Figure 3. Clk2 phosphorylates and represses PGC-1α transcriptional coactivator.

(A) Domain map and in vitro phosphorylation of PGC-1α with indicated deletion by purified recombinant Clk2 kinase (rClk2) in the presence of γ³²P-ATP reaction conditions described in Experimental Procedures. (B) Clk2 induces PGC-1α SR domain phosphorylation recognized by P-Akt substrate antibodies. Western blot analysis of Flag immunoprecipitates from Primary Hepatocytes infected with indicated adenoviruses, all viral loads were normalized with Ad-GFP. (C) Clk2 shRNA block insulin induction of PGC-1α phosphorylation. Western blot analysis of flag immunoprecipitates (IP:Flag) and whole cell extracts of H2.35 hepatocytes infected with indicated adenoviruses. Cells were serum starved overnight prior to insulin stimulation as indicated. PGC-1α transcription co-activation assay in (D) HE3K293 cells and (E) H2.35 hepatocytes using HNF-4α and gAF1-luiferase reporter performed as described in Experimental Procedures. (F) Q-RT-PCR analysis of G6Pase expression in total RNA from primary hepatocytes infected with indicated adenoviruses. Data is presented as average +/- SEM. Significance determined by unpaired two-tailed Students T-Test, ** P<0.01.