Regulation of LIM-kinase 1 and cofilin in thrombin-stimulated platelets

Abstract

Cofilin is a regulator of actin filament dynamics. We studied whether during platelet activation Rho kinase stimulates LIM kinase (LIMK) leading to subsequent phosphorylation and inactivation of cofilin. Platelet shape change and aggregation/secretion were induced by low and high concentrations of thrombin, respectively. We found that during these platelet responses Rho kinase activation was responsible for mediating rapid Thr508 phosphorylation and activation of LIMK-1 and for the F-actin increase during shape change and, in part, during secretion. Surprisingly, during shape change cofilin phosphorylation was unaltered, and during aggregation/secretion cofilin was first rapidly dephosphorylated by an okadaic acid-insensitive phosphatase and then slowly rephosphorylated by LIMK-1. LIMK-1 phosphorylation and cofilin dephosphorylation and rephosphorylation during aggregation were independent of integrin ␣ IIb ␤ 3 engagement. Cofilin phosphorylation did not regulate cofilin association with F-actin and was unrelated to the F-actin increase in thrombin-activated platelets. Our study identifies LIMK-1 as being activated by Rho kinase in thrombinstimulated platelets. Two counteracting pathways, a cofilin phosphatase and LIMK-1, are activated during platelet aggregation/secretion regulating cofilin phosphorylation sequentially and independently of integrin ␣ IIb ␤ 3 engagement. Rho kinasemediated F-actin increase during platelet shape change and secretion involves a mechanism other than LIMK-1-mediated cofilin phosphorylation, raising the possibility of another LIMK substrate regulating platelet actin assembly. IntroductionDynamic remodeling of actin structures underlies the different morphologic and functional platelet responses such as shape change, spreading, secretion, and aggregation. 1,2 The remodeling of actin is mediated by factors that regulate actin polymerization and depolymerization, disassembly of existing filaments, formation of new filaments, crosslinking of filaments to networks, and bundling of actin filaments. [3][4][5] They include signaling proteins that regulate actin dynamics as well as proteins that bind directly to actin and modulate the diverse actin structures. A key protein regulating actin remodeling is cofilin, 6,7 an essential, ubiquitously expressed and highly conserved actin-binding protein. Binding of cofilin to actin filaments stabilizes a twisted form of F-actin, thereby weakening lateral subunit interactions and promoting filament severing and depolymerization. 8,9 However, filament severing by cofilin also results in the generation of free barbed ends, which in turn is crucial for efficient enhancement of actin polymerization. [10][11][12] Cofilin is therefore an actin dynamizing protein, which favors depolymerization or polymerization of actin, depending on the cellular content of actin filaments relative to actin monomers and free barbed ends. 13 In unstimulated cells cofilin is present both in a phosphorylated and a nonphosphorylated form. 14,15 The depolymerizing activity of co...

Nischarin Inhibits LIM Kinase To Regulate Cofilin Phosphorylation and Cell Invasion

“…It is interesting that nischarin has a stronger effect on LIMK phosphorylation than cofilin phosphorylation, and the reasons for this are unclear. However, similar situations have been reported (41,61). Overall, these data indicate nischarin decreases LIMK activation and blocks LIMK activity, which in turn regulates cofilin phosphorylation.…”

Cdc6 Protein Activates p27KIP1-bound Cdk2 Protein Only after the Bound p27 Protein Undergoes C-terminal Phosphorylation

“…Both cells were incubated in MC and analyzed for the levels of the four kinases and additionally, Ser 473 -phosphorylated AKT and Thr 573 -phosphorylated Rsk1 because these phosphorylated forms are absolutely essential for their activity (26), whereas ROCK activity was monitored by detecting Thr 508 phosphorylation of LIMK1, a specific ROCK substrate (27). AKT remained expressed but was inactive in REF-K6D3 because its Ser 473 phosphorylation disappeared quickly in this cell, like in REF (Fig.…”

Unraveling a novel Rac1-mediated signaling pathway that regulates cofilin dephosphorylation and secretion in thrombin-stimulated platelets

“…All other materials were obtained as reported previously. 7 Approval was obtained from the Ethic Commission of the Medical Faculty of the University of Munich.…”

“…In platelets, the regulation of cofilin during various platelet responses such as secretion, aggregation, and store-operated calcium entry received recent attention. [6][7][8][9] We unraveled in thrombin-and lysophosphatidic acid (LPA)-stimulated platelets a 2-step regulation of the cofilin phosphocycle; a rapid first phase of Ca 2ϩ -dependent cofilin dephosphorylation (activation) through an unknown cofilin phosphatase was followed by a second phase of Rho-kinase/LIMK-1-mediated slow cofilin rephosphorylation (inactivation). The rapid cofilin dephosphorylation was upstream of dense granule secretion and aggregation.…”

“…The rapid cofilin dephosphorylation was upstream of dense granule secretion and aggregation. 7,8 The ability of active cofilin to dynamize actin (ie, to sever and depolymerize actin filaments as well as to stimulate actin polymerization) might be critical for platelet granule secretion.…”