The authors declare that they have no conflicts of interest with the contents of this article. in pancreatic cancer cells and to a greater extent than did wild-type Vgll4, suggesting that mitotic phosphorylation inhibits Vgll4’s tumor-suppressive activity. Consistent with these observations, the Vgll4-4A mutant possessed higher-binding affinity to TEAD1 than wild-type Vgll4. Interestingly, Vgll4 and Vgll4-4A markedly suppressed YAP and -catenin signaling activity. Together, these findings reveal a previously unrecognized mechanism for Vgll4 regulation in mitosis and its role in tumorigenesis. and and * mark the modest and significant inhibition of mobility upshift, respectively. Phospho-Aurora levels (in and kinase assays with GST-tagged Vgll4 proteins as substrates. Fig. 2shows that purified CDK1Ccyclin B kinase complex phosphorylated GST-Vgll4 proteins (Fig. 2kinase assays with purified kinases. kinase assays with purified CDK1Ccyclin B kinase complex. kinase assays were done as in except anti-phospho-Vgll4 antibodies were used. RO3306 (5 m) was used to inhibit CDK1 kinase activity. The phospho-Vgll4 Ser-155/Thr-159 antibody was labeled as kinase assay, suggesting that these four sites are the main phosphorylation sites for CDK1 (Fig. 2kinase assays confirmed that CDK1 robustly phosphorylates Vgll4 at all these sites (Fig. 2and and data (Fig. 2and and and and with phospho-specific antibody against Ser-280 of Vgll4. and mark some of the prometaphase cells and the interphase cells, respectively. Vgll4 phosphorylation occurred during normal mitosis Taxol or nocodazole was used to arrest cells in G2/M phase in all of GSK-2193874 the above experiments. We wanted to determine whether phosphorylation of Vgll4 occurs during normal mitosis. We performed immunofluorescence staining on cells collected from a double thymidine block and release (21). Consistent with the results in Fig. 4, very weak signals were detected in interphase or telophase/cytokinesis cells (Fig. 5, and with p-Vgll4 Ser-280 antibodies. and (in and and < 0.001; **, < 0.01; *, < 0.05 (test). and < 0.001; *, < 0.05 (test). and < 0.001; **, < 0.01 (test). Mitotic phosphorylation of Vgll4 inhibits its tumor-suppressing activity in vivo We next evaluated the influence of mitotic phosphorylation of Vgll4 on tumor growth in animals. BxPC3 cells expressing wild-type Vgll4 or Vgll4-4A were subcutaneously inoculated into immuno-deficient mice. Interestingly, tumors from mice harboring Vgll4-4A-expressing cells were significantly smaller when compared with those from mice injected with wild-type Vgll4-expressing cells (Fig. 7, and and data not shown). Immunohistochemistry staining with cleaved caspase-3 (an apoptosis marker) showed that expression of Vgll4-4A significantly promoted tumor cell death (Fig. 7< 0.001; **, < 0.01; *, < 0.05 (test). < 0.001; **, < 0.01(test). and and < 0.001; **, < 0.01; *, < 0.05 (test). Mitotic phosphorylation of Vgll4 affected YAP and -catenin activity Vgll4 competes with YAP to associate with TEADs (7). The association between Vgll4 and TEAD1 was confirmed with transfected proteins (Fig. 8, and and and and < 0.05 (test). < 0.01; ***, < 0.001 (test). < 0.05 (test). and at Ser-58, Ser-155/Thr-159, and Ser-280 during mitosis (Figs. 2?2?C5). Recently, we reported that CDK1-mediated phosphorylation of YAP promotes mitotic defects, including GSK-2193874 centrosome amplification and chromosome missegregation, and potentiates oncogenic functions of YAP (16, 22). Considering that CDK1 phosphorylates both YAP and Vgll4 during mitosis and these proteins function together in regulating tumorigenesis, one question is whether these phosphorylation events affect each other during mitosis. Mechanistic elucidation of this unanswered question will help us further understand the regulation and GSK-2193874 role of Vgll4 in normal and cancer cells. Many members of the Hippo-YAP pathway, including YAP, have been shown previously to be associated GSK-2193874 with the mitotic machinery and to cause mitotic defects when dysregulated. Therefore, future studies are required to define the role of Vgll4 and its phosphorylation in cell cycle progression, especially in mitosis-related events. Another interesting finding from this study is that Vgll4 is a phospho-protein (multiple bands were observed on Phos-tag gels) (Fig. 1and xenograft studies, S2.013 cells expressing wild-type Vgll4 or Vgll4-4A or BxPC3 cells expressing TetOn-Vgll4 or TetOn-Vgll4-4A (non-phosphorylatable mutant) (1.0 106 cells each line) were subcutaneously injected into both flanks Rabbit polyclonal to V5 of 6-week-old male athymic nude mice (Ncr-nu/nu, Harlan). S2.013 cells were suspended in PBS, and BxPC3 cells were mixed with Matrigel at 1:1 ratio (volume). Five animals were used per group. Doxycycline (0.5 mg/ml in 5% sucrose water) was administered beginning GSK-2193874 at the time of.