Phosphorylation of profilin regulates its interaction with actin and poly (L-proline).
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 (Cc) 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)-P2] 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)-P2 binding between profilin and phosphoprofilin (Kd=20.4 microM), while poly (L-proline)-binding studies indicated a sixfold decrease (27.34 microM for profilin and 4.73 microM for phosphoprofilin) in Kd with phosphoprofilin. In vivo studies with platelets indicated an increased association of p85alpha, the regulatory subunit of PI 3-kinase 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.
Sathish
K
K
Department of Biochemistry, University College of Science, Osmania University, Hyderabad 500 007, AP, India.
Padma
B
B
Munugalavadla
Veerendra
V
Bhargavi
V
V
Radhika
K V N
KV
Wasia
R
R
Sairam
M
M
Singh
Surya S
SS
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002417:Cattle; D002867:Chromones / Q000494:pharmacology; D003285:Contractile Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008840:Microfilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D051304:Profilins; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology
D000199:Actins; D002867:Chromones; D003285:Contractile Proteins; D008840:Microfilament Proteins; D009025:Morpholines; D010455:Peptides; D010716:Phosphatidylinositols; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051304:Profilins; C011083:polyproline; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D015240:Phorbol 12,13-Dibutyrate; D013917:Thrombin