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.
Fig 2: SIK2 loss is critically required for LKB1 loss‐driven metastatic uveal melanoma cell proliferation Immunoblot of SIK2 in the indicated whole‐cell lysates of OMM1.3 Ctl or SIK2‐KO cells. Three different OMM1.3 Ctl and SIK2‐KO cell lines are shown. B‐actin was used as a loading control.Colony formation assay of OMM1.3 Ctl and SIK2‐KO cells grown for 10 days. Cells were seeded at low density. Representative images of three independent experiments and crystal violet quantification at OD 561 nm are shown.LKB1‐KO1 cells were transfected with an empty vector (EV) or vectors encoding either a kinase‐dead (SIK2 K49M) or a constitutively active (SIK2 T175D) form of SIK2. Immunoblot (bottom) of SIK2 for the indicated cell lines and colony formation assay (top) are shown. B‐actin was used as a loading control. Representative images of three independent experiments are shown.RT–qPCR analysis of SLC8A1 mRNA level in OMM1.3 Ctl1 and SIK2‐KO1 noninfected (left) or infected with an empty vector (EV) or a vector‐encoding SIK2‐WT. Data represent mean ± SD of three biological replicates (unpaired t‐test with Welsch's correction); ****P = 0.000057/**P = 0.0018; NS, not significant, P = 0.2765.RT–qPCR analysis of SLC8A1 mRNA level in OMM1.3 Ctl1 and LKB1‐KO1 noninfected (left) or infected with an empty vector (EV) or a vector‐encoding SIK2‐WT. Data represent mean ± SD of three biological replicates (unpaired t‐test with Welsch's correction); **P = 0.0036/***P = 0.0005, **P = 0.0040.RT–qPCR analysis of SLC8A1 mRNA level in OMM1.3 SIK2‐KO1 cells treated with control siRNA (siCtl) or two different SLC8A1 siRNA (siSLC8A1). Data represent mean ± SD of three biological replicates (unpaired t‐test with Welsch's correction); ***P = 0.0008/**P = 0.0013.Colony formation assay of OMM1.3 SIK2‐KO1 cells grown for 10 days in the same conditions. A total of 75,000 cells were seeded. Representative images of three independent experiments and crystal violet quantification at OD 561 nm are shown. Data represent mean ± SD of three biological replicates (unpaired t‐test with Welsch's correction); ****P = 0.00000085/****P = 0.000002. Source data are available online for this figure.
Supplier Page from Abcam for Anti-SIK1 (phospho T182) + SIK2 (phospho T175) + SIK3 (phospho T163) antibody [EPR19196]