Identification and functional investigation of proteins that interact with Cdc42 and DUSP12 enzymes in human cells under conditions of genomic instability: a proteomic approach

Abstract

Since the last five years our laboratory investigates the participation of enzymes of the Rho GTPases and the dual-specificity phosphatase (DUSP) families in mechanisms of genomic stability maintenance that provide to the cells resistance and/or sensitivity to genotoxic agents. For example, we have shown the involvement of the DUSP3 (or VHR) in the repair of DNA damage induced by gamma ionizing radiation in tumor cells, including the identification of new protein targets/substrates for which this phosphatase could be acting to mediate genomic stability. In other cases we have shown that both the typical small GTPases RhoA and Rac1 are also mediating genomic stability through the DNA damage response pathway (DDR) acting indirectly on the phosphorylation of the Chk1/2 protein kinases and on the ³H2AX damage sensor, which lead to a lower proliferation and survival when defective cells for these GTPases are subjected to genotoxic stress by ultraviolet radiation. Surprisingly, the GTPase Cdc42 acts inversely, its superactivation leads to the greater sensitivity of tumor cells to genotoxic stress, that is, the mechanisms regulated by this enzyme are acting in opposite manner to DDR proteins. Cdc42 was previously described as a strictly cytosolic and/or membrane-associated enzyme with well-defined cytoskeletal regulatory functions, but Cdc42 may have unknown downstream protein effectors and/or regulators that would be correlating this enzyme with genomic stability mechanisms.Another dual phosphatase that we intend to investigate, due to the high homology of its catalytic domain with DUSP3 and the analogous biological functions they mediate, is the DUSP12 or hYVH1. This enzyme and its orthologs have already been implicated in several processes showing a cytoprotective effect in cells exposed to oxidative stress (through physical interaction with the heat shock protein HSP70), as well as their presence and influence in stress granules, particles containing mRNA, translation initiation factors and ribosomal particles, among others. However, what would be the protein targets or substrates of this phosphatase and the molecular mechanisms regulated by them are still open questions in this field.Although Cdc42 and DUSP12 are enzymes with totally different biochemical functions, they appear to be mediating similar biological functions in genomic instability induced by genotoxic agents that reduce cell proliferation. Thus, according to the previous experience of our group, this project proposes to answer the following hypotheses: 1º) what would be the protein partners and probable signaling pathways controlled by the GTPase Cdc42 under cellular conditions of genomic instability that would involve it in sensitizing tumor cells to genotoxic stress?; 2) what would be the enzymatic substrates and/or protein partners of the dual phosphatase DUSP12 under conditions of genomic instability promoted oxidative stress and/or genotoxic agents, for example, that would be responsible for the new or already known biological functions attributed to this enzyme?We intend to identify molecules that interact with these enzymes, to validate these interactions and to show that there may be precise and unknown molecular mechanisms involving them, physically and/or chemically, in mechanisms of genomic stability, and that perturbation on them would lead to cellular disorders under conditions of different types of stress. This research should be conducted in different human cell lines (transformed or not) specifically selected for each enzyme (Cdc42 and DUSP12), and the experimental approach should include basic techniques of: molecular and cellular biology, biochemistry, proteomics, confocal fluorescence microscopy, bioinformatics, and others. Whit it we thus intend to show that GTPase-regulating cytoskeleton and tyrosine phosphatase enzymes might also be components of the complex functional network behind the difficult task of genomic maintenance. (AU)