Profilin oligomerization and its effect on poly (L-proline) binding and phosphorylation.

Profilin is a cytoskeletal protein that interacts specifically with actin, phosphoinositides and poly (l-proline). Experimental results and in silico studies revealed that profilin exists as dimer and tetramer. Profilin oligomers possess weak affinity to poly (l-proline) due to unavailability of binding sites in dimers and tetramers. Phosphorylation studies indicate that profilin dimers are not phosphorylated while teramers are preferentially phosphorylated over monomers. In silico studies revealed that PKC phosphorylation site, S137 is buried in dimer while it is accessible in tetramer.

Phorbol ester mediated activation of inducible nitric oxide synthase results in platelet profilin nitration.

Nitric oxide is an important precursor for peroxynitrite production under in vivo conditions leading to cell injury and cell death. In platelets, a number of cytosolic and actin binding proteins were shown to be nitrated [K.M. Naseem, S.Y. Low, M. Sabetkar, N.J. Bradley, J. Khan, M. Jacobs, K.R. Bruckdorfer, The nitration of platelet cytosolic proteins during agonist-induced activation of platelets. FEBS Lett. 473 (1) (2000) 199-122 and M. Sabetkar, S.Y. Low, K.M. Naseem, K.R. Bruckdorfer, The nitration of proteins in platelets: significance in platelet function, Free Radic. Biol. Med. 33 (6) (2002) 728-736]. We investigated the possible mechanism that regulates profilin (an actin binding protein) nitration in platelets. Activation of bovine platelets with arachidonic acid, thrombin, and phorbol 12,13-dibutyrate resulted in nitration of profilin on tyrosine residue. In vivo profilin nitration showed a four- and eight-fold increase in the presence of thrombin and phorbol 12,13-dibutyrate, respectively. Analysis of nitroprofilin levels in the presence of NOS inhibitors (1400W and EGTA), indicated that profilin nitration in phorbol 12,13-dibutyrate treated platelets is mediated by inducible nitric oxide synthase. Phorbol ester treated platelets exhibited higher levels by inducible nitric oxide synthase (491% over control), while total nitric oxide synthase activity increased by 5% over control. Higher levels of peroxynitrite in platelets treated with phorbol 12,13-dibutyrate indicated that profilin nitration is mediated by peroxynitrite. Increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity in platelets treated with thrombin and phorbol 12,13-dibutyrate indicates that nitration of platelet profilin could be mediated by PI 3-kinase. A decrease in the level of nitroprofilin in PDBu treated platelets in the presence of inducible nitric oxide synthase inhibitor, 1400W, was observed suggesting that profilin nitration is mediated by PI 3-kinase dependent activation of inducible nitric oxide synthase.

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.