MARK3-mediated Slingshot-1 phosphorylation is essential for polarized lamellipodium formation

Cofilin acts as a key regulator of actin cytoskeletal remodeling via stimulating actin filament disassembly. Cofilin is inactivated by Ser-3 phosphorylation and reactivated by cofilin-phosphatase Slingshot-1 (SSH1). SSH1 is activated upon binding to F-actin, and this activation is inhibited by its phosphorylation at Ser-937 and Ser-978 and the subsequent binding of 14-3-3 proteins. In this study, we identified MARK3 (also named Par-1a and C-TAK1) as a kinase responsible for Ser-937/Ser-978 phosphorylation of SSH1. MARK3-mediated phosphorylation promoted SSH1 binding to 14-3-3 proteins and suppressed its F-actin-assisted cofilin-phosphatase activity. When Jurkat cells were stimulated with SDF-1α, actin filaments formed multidirectional F-actin-rich lamellipodia around the cells in the initial stage, and thereafter, they were rearranged as a single polarized lamellipodium to the direction of cell migration. Upon SDF-1α stimulation, SSH1 was translocated into F-actin-rich lamellipodia, but its Ser-937/Ser-978 non-phosphorylatable mutant SSH1(2SA) was retained at the location of the original cortical F-actin. Knockdown of MARK3 or overexpression of SSH1(2SA), similar to SSH1 knockdown, impaired the conversion of multiple lamellipodia to a single polarized lamellipodium. These results indicate that MARK3-mediated Ser-937/Ser-978 phosphorylation is required for SSH1 liberation from F-actin and translocation to lamellipodia, and hence, facilitates the formation of a single polarized lamellipodium for directional cell migration. Our results suggest that the phosphorylation-dephosphorylation cycle of SSH1 is crucial for its localization to lamellipodia via promoting the dissociation-reassociation cycle of SSH1 to F-actin, and thereby the stimulus-induced lamellipodium formation to the direction of cell movement. ### Competing Interest Statement The authors have declared no competing interest. * ### Abbreviations CFP : cyan fluorescence protein F-actin : filamentous actin FCS : fetal calf serum GST : glutathione S-transferase IPTG : isopropyl β-D-1-thiogalactopyranoside LIMK : LIM-kinase MARK : microtubule affinity-regulating kinase 3 Ni-NTA : nickel nitrilotriacetic acid PAK : p21-activated kinase PAR-1 : partitioning defective-1 PBS : phosphate-buffered saline P-cofilin : Ser-3-phosphorylated cofilin PKC : protein kinase C PKD : protein kinase D pS-978 : phospho-serine 978 SDF-1α : stromal cell-derived factor 1α shRNA : short hairpin RNA SSH1 : Slingshot 1 SSH1(2SA) : SSH1 mutant in which Ser-937 and Ser-978 are replaced by alanine WT : wild-type