| Summary: | 博士 === 國立成功大學 === 基礎醫學研究所 === 101 === MST3 (mammalian STE20-like kinase 3) belongs to the Ste20 serine/threonine protein kinase family. The downstream signaling includes protein tyrosine phosphatase PEST (PTP-PEST), paxillin, and nuclear Dbf2-realated (NDR) proteins; however, the role of MST3 in cancer progression has not been studied as thoroughly. Initial examination of the expression of MST3 by immunoblotting revealed overexpression of MST3 in breast cancer tissues. The online Kaplan-Meier plotter analysis revealed that overexpression of MST3 predicts poor prognosis in breast cancer patients. Expression of shMST3 inhibited proliferation and anchorage-independent growth in vitro. Downregulation of MST3 in MDA-MB-231 breast cancer cells decreased tumor formation in NOD/SCID mice. To identify the pathway through which MST3 influences tumor progression, we demonstrated, by co-immunoprecipitation and confocal microscopy, that MST3 interacted with VAV2. By truncating different domains of MST3, we determined that the proline-rich region of MST3 (353KDIPKRP359) interacted with the SH3 domain of VAV2. Overexpression of wild-type MST3 (WT-MST3), but not proline-rich-deleted MST3 (∆P-MST3), enhanced proliferation rates and anchorage-independent growth in SK-Br-3 cells. Overexpression of WT-MST3 increased VAV2 phosphorylation and GTP-Rac1 activation, whereas downregulation of MST3 and ∆P-MST3 resulted in a reduction of VAV2 and Rac1 activation. Knockdown of MST3 inhibited cyclin D1 protein expression. Rac1 inhibitor EHop-016 attenuated induction of cell proliferation and cyclin D1 by WT-MST3 in SK-Br-3 cells. These data indicated that MST3 interacted with VAV2 to induce cyclin D1 to promote the tumorigenicity of breast cancer.
The upstream activators of MST3 remain largely unknown at present,. Our study revealed that MST3 translocated to the membrane when stimulated by EGF in A431 cells. In addition, MST3 kinase activity was induced by EGF. To determine whether membrane translocation plays a role in kinase activation, MST3 was fused with myristoylation (myr) signal peptide to mimic membrane translocation, and it was found that myr-MST3 exhibits higher kinase activity than WT-MST3. Co-immunoprecipitation demonstrated that MST3 formed a complex with EGFR during EGF treatment. Mutations of Y992, Y1086, and Y1148-EGFR weakened the complex formation of EGFR and MST3. The tyrosine residues of MST3 were phosphorylated by treatment with EGF or the phosphatase inhibitor pervanadate. Recombinant EGFR directly phosphorylated MST3 in an in vitro assay. The public domain software NetPhos (ver. 2.0) predicted the phosphorylation sites of MST3 at Y81, Y196 and Y283. Mutations of these three tyrosine residues into phenylalanine revealed that Y196F and Y283F-MST3 exhibited lower tyrosine phosphorylation, but only Y196F-MST3 lost its kinase activity. Overexpression of Y196F- or Y283F-MST3 decreased pAKT and pERK and attenuated cell growth in H1299 lung cancer cells. These data suggest that the membrane location of MST3 was phosphorylated, and that it enhanced the activation of downstream pathways of EGFR to promote cell growth upon EGF stimulation.
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