Phosphorylation of profilin regulates its interaction with actin and poly ( l-proline)

Abstract

Activation of bovine platelets with thrombin and phorbol 12,13-dibutyrate (PDBu) resulted in phosphorylation of profilin on serine. The phosphorylation was inhibited when platelets were pretreated with the PI 3-kinase inhibitor, LY294002, indicating that profilin phosphorylation is a downstream event with respect to PI 3-kinase activation. Phosphorylation of profilin resulted in significant decrease in actin polymerization (16.5%), indicating an increased affinity of phosphoprofilin towards actin. The critical actin monomer concentration ( C c) increased to 260 nM in the presence of phosphoprofilin in comparison with 200 nM in the presence of profilin. The interaction of phosphoprofilin with phosphatidylinositol 4,5-bisphosphate [PI (4,5)-P 2] and poly ( l-proline) (PLP) was examined by monitoring the quenching of tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no difference in PI (4,5)-P 2 binding between profilin and phosphoprofilin ( K d=20.4 μM), while poly ( l-proline)-binding studies indicated a sixfold decrease (27.34 μM for profilin and 4.73 μM for phosphoprofilin) in K d with phosphoprofilin. In vivo studies with platelets indicated an increased association of p85α, the regulatory subunit of PI 3-kinse with phosphoprofilin over profilin. Overall, the data presented conclude that profilin phosphorylated under in vivo conditions and phosphorylation depends upon activation of PI 3-kinase. Phosphoprofilin exhibited increased affinity to poly ( l-proline) sequences both in vitro and in vivo.