Abstract: Objective To investigate the protein-protein interactions between the novel candidate tumor suppressor SAM and SH3 domain containing 1 (SASH1) with MAP2K2 and MAP4K4，respectively Methods Retrovirus vector of SBP-Flag- SASH1-pBABE-puro was generated by subcloning SASH1 gene into Xho? and Hpa? sites and transfected into HEK-293T cells for the construction of stable cells expressing exogenous SASH1 after puromycin selection. Exogenous fusion protein expression of Flag-SASH1 was identified by western blot. Pull-down assay, LC-MS/MS analysis and immunoprecipitation were performed to explore the key SASH1 binding proteins which might regulate the proliferation, apoptosis and metastasis of tumor cells. SASH1-siRNA1 and SASH1-siRNA2 was transfected into MDA-MB-231 cell lines, compared with blank group and Negativ - siRNA. Western blot detection the interference effect of SASH1 protein and P-ERK1/2 levels after72 hours.Results Recombinant plasmid of SBP-Flag-SASH1-pBABE-puro was successfully constructed and stably expressed in HEK-293T cells. SASH1 had binding with both MAP2K2 and MAP4K4. The SASH1-siRNA can efficiently block the expression of SASH1, and the expression of P-ERK1/2 was increased in group of SASH1-siRNA. Conclusion MAP2K2 and MAP4K4 is an important candidate binding proteins of SASH1 and SASH1 may cross-talk with ERK1/2 signaling pathways probably through the mediation of them to promote many cell processes including cell proliferation and migration.

Key words: SASH1(SAM and SH3 domain containing 1), extracellular regulated protein kinase, mitogen-activated protein kinase kinase 2, mitogen-activated protein kinase kinase kinase kinase 4.