PKC substrates tested, neurogranin peptide analog, neurogranin, and histone III-S, formed aggregates with PS/DG/NP-40/Ca2+ mixed micelles in a time-dependent fashion. The poly(cationic-anionic) PKC substrate protamine sulfate also forms aggregates with the mixed micelles in the presence of Ca2+, but without affecting the substrate phosphorylation by the kinase. Under similar conditions, but at 4 degrees C, neither aggregation nor loss of cationic substrate phosphorylation was observed. Another nonionic detergent, octyl glucoside, behaved similarly to NP-40. Phosphatidylinositol (PI) and phosphatidylglycerol like PS, were effective in forming aggregates with NP-40/cationic polypeptide/DG/Ca2+ as monitored by light scattering, yet without affecting substrate phosphorylation. Phosphorylation of cationic substrates by M-kinase, derived from trypsinized PKC beta, was also greatly diminished by the aggregation. In contrast, [3H]phorbol 12,13-dibutyrate binding to PKC beta was unaffected. Formation of the aggregates that were selectively utilized by the Ca(2+)-independent PKCs was dependent on the ratio of cationic substrate to the number of mixed micelles.(ABSTRACT TRUNCATED AT 250 WORDS)
Mahoney
C W
CW
Section on Metabolic Regulation, National Institutes of Health, Bethesda, Maryland 20892-4510.
Huang
K P
KP
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium; D002148:Calmodulin-Binding Proteins / Q000235:genetics; D002417:Cattle; D003902:Detergents; D004075:Diglycerides; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D008823:Micelles; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D051579:Neurogranin; D017830:Octoxynol; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010718:Phosphatidylserines; D010766:Phosphorylation; D011092:Polyethylene Glycols; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats
D002148:Calmodulin-Binding Proteins; D003902:Detergents; D004075:Diglycerides; D007527:Isoenzymes; D008823:Micelles; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins; C496651:Nrgn protein, rat; D010446:Peptide Fragments; D010455:Peptides; D010718:Phosphatidylserines; D051579:Neurogranin; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D011493:Protein Kinase C; D002118:Calcium