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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Combined experimental and bioinformatics analysis for the prediction and identification of VHR/DUSP3 nuclear targets related to DNA damage and repair

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Author(s):
Forti, Fabio Luis [1]
Total Authors: 1
Affiliation:
[1] Univ Sao Paulo, Inst Chem, Dept Biochem, Lab Signalling Biomol Syst, BR-05508900 Sao Paulo - Brazil
Total Affiliations: 1
Document type: Journal article
Source: INTEGRATIVE BIOLOGY; v. 7, n. 1, p. 73-89, 2015.
Web of Science Citations: 7
Abstract

The atypical dual-specificity phosphatases (aDUSPs) are a group of protein tyrosine phosphatases (PTPs) that have been increasingly studied recently, but little is known about their substrates or their roles and regulation. aDUSPs are typically low-molecular-weight enzymes that are distinct from the mitogenactivated protein kinase phosphatases (MKPs) but that still function in the regulation of the MAPK signalling cascade. aDUSPs may also have non-MAPK substrates, based on homologies observed in the sequences flanking potential phosphotyrosine target sites of other proteins and the cell type-specific characteristics of certain aDUSPs. Here, we combined experimental and computational tools to identify new substrates and protein partners of VHR (DUSP3) phosphatase in HeLa cells exposed to genotoxic stress. Experimental approaches confirmed the good stability of VHR and its nuclear co-localisation with classical MAPK substrates. The bioinformatics analysis of 4539 human nuclear proteins to identify a subset with functions related to DNA damage response and repair or to checkpoints and cell cycle control, that contain the phosphorylatable Thr-X-Tyr motif of MAPK with a high probability of dual phosphorylation, and that have structural homology to the MAPK activation loop resulted in a list of 57 putative VHR substrates. Fluorescence confocal microscopy and pull-down experiments followed by immunoblots revealed that VHR co-localised and interacted with components of the MRN complex and pH2AX, a DNA double-strand break sensor. Our platform, which combines experimental data from structure-function and bioinformatics analyses based on MAPK substrate similarities, provides a lowcost and rapid approach for the identification of novel aDUSP-interacting proteins with unknown roles in genotoxic stress response and genome stability maintenance. (AU)