Figure 4. Pask directly interacts with and phosphorylates Wdr5 at Ser49.

(A) Endogenous Pask was immunoprecipitated from C2C12 cells, either before (day -1, 0) or after induction of differentiation (Day 1). Immunoprecipitates were analyzed by western blot for Pask and Wdr5, indicating an enrichment of co-immunoprecipitation at Day 1 of differentiation. (B) V5-tagged LacZ, WT Pask or KD (K1028R) Pask was co-expressed with Flag-YFP or Flag-Wdr5 in 293T cells. V5 or Flag-tagged proteins were immunoprecipitated and examined by western blot using anti-Flag or V5 antibody. (C) V5-hPask was expressed in HEK293T cells with control or Flag-Wdr5 vector. V5 and Flag were stained using Alexa Flour 568 or Alexa Flour 488, respectively. The fraction of cells with nuclear Pask localization was scored as a function of the presence (+) or absence (−) of Wdr5. (D) In vitro phosphorylation of purified His-Wdr5 was performed using WT or KD Pask and analyzed by autoradiogram of the reaction mixture after western blotting, with total protein visualized by Ponceau S staining. pPask indicates autophosphorylation of WT-Pask during kinase reaction. (E) The Pask-Wdr5 complex was immunoprecipitated from cells incubated with ³²P in the presence of DMSO or 25 µM BioE-1197. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography or western blot. (F) Endogenous Pask was immunoprecipitated from C2C12 cells growing in growth media or 12 hr after replacement with differentiation media containing 10 nM Insulin and was incubated with purified Flag-Wdr5 and [γ-³²P] ATP. Autoradiogram shows incorporation of ³²P into Pask (p-Pask) and Wdr5 (p-Wdr5). (G) Schematic showing Ser49 and upstream sequence in Wdr5, compared to the site of Pask phosphorylation in Ugp1, a bona fide substrate of S. cerevisiae Pask. (H) GST-tagged WT, S49A or S49E Wdr5 was incubated with Pask and [γ-³²P] ATP and phosphorylation was detected by autoradiography after SDS-PAGE. (I) WT or S49A Wdr5 was co-immunoprecipitated with Pask from cells incubated with ³²P-phosphate and analyzed as in (E).

DOI: http://dx.doi.org/10.7554/eLife.17985.016

Figure 4—figure supplement 1. Pask directly interacts with and phosphorylates Wdr5 at Ser49.

(A) Flag-tagged YFP or indicated members of various protein complexes of which Wdr5 is a member were expressed in 293T cells. Flag tagged proteins were immunoprecipitated using anti-Flag antibody. Co-immunoprecipitation of Pask was detected via western blotting of anti-Flag immunoprecipitates. (B) Bacterially purified GST-Wdr5 or GST control was attached to glutathione sepharose and purified His-tagged Pask was passed over and the beads were washed extensively. Bound protein was detected by Coomassie staining or anti-Pask immunoblot. (C) Domain truncation of Pask to determine Wdr5 binding region. V5-tagged versions of various Pask truncation mutants were co-expressed with Flag-Wdr5 in HEK293T cells. Wdr5 was detected using anti-Flag antibody after V5 immunoprecipitation. (D) In vitro phosphorylation of Wdr5 by Pask in the presence of DMSO or 25 µM 1197 as described in the Experimental Procedures. (E) Bacterially purified GST-(Flag)-WT, S49A or S49E Wdr5 were used for GST pull-down assays of Pask as in (B). (F) HEK293T cells expressing Flag tagged YFP or the indicated Wdr5 variants were lysed and Flag-tagged proteins were imunoprecipitated. Endogenous interaction of various members of protein complexes with WT or mutant Wdr5 were determined by Western blotting using the indicated antibodies.

DOI: http://dx.doi.org/10.7554/eLife.17985.017