CDK4 phosphorylates zFoxj1a at T102 to suppress its degradation.

Abstract

(A) zFoxj1a and CDK4 show evident colocalization in HeLa cells. HeLa cells were transfected with Flag-zFoxj1a and Myc-CDK4 and immunostained with anti-Flag (red) and anti-Myc (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (B) Overexpressed CDK4 interacts with endogenous zFoxj1a. WT embryos were injected with 200 pg Myc-CDK4 mRNA at midgastrulation and then harvested at the bud stage for immunoprecipitation with anti-Myc antibody or normal mouse IgG. (C) CDK4, but not its kinase-deficient mutant, interacts with zFoxj1a. HEK293T cells were transfected with plasmids as indicated, followed by treatment with MG132 for 5 hours prior to harvest for immunoprecipitation. (D) Direct binding of CDK4 to zFoxj1a in vitro. GST, GST-zFoxj1a, and Myc-CDK4 were expressed in bacterial cells and purified. Myc-CDK4 proteins were incubated with GST or GST-zFoxj1a. The presence of Myc-CDK4 in the protein complex pulled down by glutathione agarose was assessed using an anti-Myc antibody. Input proteins were examined by Coomassie blue staining. (E) Conserved CDK substrate motifs in Foxj1 proteins from different species. Red stars indicate critical residues in the CDK substrate motifs. (F–G) CDK4 phosphorylates zFoxj1a at T102. HEK293T cells were transfected with the indicated plasmids. CDK substrates were immunoprecipitated using a phospho-threonine–proline antibody and blotted with anti-Flag antibody to detect phosphorylated zFoxj1a or zFoxj1a-T102A. zFoxj1a-T102A is an unphosphorylated form of zFoxj1a. Note that WT zFoxj1a could be phosphorylated by CDK4 but not by the CDK4-K38M mutant (F). CDK4-mediated phosphorylation was nearly abolished in zFoxj1a-T102A (G). (H) In vitro kinase assays revealed that CDK4 phosphorylates WT zFoxj1a but not the zFoxj1a-T102A mutant. zFoxj1a and zFoxj1a-T102A proteins were purified from bacterial cells and incubated with recombinant Cyclin D1 and CDK4 proteins in the presence or absence of ATP. Phosphorylation of zFoxj1a and zFoxj1a-T102A was detected by western blot using a phospho-threonine–proline antibody, and input proteins were examined by Coomassie blue staining. (I) Ectopic CDK4 expression is unable to stabilize zFoxj1a-T102 mutant. Lysates from HEK293T cells transfected with the indicated plasmids were subjected to immunoblotting. CDK, cyclin-dependent kinase; Foxj1a, forkhead box j1a; GST, glutathione S-transferase; HA, hemagglutinin; HEK, human embryonic kidney; IgG, immunoglobulin G; IP, immunoprecipitation; T102, threonine 102; WB, western blot; WT, wild-type; zFoxj1a, zebrafish Foxj1a; α-Tubulin, acetylated tubulin.</p