Caspase 3 Cleavage of the Ste20-Related Kinase SLK Releases and Activates an Apoptosis-Inducing Kinase Domain and an Actin-Disassembling Region

Abstract

We have demonstrated that a novel Ste20-related kinase, designated SLK, mediates apoptosis and actin stress fiber dissolution through distinct domains generated by caspase 3 cleavage. Overexpression of SLK in C2C12 myoblasts stimulated the disassembly of actin stress fibers and focal adhesions and induced apoptosis, as determined by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling analysis. SLK was cleaved by caspase 3 in vitro and in vivo during c-Myc-, tumor necrosis factor alpha, and UV-induced apoptosis. Furthermore, cleavage of SLK released two domains with distinct activities: an activated N-terminal kinase domain that promoted apoptosis and cytoskeletal rearrangements and a C-terminus domain that disassembled actin stress fibers. Moreover, our analysis has identified a novel conserved region (termed the AT1-46 homology domain) that efficiently promotes stress fiber disassembly. Finally, transient transfection of SLK also activated the c-Jun N-terminal kinase signaling pathway. Our results suggest that caspase-activated SLK represents a novel effector of cytoskeletal remodeling and apoptosis.

Ste20-like kinase SLK, at the crossroads

“…14,23 A putative caspase 3 consensus cleavage site (DXXD) is also contained within the central domain at amino acid residue 436 of the mouse sequence and is loosely conserved in several other species. 17 Mutation of the putative cleavage site did not however affect the ability of SLK to be cleaved by caspase3 in vitro, suggesting the presence of additional sites and more complex regulation 17 The N-terminal catalytic domain is most similar to LOK (74%) and mammalian sterile 20 1 (MST1) (26%) from amino acids 40-307 (Fig. 1).…”

“…Supporting this, T183 and S189 are located within the activation segment; subdomains VII and VIII, respectively. 24,25 Albeit not well conserved, 17,26 a putative consensus SH3 binding site (PXXPX) is found at position 735 of the murine SLK sequence, suggesting that SLK may interact with SH3 domain containing proteins that have yet to be identified. 17,26 A number of protein kinases regulate themselves by autophosphorylation on at least one key residue within the activation segment, usually contained within the kinase lobe of the protein.…”

“…24,25 Albeit not well conserved, 17,26 a putative consensus SH3 binding site (PXXPX) is found at position 735 of the murine SLK sequence, suggesting that SLK may interact with SH3 domain containing proteins that have yet to be identified. 17,26 A number of protein kinases regulate themselves by autophosphorylation on at least one key residue within the activation segment, usually contained within the kinase lobe of the protein. 27 Autophosphorylation sites on SLK have been mapped to T183 and S189 through electron density map analysis obtained from mass spectrometry, however neither site appeared to be completely phosphorylated.…”

“…14,16 SLK was first identified and characterized as a caspase3-activated kinase which played a role in the induction of apoptosis. 14,[17][18][19] SLK is classified as a GCK-related kinase that shares extensive sequence similarity with lymphocyte oriented kinase (LOK). 20 SLK is comprised of an N-terminal Ste20 catalytic kinase domain (amino acids 1-338) and predicted coil regions between amino acids 339-788 and residues 825-1,180, respectively (Fig.…”

“…The carboxy-terminus of SLK shares a high degree of identity with AT1-46 at amino acids 788-936 (63%) and 957-1171 (71%) as well as with LOK (56%) between amino acids 867 and 1178. 14,17,21,22 This C-terminal coil motif was termed the ATH domain, for AT1-46 homology domain. 14,23 A putative caspase 3 consensus cleavage site (DXXD) is also contained within the central domain at amino acid residue 436 of the mouse sequence and is loosely conserved in several other species.…”