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Fig. 4. Enhancement of QSK and SIK activity by 14-3-3 binding. 293 cells were transfected with constructs encoding GST-QSK (A,C) or GST-SIK (B,D). Thirty-six hours post-transfection, the GST-QSK or GST-SIK was absorbed onto glutathione-Sepharose and the beads were incubated with buffer containing no peptide, non-phosphorylated QSK T-loop peptide TPGQLIKTWCGSPPY (non-phos peptide) or phosphorylated QSK T-loop peptide TPGQLIKT(p)WCGSPPY (phos peptide). The beads were then washed in buffer A and GST-QSK or GST-SIK was eluted with glutathione. Similar amounts of the purified GST-QSK (A) or GST-SIK (C) were subjected to immunoblot analysis or assayed with either TORC2 protein substrate (upper panel), or the AMARA peptide (lower panel). The QSK and SIK levels were assessed in the immunoblot analysis using anti-GST antibodies. (C,D) GST-QSK (C) or GST-SIK (D) was also assayed using the TORC2 substrate (upper panel) or AMARA peptide (lower panel) in the presence (+) or absence (-) of wild-type GST-14-3-3 or mutant GST-14-3-3[E180K]. In addition, the samples were immunoblotted with anti-GST antibodies, to ensure similar amounts of GST-QSK and GST-SIK were present in each assay, and with 14-3-3 antibody, to ensure removal of endogenously associated 14-3-3. The immunoblot analysis is representative of at least three separate experiments performed in duplicate. The kinase activities are presented as the mean±s.e.m. for triplicate samples and the results are representative of at least three separate experiments.

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