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

Abstract

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.

Fig. 1.

Ste20-like kinase, SLK, constructs employed in this study. See materials and methods for details. SLK (full-length; wild type), SLK 1–373, and Fv-SLK 1–373 contain hemagglutinin (HA) tags (not illustrated). CTC, C-terminal coil; NTC, N-terminal coil; GFP, green fluorescent protein; GST, glutathione S-transferase (GST).

Fig. 2.

Homodimerization of SLK. A: COS-1 cells were transfected with GFP-N-terminal coil, GFP-C-terminal coil, or GFP-double coil. Lysates were mixed with GST-SLK(CT)-glutathione-agarose or agarose alone. Pull-down with GST-SLK(CT)-glutathione-agarose (but not agarose alone) demonstrated prominent association with the SLK C-terminal coil and weaker interactions with the N-terminal coil and the double coil. The lysates are presented at a lower exposure compared with the pull-downs. B: following expression of GFP-N-terminal coil, C-terminal coil, or double coil, GST-SLK(CT) was added to some of the COS-1 cell lysates (+), and the mixtures were immunoprecipitated with anti-GFP antibody, or were subjected to pull-down with glutathione-agarose. GST-SLK(CT) was recovered by both anti-GFP immunoprecipitation and glutathione-agarose pull-down. C: gel-filtration chromatography. Top: anti-HA antibody immunoblot of eluted fractions (HA-SLK-transfected COS-1 cells). Bottom: anti-SLK antibody immunoblot (untransfected cells, i.e., endogenous SLK). In cells transiently transfected with HA-SLK, SLK eluted in fractions at or just below the size of thyroglobulin (669 kDa), and there was an absence of SLK in fractions the size of IgG (150 kDa). The elution profile of endogenous SLK in COS-1 cells was similar to that of ectopic HA-SLK. In A and B (and in certain panels of the other figures below), the white spaces denote reassembly of noncontiguous gel lanes; there were no adjustments made to the digital images among the lanes that would alter the information in the panels.

Fig. 3.

Effect of the C-terminal domain on the kinase activity of SLK. A and B: COS-1 cells were transiently transfected with HA-SLK (full-length) or HA-SLK 1–373 cDNAs. After 48 h, lysates were immunoprecipitated (IP) with anti-HA antibody (+) or normal IgG in controls (−) and were then subjected to a kinase assay with myelin basic protein (MBP) as substrate, or were immunoblotted with anti-HA antibody. A: representative autoradiogram of ³³P incorporation into MBP and anti-HA immunoblot. B: densitometric quantification of phosphorylated MBP normalized for protein expression. Values are means ± SE of 4 experiments. *P < 0.04 SLK (full-length) vs. SLK 1–373. C and D: COS-1 cells were transiently transfected with HA-SLK 1–373 cDNA. After 48 h, cells were lysed, and GST-SLK(CT) was added to some samples (+). Samples were immunoprecipitated with anti-HA antibody (+) or normal IgG in controls (−) and were then subjected to a kinase assay with MBP as substrate (C) or were immunoblotted with anti-HA or anti-SLK(CT) antibodies (D). GST-SLK(CT) coimmunoprecitated with HA-SLK 1–373 (D) but had no effect on the kinase activity of SLK 1–373 (C).

Fig. 4.

Homodimerization increases the kinase activity of SLK. A and B: COS-1 cells were transiently transfected with Fv-SLK 1–373 cDNA and were treated after 48 h with (+) or without (−) 1 μM AP20187 for 24 h to induce homodimerization of Fv-SLK 1–373. Lysates were immunoprecipitated with anti-HA antibody (normal IgG in controls) and were then subjected to a kinase assay with MBP as substrate. A: representative autoradiogram of phosphorylated MBP (duplicate lanes). B: densitometric quantification of phosphorylated MBP in the anti-HA antibody immunoprecipitations. Values are means ± SE of 4 experiments. *P < 0.0001 AP20187 vs. untreated. C and D: COS-1 cells were transfected with Fv-SLK 1–373 cDNA and were then treated with (+) or without (−) 1 μM AP20187 as above. Lysates were immunoprecipitated with anti-HA antibody, and the immune complexes were then immunoblotted with anti-HA antibody (C, top). Bottom: lysates immunoblotted with anti-HA antibody. D: densitometric quantification of anti-HA antibody immunoprecipitations/immunoblots. Values are means ± SE of 5 experiments. *P < 0.0001 AP20187 vs. untreated.

Fig. 5.

Effect of AP20187 on the homodimerization of Fv-SLK 1–373. COS-1 cells were transiently transfected with Fv-SLK 1–373 cDNA and were treated after 48 h with or without 0.1 μM AP20187 for 24 h. Lysates were subjected to gel-filtration chromatography. A: representative anti-HA antibody immunoblots. B: densitometric quantification (3–5 experiments). D, dimer; M, monomer.

Fig. 6.

Homodimerization of Fv-SLK 1–373 increases phosphorylation of p38. A: glomerular epithelial cells were transiently transfected with Fv-SLK 1–373 cDNA, and after 48 h, were treated with (+) or without (−) 1 μM AP20187 for 3 h. Empty vector transfections were used as a control. Transfection with full-length, wild-type SLK (SLK WT), untransfected cells (Un), and exposure of cells to UV are shown for comparison. Cell lysates were immunoblotted with antibodies to phospho-p38 (pp38; top), HA (middle), and p38 (bottom). B and C: densitometric quantification of p38 phosphorylation. B: transfected cells were incubated with AP20187 (AP) for 3 h. Values are means ± SE of 6 experiments. *P = 0.0035 AP20187 vs. untreated. C: cells were incubated with AP20187 for 24 h. Values are means ± SE of 8 experiments. *P = 0.002 AP20187 vs. untreated.

Fig. 7.

Homodimerization of Fv-SLK 1–373 increases phosphorylation of JNK (46-kDa isoform). A: glomerular epithelial cells were transiently transfected with Fv-SLK 1–373 cDNA, and after 48 h, were treated with (+) or without (−) 1 μM AP20187 for 3 h. Empty vector transfections were used as a control. Transfection with full-length SLK WT and exposure of cells to UV are shown for comparison. Cell lysates were immunoblotted with antibodies to phospho-JNK (pJNK; top), HA (middle), and JNK (bottom). B and C: densitometric quantification of JNK phosphorylation (46 kDa). B: transfected cells were incubated with AP20187 (AP) for 3 h. Values are means ± SE *P = 0.05 AP20187 vs. untreated, 6 experiments. C: cells were incubated with AP20187 for 24 h. Values are means ± SE of 8 experiments. *P = 0.05 AP20187 vs. untreated.

Fig. 8.

Homodimerization of Fv-SLK 1–373 enhances apoptosis. COS-1 cells were transiently transfected with Fv-SLK 1–373 cDNA or empty vector (control) and were then treated with (+) or without (−) 3 μM AP20187 (materials and methods). A: cells were analyzed by Hoechst H33342 staining. Values are means ± SE of 4 experiments. *P = 0.03 group 1 vs. group 3. **P = 0.004 group 3 vs. group 4. B: number of viable COS-1 cells. Values are means ± SE of 3 experiments. *P = 0.057 group 1 vs. group 3. **P < 0.0001 group 1 vs. group 4. **P < 0.0001 group 2 vs. group 4. **P < 0.004 group 3 vs. group 4.

Fig. 9.

Homodimerization of Fv-SLK 1–373 enhances Bax promoter-luciferase reporter activities. Glomerular epithelial cells were transfected with a firefly luciferase reporter plasmid that contains the Bax promoter (−680 bp to −317 bp), together with Fv-SLK 1–373 or empty vector. Then, cells were treated with (+) or without (−) 2 μM AP20187 (AP). Cells were harvested 72 h after transfection. Luciferase activity is expressed in arbitrary units. Values are means ± SE of 4 experiments. *P = 0.02 group 1 vs. group 3. *P = 0.0006 group 1 vs. group 4. **P = 0.02 group 3 vs. group 4.