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Fig. 3. Binding of 14-3-3 to the phosphorylated T-loop of QSK and SIK. (A) 293 cell lysates were incubated with the indicated N-terminal biotinylated peptides encompassing the T-loop of QSK. The peptides were affinity purified on streptavidin-Sepharose and immunoblotted with an antibody recognizing 14-3-3 isoforms. The results are representative of at least two separate experiments performed in duplicate. (B) 1 µg of the indicated biotinylated peptides was conjugated to streptavidin-Sepharose and incubated with indicated amounts of GST-14-3-3
. Following washing, the beads were subjected to immunoblot analysis with a 14-3-3 antibody. The RAF-phospho peptide LSQRQRSTS(P)TPNVHMV binds 14-3-3 with high affinity (Muslin et al., 1996). (C,E) 293 cells were transfected with constructs encoding GST fusion proteins of wild-type (WT), T-loop mutant (T/A) or kinase-inactive (KI) mutants of the indicated AMPK-related kinases. Thirty-six hours post-transfection, the AMPK-related kinases were affinity purified from the cell lysates using glutathione-Sepharose. Similar amounts of the purified GST-fusion proteins were assayed for AMARA peptide kinase activity (activity) or subjected to immunoblot analysis (GST-pull-down). The QSK, SIK and MARK isoform levels were assessed using anti-GST antibodies. The kinase activities are presented as the mean±s.e.m. for triplicate samples relative to the activity observed for the wild-type kinase (100% activity). The results are representative of at least two separate experiments. (D) Equal amounts of wild-type and T-loop mutant of QSK and SIK GST fusion proteins isolated from 293 cells, as described in panel C, were electrophoresed on a polyacrylamide gel and the protein bands visualized by colloidal Coomassie Blue staining. The identity of the bands indicated with an arrow was determined by mass spectrometry. EF1b2 (elongation factor-1b2) is found as a contaminant in most preparations of GST-fusion derived from 293 cells. (F) 500 ng wild-type and T-loop mutant forms of GST-QSK and GST-SIK proteins, as well as wild-type GST-MARK3, were subjected to polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and subjected to a 14-3-3 overlay assay (upper panel) as described in the Materials and Methods. 50 ng of each of the GST-fusion proteins was also subjected to immunoblot analysis with GST antibody (lower panel).
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