Mass Spectrometric Analysis of Post-Translational Modifications of Xenopus laevis Cytoskeletal Protein Zyxin

Abstract

Objective: Zyxin, a LIM-domain protein within the cytoskeletal system, is actively researched due to its involvement in fundamental cellular processes, from mechanosensory functions and regulation of actin polymerization at cell junctions to gene expression modulation. Disruption of zyxin expression and processing has been linked to carcinogenesis and cardiovascular diseases, and it plays a crucial role in tumor invasion and metastasis. Additionally, the post-translational modifications of zyxin in mammals regulate its activity and subcellular localization. Methods: Given highly conserved evolutionary nature of zyxin, we conducted a search for post-translational modifications of the zyxin homolog in Xenopus laevis using chromatographic mass spectrometry. To identify modified peptides, we utilized an enrichment method involving the co-immunoprecipitation of endogenous zyxin from gastrula-stage embryonic cell lysates. Furthermore, to identify zyxin isoforms with varying electrophoretic mobilities, separation was achieved through polyacrylamide gel electrophoresis. Results and Discussion: Our findings revealed previously unknown modifications of zyxin, specifically, N-terminal acetylation at methionine position 1 and phosphorylation at Ser197 and Ser386. Zyxin was observed in bands corresponding to electrophoretic mobilities of 70 and 105 kDa. This study thus presents novel data regarding the post-translational modifications of zyxin in X. laevis. Conclusions: Since defects in mechanical signal transduction are associated with developmental disorders, oncogenesis, and metastasis, the study of mechanosensitive protein zyxin modifications and processing on the model organism X. laevis opens up opportunities for diagnostic studies at the molecular level, which can be used in the future to determine the prospects for drugs use in pharmacology.