Molecular regulation of the Ste20-like kinase (SLK) by SRC-dependent pathways

Abstract

The Ste20-like kinase (SLK) is a conserved serine/threonine kinase that induces the dissociation of actin stress fibers in a Rac1-dependent manner. Recently, our laboratory has shown that SLK is a microtubule-associated protein regulating migratory components at the cell periphery in membrane ruffles and lamellipodia (Storbeck et al.; Wagner et al. unpublished data). To investigate the role of SLK during cell adhesion and migration, we used the Polyomavirus Enhancer Activator 3-null (PEA3(-/-/)) cell line, known to exhibit migration deficiencies. We observed that SLK redistribution to the cell periphery is altered in the PEA3(-/-) cells and that this phenotype could be rescued by the re-expression of PEA3 cDNA into PEA3(-/-) cells. Further analysis revealed that the PEA3(-/-) cells exhibit impaired c-src activation highlighted by decreased focal adhesion kinase (FAK) cleavage and reduced p130Cas tyrosine phosphorylation following fibronectin stimulation, suggesting that c-src activity and proper adhesion signaling may be required for SLK recruitment to the cell periphery. To further investigate FAK and c-src involvement in SLK redistribution to the cell periphery, we used FAK- or src, yes and fyn (SYF)-deficient cells. We have shown that SLK redistribution to the cell periphery is also altered in SYF deficient cells but not in FAK-deficient cells, suggesting that c-src mediates SLK redistribution to the cell periphery independently of FAK. SLK in vitro kinase assays performed on PEA3(-/-), FAK- and SYF-deficient cells revealed that SLK kinase activity is unaffected. However, v-src and c-srcY527F transformed cells displayed a 2- to 3-fold decrease in SLK kinase activity. Biochemical analysis revealed that SLK is hyperphosphorylated on serine residues in these cells with the kinase domain being the main target. Further analysis revealed that casein kinase II (CK2) phosphorylates the SLK kinase domain on serine 347/348 to downregulate SLK kinase activity. In addition, we also showed that CK2 kinase activity is dramatically increased in v-src-transformed cells and that the inhibition of CK2 restores SLK kinase activity. Overall, we showed that SLK redistribution to the cell periphery requires c-src, which can also regulate SLK kinase activity via CK2 to allow the turnover of adhesion sites and cytoskeletal rearrangements.