Phosphoinositide-dependent in vitro phosphorylation of profilin by protein kinase C. Phospholipid specificity and localization of the phosphorylation site.
Phosphoinositides bind to profilin and regulate actin-based cytoskeletal protein assembly. We report here that profilin is phosphorylated in vitro by protein kinase C (PKC) in the presence of phosphoinositides and micromolar concentrations of calcium. PKC-mediated phosphorylation of profilin was observed only in the presence of phosphoinositides; phosphatidylserine and diacylglycerol (known activators of PKC) and other lipids, including phosphatidic acid and phosphatidylglycerol phosphate, did not activate the phosphorylation. The activation of PKC-mediated phosphorylation of profilin by phosphoinositides was as follows: phosphatidylinositol (PI) 4-phosphate (K(m) = 18 microM) > PI 4,5-bisphosphate (K(m) = 30 microM) > PI (no activation). About 0.5 mol phosphate was incorporated per mol of profilin. Phosphorylation of profilin by PKC was not affected by the presence of various concentrations of actin. Phospho-amino acid analysis showed serine to be the only amino acid phosphorylated. The amino acid sequence of a phosphopeptide from CNBr-digested profilin corresponded to the COOH-terminal peptide of profilin (Ala-Ser-His-Leu-Arg-Ser-Gln-Tyr). Further digestion of this phosphopeptide by trypsin generated two phosphopeptides (Arg-Ser-Gln-Tyr and Ser-Gln-Tyr), thereby confirming that the phosphorylation site was the antepenultimate Ser (Ala-Ser-His-Leu-Arg-Arg-Ser(P)-Gln-Tyr).
Singh
S S
SS
New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314, USA.
Chauhan
A
A
Murakami
N
N
Styles
J
J
Elzinga
M
M
Chauhan
V P
VP
D000199:Actins / Q000494:pharmacology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D003285:Contractile Proteins; D008081:Liposomes / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation / Q000187:drug effects; D051304:Profilins; D011493:Protein Kinase C / Q000378:metabolism*; D017421:Sequence Analysis; D013379:Substrate Specificity
D000199:Actins; D000596:Amino Acids; D003285:Contractile Proteins; D008081:Liposomes; D008840:Microfilament Proteins; D010716:Phosphatidylinositols; D010748:Phosphopeptides; D051304:Profilins; D011493:Protein Kinase C