NES1, nuclear export transmission 1; NS, not significant; Ser, serine. Since Ser-169 is close to NES1, we hypothesized that O-GlcNAcylation of Zyxin might regulate its nuclear shuttling by affecting the function of NES1. is usually reported to bind with phosphorylated Zyxin. Furthermore, we found that 14-3-3 could promote the nuclear localization of Zyxin after Ser-169 O-GlcNAcylation by affecting the function of the N-terminal nuclear export transmission sequence; functionally, UV treatment increases the O-GlcNAcylation of Zyxin, which may enhance the nuclear location of Zyxin. Finally, Zyxin in the nucleus maintains homeodomain-interacting protein kinase 2 stability and promotes UV-induced cell death. In conclusion, we uncover that this nuclear localization of Zyxin can be regulated by its O-GlcNAcylation, and that this protein may regulate UV-induced cell death. and S1and and converted into rate measurements. DMSO, dimethyl sulfoxide; HEK293T, human embryonic kidney 293T cell collection; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase; Ser, serine; SFB, S protein-Flag-Streptavidin binding peptide; TG, thiamet-G. To identify the O-GlcNAcylated sites on Zyxin, the protein sequence of Zyxin was analyzed using YinOYang 1.2 Server (http://www.cbs.dtu.dk/services/YinOYang/) to find Chelerythrine Chloride the possible O-GlcNAcylation sites (Fig.?S1, and and S1and S1O-GlcNAcylation assay to determine the O-GlcNAcylation on Zyxin. The result showed that Zyxin was directly O-GlcNAcylated by GST-OGT, and the O-GlcNAcylation level of wildtype Zyxin was much Chelerythrine Chloride higher than that of S169A mutant (Fig.?S1and and and test. The scale bars represent 200?m. NS, not significant; OGT, O-GlcNAc transferase. The O-GlcNAcylation of Zyxin Ser-169 enhances its interaction with 14-3-3 The N terminus of Zyxin contains four proline-rich ActA repeats that mediate the interaction between Zyxin and the actin regulator VASP (6, 7). We wondered whether O-GlcNAcylation of Zyxin, which also locates at the N terminus, would affect the interaction between Zyxin and VASP. We examined the interactions between Zyxin and VASP at different O-GlcNAcylation levels and found that there was no significant change (Fig.?S2), indicating that Zyxin O-GlcNAcylation did not affect ActA repeatCmediated protein interactions. Meanwhile, we found that Zyxin O-GlcNAcylation did not affect its interaction with SIAH2 neither (Fig.?S2), Chelerythrine Chloride which mediates the role of Zyxin in regulating Hippo pathway (9). It has been reported that protein O-GlcNAcylation could be recognized by 14-3-3 (34). As 14-3-3 is a Zyxin-associated protein, we conjecture whether the O-GlcNAcylation of Zyxin regulates its interaction with 14-3-3. To confirm this hypothesis, we purified the GST-tagged 14-3-3 proteins and found that GST-14-3-3 could pull down SFB-Zyxin (Fig.?3and S3and of each figure. GST, glutathione-and S3and S3and S3and S3and S3and S3GST pull-down experiments using GST-14-3-3 and His-Zyxin protein. We found that GST-14-3-3 but not GST protein could pull down wildtype His-Zyxin, suggesting that these two proteins did interact directly (Fig.?S3and S3and S4and S4test. Rabbit Polyclonal to LAMA5 The scale bars represent 20?m. C, cytoplasm; DMSO, dimethyl sulfoxide; N, nuclear; NS, not significant; OGT, O-GlcNAc transferase; SFB, S protein-Flag-Streptavidin binding peptide; TG, thiamet-G; TRITC, tetramethylrhodamine isothiocyanate. As Zyxin Ser-169 O-GlcNAcylation promotes its association with 14-3-3, we examined Chelerythrine Chloride whether 14-3-3 participated in the O-GlcNAcylation-mediated Zyxin nuclear localization. Overexpression of 14-3-3 increased the nuclear fraction of endogenous Zyxin (Fig.?4and S4and (36). and test. The scale bars represent 20?m. NES1, nuclear export signal 1; NS, not significant; Ser, serine. Since Ser-169 is close to NES1, we hypothesized that O-GlcNAcylation of Zyxin might regulate its nuclear shuttling by affecting the function of NES1. As Zyxin (1C350 amino acids) only contains NES1, we analyzed the effect of O-GlcNAcylation on Zyxin (1C350 amino acids) nuclear export in the following experiments. The nuclear distribution of Zyxin (1C350 amino acids) was slightly affected by S169A mutant (Figs.?5and S5and S5and S5and S5and S5and S5and S5and S6and and and in HCT116?cell lines was determined by RTCqPCR. test. APC, allophycocyanin; HEK293T, human embryonic kidney 293T cell line; HIPK2, homeodomain-interacting protein kinase 2; NS, not significant; qPCR, quantitative PCR; SFB,.
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