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.
Department of Medicine, McGill University Health Centre, McGill University, Montreal, Quebec H3A 1A1, Canada.
Artem Y
AY
Luhovy
Aala
A
Jaberi
Joan
J
Papillon
Julie
J
Guillemette
Andrey V
AV
Cybulsky
D000818:Animals; D055162:Biocatalysis; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D051381:Rats; D018808:Transcription Factor AP-1 / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D018808:Transcription Factor AP-1; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases