Fig 1: Phosphorylation of SIK3-T221 by recombinant MST3 in vitro. All experiments in this figure except that in H were performed with recombinant proteins expressed in Escherichia coli (Fig. S7 in the companion article). rSIK3 WT, rSIK3 T221A, and rSIK3 T221E were SIK3 59 to 558 WT and its mutant forms. rMST3 was recombinant human MST3 protein expressed in E. coli. MST3b is human MST3b. mMST3 is mouse MST3. rHDAC4 is recombinant human HDAC4 400 to 655. pSIK3 represents SIK3 phosphorylated at T221, whereas pHDAC4 represents HDAC4 phosphorylated at S632. A, recombinant human MST3 protein phosphorylated T221 of recombinant SIK3 (rSIK3) but not T221A or T221E mutants of SIK3. B, recombinant human MST3b protein phosphorylated rSIK3 at T221 but not its T221A or T221E mutants. C, recombinant mouse MST3 protein (r-mMST3) phosphorylated rSIK3 at T221 but not its T221A or T221E mutants. D, HDAC4 is a known target for SIK3. Phosphorylation of recombinant HDAC4 by rSIK3 was enhanced by recombinant MST3. Phosphorylation reactions were 1 h at 37 °C with a final concentration of 1 mM ATP. HDAC4 phosphorylation was detected by an anti–phospho-HDAC4 antibody. E, the Michaelis–Menten constant of MST3 on SIK3. About 0.2 µg rMST3 was incubated with the rSIK3 substrate of different concentrations at 37 °C for 30 min. Results from Western analysis were quantified by the ImageJ software, and the Michaelis–Menten constant (Km) was calculated by the GraphPad software. F, recombinant SIK1 could be phosphorylated at T182 by recombinant human MST3, human MST3b, and mouse MST3. G, recombinant SIK2 could be phosphorylated at T175 by recombinant human MST3, human MST3b, and mouse MST3. H, FLAG-tagged GFP and human MST1 to MST5 were individually expressed in HEK cells and immunoprecipitated by the anti-FLAG antibody. Immunoprecipitated GFP did not phosphorylate rSIK3. SIK3-T221 was phosphorylated strongly by immunoprecipitated MST1, MST2, MST3, and MST5 and weakly by immunoprecipitated MST4. HDAC4, histone deacetylase 4; HEK, human embryonic kidney cell; MST3, mammalian sterile 20–like kinase 3; SIK3, salt-inducible kinase 3; T221, threonine 221.