Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over

Abstract

BackgroundInitially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion.Methodology/Principal FindingsTo study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-δ isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process.Conclusions/SignificanceAltogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-δ signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion.

Angiotensin II regulates phosphorylation of actin‐associated proteins in human podocytes

“…Furthermore, phosphomimicking LCP1-S5E was observed to accumulate in actin-rich regions in monkey kidney epithelial cells (Vero cells) (27). Ser5 phosphorylation has been described to modulate the actin dynamics in focal adhesions, which suggests that LCP1 redistribution to de novo assembled actin-rich structures depends on phosphorylation (40). However, the authors admitted that nonphosphorylatable LCP1-S5A also shifted to cell margins after treatment with phorbol 12-myristate 13-acetate, but to a lesser degree (40).…”

“…A correlation of LCP1 redistribution, membrane ruffling, and actin binding has previously been observed by other groups. For example, treatment of LCP1 MCF-7 breast carcinoma cells with phorbol 12-myristate 13-acetate, which is a potent activator of conventional and novel PKC family members (55), also led to increased LCP1 phosphorylation, LCP1 relocalization, and membrane ruffling (40). Furthermore, phosphomimicking LCP1-S5E was observed to accumulate in actin-rich regions in monkey kidney epithelial cells (Vero cells) (27).…”

“…This Ab bound to the second actin-binding domain of LCP1, which led to the conclusion that the actin-bundling activity of LCP1 plays a fundamental role in filopodia integrity. Actin bundling activity is influenced by LCP1 phosphorylation (40). In general, actin-bundling proteins, among other plastins, are involved in the assembly of filopodia (56).…”

“…It has been previously shown that phosphorylation of LCP1 at Ser5 promotes the targeting of LCP1 to actin assembly sites (27,40). To address this, we visualized endogenously phosphorylated LCP1 (p-Ser5-LCP1) in podocytes.…”

“…LPS is known as a podocyte-affecting substance, which leads in vivo to foot process effacement and damage to the slit diaphragm (38). To examine whether podocyte damage in general leads to LCP1 It has been demonstrated in breast cancer cell lines that ERK, RSK, PKA, and PKC are involved in the regulation of LCP1 phosphorylation at Ser5 (27,39,40); therefore, we investigated whether these kinases are also responsible for LCP1 phosphorylation in podocytes. Treatment with the specific RSK inhibitor, BID1870 (41), or the ERK inhibitor, U0126 (42), completely blocked AngII-induced LCP1 phosphorylation, which demonstrated that ERK and RSK are involved in LCP1 phosphorylation also in podocytes.…”