Protein Post-translational Modifications And Cross-talks Between Different Modifications

Csk was originally purified as a non-receptor tyrosine kinase that specifically phosphorylates tyrosine527on the carboxyl-tail of proto-oncogene tyrosine kinase Src, which in turn inhibits Src activity. However, both genetic studies and molecular cellular biological investigations predict that Csk has other protein targets. Considering Csk’s vital function in mouse development, it is important to identify the protein substrates of Csk and to investigate the effects of Csk-mediated phosphorylation on the biological functions of these protein targets.In this study, we identified Eukaryotic elongation factor2(eEF2) as a noval Csk substrate and discovered several modulating mechanisms on eEF2modification and function. We found that in addition to phosphorylation, eEF2can be SUMOylated. eEF2tyrosine phosphorylation was dependent on its SUMOylation, and the SUMOylation in return was enhanced by tyrosine phosphorylation. We provided evidences shown that ubiquitination also present on eEF2, and the ubiquitination and SUMOylation on eEF2were mutually inhibitive. In addition, we discovered that eEF2not only present in the cytoplasm, it also can enter the nuclear. eEF2can be cleaved into many fragments with some fragments entered nuclear. The presence of these nuclear fragments were positively regulated by SUMOyation and tyrosine phosphorylation, but negatively regulated by ubiquitination. We showed that these nuclear fragments played roles in modulating apoptosis, which may be connected with p53in the nuclear. Our study demonstrated that eEF2, as an essential factor in protein synthesis, may have other cellular functions though complex post-translational modifications and the related regulatory network.Using eEF2as an example, we demonstrated above that various protein modifications are critical for the regulation of protein functions. Cross-talks among different types of protein modifications should yield concerted and coordinated regulatory networks for physiological functions. To illustrate the cross-talks between different post-translational modifications at proteome level, we conducted quantitative phosphoproteomics studies on SUMUylation dependent protein phosphorylation. We revealed a global cross-talk for SUMOylation-modulated phosphorylation. Furthermore, as specific examples, we have shown that the subunit of casein kinase Ⅱ is SUMOylated and this affects the phosphorylation of its substrates. SUMO-regulated phosphorylation is involved in cell cycle control. Our data demonstrate interplay between protein SUMOylation and phosphorylation, and imply a regulatory role for this SUMOylation-modulated phosphorylation.