Fig. 4: LIMK2 decreases SPOP stability via direct phosphorylation at three sites.

a Figure shows protein domains of SPOP. The SPOP protein was fragmented into three pieces: SPOP-1–180 (first fragment), which contains the MATH domain; SPOP-181–300 (second fragment), which contains the BTB domain, and SPOP-301–374 (third fragment), which consisted of the C-terminal region. b LIMK2 phosphorylates the first (SPOP-1–180) and the second fragment (SPOP-181–300) and not the third fragment (SPOP-301–374). c LIMK2 phosphorylates SPOP at S59, S171 and S226 positions. All SPOP proteins were 6x-His-tagged. Kinase reaction was performed for 30 min. The top panel shows autoradiography and the bottom panel shows Coomassie staining. d LIMK2 phosphorylates SPOP at only the aforementioned three sites (S59, S171 and S226), as the corresponding 3A-phospho-resistant mutant did not show any phosphorylation. e Phospho-resistant SPOP is expressed at higher level compared to WT-SPOP in C4-2 cells. C4-2 cells were infected with HA-tagged wild-type SPOP or 3A-SPOP retrovirus for 36 h, and the protein levels of SPOP, LIMK2, HA and actin were analysed using their respective antibodies. f The bar graph shows quantification of WT and mutant SPOP levels obtained from three experiments. Data shown are mean ± SEM of three experiments. *P < 0.05 compared to control cells. g SPOP protein levels in 22Rv1 cells infected with HA-tagged SPOP or 3A-SPOP. h Quantification of SPOP levels obtained from three independent experiments. i LIMK2-mediated phosphorylation of SPOP decreases its stability in C4-2 cells. SPOP levels were analysed in C4-2, SPOP-C4-2 and 3A-SPOP-C4-2 cells treated with cycloheximide for 3 and 6 h. j Graphical depiction of SPOP half-life in C4-2, SPOP-C4-2 and 3A-SPOP-C4-2 cells. k LIMK2-mediated phosphorylation of SPOP decreases its stability in 22Rv1 cells. 22Rv1, SPOP-22Rv1 and 3A-SPOP-22Rv1 cells were treated with cycloheximide for 3 and 6 h, and SPOP levels were analysed. l Graphical depiction of SPOP half-life in 22Rv1, SPOP-22Rv1 and 3A-SPOP-22Rv1 cells. m LIMK2 overexpression augmented the ubiquitylation of WT-SPOP but not of 3A-SPOP in C4-2 cells. WT-SPOP-C4-2 and 3A-SPOP-C4-2 cells were infected with 6x-His-Ubiquitin with or without LIMK2 for 30 h, followed by MG132 treatment for 12 h. SPOP was immunoprecipitated using HA antibody and ubiquitylation analysed. n LIMK2 overexpression augmented the ubiquitylation of WT-SPOP but not of 3A-SPOP in 22Rv1 cells.