Proteomic and Interactome Approaches Reveal PAK4, PHB-2, and 14-3-3 eta as Targets of Overactivated Cdc42 in Cellular Responses to Genomic Instability

Author(s)

Silva, Luiz E. ^[1] ; Souza, Renan C. ^[1] ; Kitano, Eduardo S. ^[2] ; Monteiro, Lucas F. ^[1] ; Iwai, Leo K. ^[2] ; Forti, Fabio L. ^[1]

Affiliation

^[1] Univ Sao Paulo, Inst Chem, Dept Biochem, Lab Signaling Biomol Syst LSSB, BR-05508900 Sao Paulo, SP - Brazil

^[2] Butantan Inst, Ctr Toxins Immune Response & Cell Signaling CeT, Special Lab Appl Toxicol LETA, BR-05503000 Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: JOURNAL OF PROTEOME RESEARCH; v. 18, n. 10, p. 3597-3614, OCT 2019.
• Web of Science Citations: 0

Abstract

Cdc42, a member of the Rho GTPase family, is an intracellular signaling protein known for its roles in cytoskeleton rearrangements and, more recently, in apoptosis/senescence triggered by genotoxic stress. In some tumor cells, the overactivation of Cdc42 through the expression of constitutively active mutants (G12V or Q61L), GEF activation, or GAP downregulation functions as an antiproliferative or pro-aging mechanism. In this study, human cell lines with different P53 protein profiles were exposed to UV radiation, and the interactions between Cdc42 and proteins that are putatively involved in the DNA damage response and repair mechanisms were screened. The affinity-purified proteins obtained through pull-down experiments of the cell lysates using the recombinant protein baits GST, GST-Cdc42-WT, or GST-Cdc42-G12V were identified by mass spectrometry. The resulting data were filtered and used for the construction of protein-protein interaction networks. Among several promising proteins, three targets, namely, PAK4, PHB-2, and 14-3-3q, which are involved in the cell cycle, apoptosis, DNA repair, and chromatin remodeling processes, were identified. Biochemical validation experiments showed physical and proximal interactions between Cdc42 and the three targets in the cells, particularly after exposure to UV. The results suggest that the molecular mechanisms coordinated by overactivated Cdc42 (with the G12V mutation) to increase the cellular sensitivity to UV radiation and the susceptibility to cell death are collectively mediated by these three proteins. Therefore, the Cdc42 GTPase can potentially be considered another player involved in maintenance of the genomic stability of human cells during exposure to genotoxic stress. (AU)

• FAPESP's process: 13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling
• Grantee: Hugo Aguirre Armelin
• Support type: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 18/01753-6 - Identification and functional investigation of proteins that interact with Cdc42 and DUSP12 enzymes in human cells under conditions of genomic instability: a proteomic approach
• Grantee: Deborah Schechtman
• Support type: Regular Research Grants