Effect and mechanism of phosphorylation modification of Thr592 site in SAMHD1 protein on gastric cancer / 安徽医科大学学报

ABSTRACT

Objective @#To elucidate the effect of phosphorylation modification at the threonine 592 (Thr592) site on the inhibition of gastric cancer proliferation by sterile alpha motifs and HD structural domain－containing protein 1 (SAMHD1) and the potential mechanism of action．@*Methods@# Post-translational modifications (PTMs) of SAMHD1 protein in gastric cancer tissues and cell lines in the database were analyzed，and immunohistochemical stai- ning was performed to detect SAMHD1 Thr592 phosphorylation in paired tissues of gastric cancer patients．In gastric cancer cells，SAMHD1 Thr592 variants were constructed and transiently transfected，and cell proliferation was detected using the cell counting kit 8 ( CCK-8 ) method． The phosphorylation of the cyclin-dependent kinases ( CDK) 2 protein threonine 160 (Thr160) site was inhibited by the addition of different concentrations of the CDK6 inhibitor，Palbociclib，which reduced the level of SAMHD1 protein Thr592 phosphorylation．Three online databases were used to analyze the SAMHD1 reciprocal proteins and take the intersection to derive the Nik-related kinase (NRK) protein．Immunoprecipitation ( Co-IP) ，mass spectrometry and Western blot were used to verify the interactions between SAMHD1 and NRK proteins and detect the effect of NRK on the phosphorylation of the SAMHD1 Thr592 site. @*Results @#Compared with PTMs such as ubiquitination，the highest level of phosphorylation modification of SAMHD1 was observed in tumors，and the difference was statistically significant (P＜0. 01) ．Immunohistochemical experiments showed that phosphorylated SAMHD1 (Thr592) was expressed higher in gastric adenocarcinoma than that in normal mucosal tissue adjacent to the cancer，and the difference was statistically significant (P < 0. 01) ．Western blot assay showed that SAMHD1 protein expression was elevated in MKN-45 cells in the overexpression wild type and mutant groups ，and phosphorylated SAMHD1 levels were also elevated in the wild type， T592E and HD / AA groups． CCK-8 assay showed that both SAMHD1 wild type and T592A could inhibit gastric cancer cell proliferation，while T592E and HD / AA had no effect on gastric cancer proliferation． On the basis of overexpression of SAMHD1，CCK-8 suggested that cell proliferation was inhibited after adding different concentrations of Palbociclib treatment，and Western blot assay suggested that the phosphorylation level was also reduced. NRK protein was obtained by Co-IP and mass spectrometry identification to screen the SAMHD1 reciprocal protein profile and database intersection，and NRK was found to interact with SAMHD1 protein and promote phosphorylation at SAMHD1 Thr592 site by Co-IP and Western blot assay．@*Conclusion @#Phosphorylation of the Thr592 site contributes to the loss of SAMHD1 's ability to inhibit gastric cancer cell proliferation，which is reversed by Palbociclib．NRK interacts with SAMHD1 protein，promoting phosphorylation of the SAMHD1 Thr592 site.