SLK Activity Research Articles

The Ste20-like kinase - a Jack of all trades?

Over the past 20 years, the Ste20-like kinase (SLK; also known as STK2) has emerged as a central regulator of cytoskeletal dynamics. Reorganization of the cytoskeleton is necessary for a plethora of biological processes including apoptosis, proliferation, migration, tissue repair and signaling. Several studies have also uncovered a role for SLK in disease progression and cancer. Here, we review the recent findings in the SLK field and summarize the various roles of SLK in different animal models and discuss the biochemical mechanisms regulating SLK activity. Together, these studies have revealed multiple roles for SLK in coupling cytoskeletal dynamics to cell growth, in muscle repair and in negative-feedback loops critical for cancer progression. Furthermore, the ability of SLK to regulate some systems appears to be kinase activity independent, suggesting that it may be an important scaffold for signal transduction pathways. These various findings reveal highly complex functions and regulation patterns of SLK in development and disease, making it a potential therapeutic target.

Ste20-like kinase, SLK, a novel mediator of podocyte integrity.

SLK is essential for embryonic development and may play a key role in wound healing, tumor growth, and metastasis. Expression and activation of SLK are increased in kidney development and during recovery from ischemic acute kidney injury. Overexpression of SLK in glomerular epithelial cells/podocytes in vivo induces injury and proteinuria. Conversely, reduced SLK expression leads to abnormalities in cell adhesion, spreading, and motility. Tight regulation of SLK expression thus may be critical for normal renal structure and function. We produced podocyte-specific SLK-knockout mice to address the functional role of SLK in podocytes. Mice with podocyte-specific deletion of SLK showed reduced glomerular SLK expression and activity compared with control. Podocyte-specific deletion of SLK resulted in albuminuria at 4-5 mo of age in male mice and 8-9 mo in female mice, which persisted for up to 13 mo. At 11-12 mo, knockout mice showed ultrastructural changes, including focal foot process effacement and microvillous transformation of podocyte plasma membranes. Mean foot process width was approximately twofold greater in knockout mice compared with control. Podocyte number was reduced by 35% in knockout mice compared with control, and expression of nephrin, synaptopodin, and podocalyxin was reduced in knockout mice by 20-30%. In summary, podocyte-specific deletion of SLK leads to albuminuria, loss of podocytes, and morphological evidence of podocyte injury. Thus, SLK is essential to the maintenance of podocyte integrity as mice age.

Regulation of Ste20-like kinase, SLK, activity: Dimerization and activation segment phosphorylation.

The Ste20-like kinase, SLK, has diverse cellular functions. SLK mediates organ development, cell cycle progression, cytoskeletal remodeling, cytokinesis, and cell survival. Expression and activity of SLK are enhanced in renal ischemia-reperfusion injury, and overexpression of SLK was shown to induce apoptosis in cultured glomerular epithelial cells (GECs) and renal tubular cells, as well as GEC/podocyte injury in vivo. The SLK protein consists of a N-terminal catalytic domain and an extensive C-terminal domain, which contains coiled-coils. The present study addresses the regulation of SLK activity. Controlled dimerization of the SLK catalytic domain enhanced autophosphorylation of SLK at T183 and S189, which are located in the activation segment. The full-length ectopically- and endogenously-expressed SLK was also autophosphorylated at T183 and S189. Using ezrin as a model SLK substrate (to address exogenous kinase activity), we demonstrate that dimerized SLK 1–373 or full-length SLK can effectively induce activation-specific phosphorylation of ezrin. Mutations in SLK, including T183A, S189A or T193A reduced T183 or S189 autophosphorylation, and showed a greater reduction in ezrin phosphorylation. Mutations in the coiled-coil region of full-length SLK that impair dimerization, in particular I848G, significantly reduced ezrin phosphorylation and tended to reduce autophosphorylation of SLK at T183. In experimental membranous nephropathy in rats, proteinuria and GEC/podocyte injury were associated with increased glomerular SLK activity and ezrin phosphorylation. In conclusion, dimerization via coiled-coils and phosphorylation of T183, S189 and T193 play key roles in the activation and signaling of SLK, and provide targets for novel therapeutic approaches.

Ste20-like kinase, SLK, activates the heat shock factor 1 – Hsp70 pathway

Expression and activation of SLK increases during renal ischemia-reperfusion injury. When highly expressed, SLK signals via c-Jun N-terminal kinase and p38 to induce apoptosis, and it exacerbates apoptosis induced by ischemia-reperfusion injury. Overexpression of SLK in glomerular epithelial cells (GECs)/podocytes in vivo induces injury and proteinuria. In response to various stresses, cells enhance expression of chaperones or heat shock proteins (e.g. Hsp70), which are involved in the folding and maturation of newly synthesized proteins, and can refold denatured or misfolded proteins. We address the interaction of SLK with the heat shock factor 1 (HSF1)-Hsp70 pathway. Increased expression of SLK in GECs (following transfection) induced HSF1 transcriptional activity. Moreover, HSF1 transcriptional activity was increased by in vitro ischemia-reperfusion injury (chemical anoxia/recovery) and heat shock, and in both instances was amplified further by SLK overexpression. HSF1 binds to promoters of target genes, such as Hsp70 and induces their transcription. By analogy to HSF1, SLK stimulated Hsp70 expression. Hsp70 was also enhanced by anoxia/recovery and was further amplified by SLK overexpression. Induction of HSF1 and Hsp70 was dependent on the kinase activity of SLK, and was mediated via polo-like kinase-1. Transfection of constitutively active HSF1 enhanced Hsp70 expression and inhibited SLK-induced apoptosis. Conversely, the proapoptotic action of SLK was augmented by HSF1 shRNA, or the Hsp70 inhibitor, pifithrin-μ. In conclusion, increased expression/activity of SLK activates the HSF1-Hsp70 pathway. Hsp70 attenuates the primary proapoptotic effect of SLK. Modulation of chaperone expression may potentially be harnessed as cytoprotective therapy in renal cell injury.

Recruitment and activation of SLK at the leading edge of migrating cells requires Src family kinase activity and the LIM-only protein 4

The Ste20-like kinase SLK plays a pivotal role in cell migration and focal adhesion turnover and is regulated by the LIM domain-binding proteins Ldb1 and Ldb2. These adapter proteins have been demonstrated to interact with LMO4 in the organization of transcriptional complexes. Therefore, we have assessed the ability of LMO4 to also interact and regulate SLK activity. Our data show that LMO4 can directly bind to SLK and activate its kinase activity in vitro and in vivo. LMO4 can be co-precipitated with SLK following the induction of cell migration by scratch wounding and Cre-mediated deletion of LMO4 in conditional LMO4fl/fl fibroblasts inhibits cell migration and SLK activation. Deletion of LMO4 impairs Ldb1 and SLK recruitment to the leading edge of migrating cells. Supporting this, Src/Yes/Fyn-deficient cells (SYF) expressing very low levels of LMO4 do not recruit SLK to the leading edge. Re-expression of wildtype Myc–LMO4 in SYF cells, but not a mutant version, restores SLK localization and kinase activity. Overall, our data suggest that activation of SLK by haptotactic signals requires its recruitment to the leading edge by LMO4 in a Src-dependent manner. Furthermore, this establishes a novel cytosolic role for the transcriptional co-activator LMO4.

Distinct roles for Ste20-like kinase SLK in muscle function and regeneration

BackgroundCell growth and terminal differentiation are controlled by complex signaling systems that regulate the tissue-specific expression of genes controlling cell fate and morphogenesis. We have previously reported that the Ste20-like kinase SLK is expressed in muscle tissue and is required for cell motility. However, the specific function of SLK in muscle tissue is still poorly understood.MethodsTo gain further insights into the role of SLK in differentiated muscles, we expressed a kinase-inactive SLK from the human skeletal muscle actin promoter. Transgenic muscles were surveyed for potential defects. Standard histological procedures and cardiotoxin-induced regeneration assays we used to investigate the role of SLK in myogenesis and muscle repair.ResultsHigh levels of kinase-inactive SLK in muscle tissue produced an overall decrease in SLK activity in muscle tissue, resulting in altered muscle organization, reduced litter sizes, and reduced breeding capacity. The transgenic mice did not show any differences in fiber-type distribution but displayed enhanced regeneration capacity in vivo and more robust differentiation in vitro.ConclusionsOur results show that SLK activity is required for optimal muscle development in the embryo and muscle physiology in the adult. However, reduced kinase activity during muscle repair enhances regeneration and differentiation. Together, these results suggest complex and distinct roles for SLK in muscle development and function.

Regulation of the Ste20-like Kinase, SLK: INVOLVEMENT OF ACTIVATION SEGMENT PHOSPHORYLATION

Expression and activation of the Ste20-like kinase, SLK, is increased during kidney development and recovery from ischemic acute kidney injury. SLK promotes apoptosis, and it may regulate cell survival during injury or repair. This study addresses the role of phosphorylation in the regulation of kinase activity. We mutated serine and threonine residues in the putative activation segment of the SLK catalytic domain and expressed wild type (WT) and mutant proteins in COS-1 or glomerular epithelial cells. Compared with SLK WT, the T183A, S189A, and T183A/S189A mutants showed reduced in vitro kinase activity. SLK WT, but not mutants, increased activation-specific phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. Similarly, SLK WT stimulated activator protein-1 reporter activity, but activation of activator protein-1 by the three SLK mutants was ineffective. To test if homodimerization of SLK affects phosphorylation, the cDNA encoding SLK amino acids 1-373 (which include the catalytic domain) was fused with a cDNA for a modified FK506-binding protein, Fv (Fv-SLK 1-373). After transfection, the addition of AP20187 (an FK506 analog) induced regulated dimerization of Fv-SLK 1-373. AP20187-stimulated dimerization enhanced the kinase activity of Fv-SLK 1-373 WT. In contrast, kinase activity of Fv-SLK 1-373 T183A/S189A was weak and was not enhanced after dimerization. Finally, apoptosis was increased after expression of Fv-SLK 1-373 WT but not T183A/S189A. Thus, phosphorylation of Thr-183 and Ser-189 plays a key role in the activation and signaling of SLK and could represent a target for novel therapeutic approaches to renal injury.

Activity of the Ste20-like kinase, SLK, is enhanced by homodimerization

The expression and activation of the Ste20-like kinase, SLK, is increased during renal development and recovery from ischemic acute renal failure. SLK promotes apoptosis, and during renal injury and repair, transcriptional induction or posttranscriptional control of SLK may, therefore, regulate cell survival. SLK contains protein interaction (coiled-coil) domains, suggesting that posttranslational homodimerization may also modulate SLK activity. We therefore expressed coiled-coil regions in the C-terminal domain of SLK as fusion proteins and demonstrated their homodimerization. By gel-filtration chromatography, endogenous and heterologously expressed SLK were detected in a macromolecular protein complex. To test the role of homodimerization in kinase activation, we constructed a fusion protein consisting of the SLK catalytic domain (amino acids 1-373) and a modified FK506 binding protein, Fv (Fv-SLK 1-373). Addition of AP20187 (an analog of FK506) enhanced the homodimerization of Fv-SLK 1-373. In an in vitro kinase assay, the dimeric Fv-SLK 1-373 displayed greater kinase activity than the monomeric form. In cells expressing Fv-SLK 1-373, homodimerization increased activation-specific phosphorylation of the proapoptotic kinases, c-Jun N-terminal kinase and p38 kinase. Compared with the monomer, dimeric Fv-SLK 1-373 enhanced the activation of a Bax promoter-luciferase reporter. Finally, expression of Fv-SLK 1-373 induced apoptosis, and the effect was increased by homodimerization. Thus the activity, downstream signaling, and functional effects of SLK are enhanced by dimerization of the kinase domain.

The Ste20-like kinase SLK is required for ErbB2-driven breast cancer cell motility

The Ste20-like kinase, SLK, is involved in the control of cell motility through its effects on actin reorganization and focal adhesion turnover. Here we investigated the role of SLK in chemotaxis downstream of the tyrosine kinase receptor, HER2/ErbB2/Neu, which is frequently overexpressed in human breast cancers. Our results show that SLK is required for the efficient cell migration of human and mouse mammary epithelial cell lines in the presence of the Neu activator, heregulin, as a chemoattractant. SLK activity is stimulated by heregulin treatment or by overexpression of activated Neu. Phosphorylation of tyrosine 1201 or tyrosines 1226/7 on Neu is a key event for SLK activation and cell migration, and cancer cell invasion mediated by these tyrosines is inhibited by kinase-inactive SLK. Signaling pathway inhibitors show that Neu-mediated SLK activation is dependent on MEK, PI3K, PLCgamma and Shc signaling. Furthermore, heregulin-stimulated SLK activity requires signals from the focal adhesion proteins, FAK and src. Finally, phospho-FAK analysis shows that SLK is required for Neu-dependent focal adhesion turnover. Together, these studies define an interaction between Neu and SLK signaling in the regulation of cancer cell motility.

Ste20-like kinase SLK displays myofiber type specificity and is involved in C2C12 myoblast differentiation.

Cell growth and terminal differentiation are controlled by complex signaling cascades that regulate the expression of specific subsets of genes implicated in cell fate and morphogenic processes. We have recently cloned and characterized a novel Ste20-like kinase termed SLK that is associated with adhesion structures during cell adhesion and spreading. However, the specific function of SLK is poorly understood. To gain further insight into the role of SLK, we have characterized its activity, expression, and distribution in skeletal muscle and during the in vitro differentiation of C2C12 myoblasts. Although SLK is expressed ubiquitously in adult tissues, our results show that it is predominantly expressed in muscle masses during development. Furthermore, SLK activity is upregulated during the differentiation of C2C12 myoblasts. In addition, we have found that SLK localizes presynaptically at neuromuscular junctions and that it is preferentially expressed in types I and IIA myofibers at major myofibrillar striations. Supporting a role in myoblast function and differentiation, SLK expression is induced in Myf5- and Pax7-positive activated satellite cells during regeneration and expression of dominant negative SLK in C2C12 cultures impairs myoblast fusion, suggesting a role for SLK in muscle cell differentiation.