Genome wide profiling of P21 activated Kinase 1 during DNA Damage response
Open AccessBackground: P21-activated kinase 1 (Pak1), a serine / threonine protein kinase, has been implicated in a variety of cellular processes by phosphorylating its downstream substrates. In addition to its well-documented kinase activity, it is increasingly being recognized that Pak1 affects nuclear events, presumably by modulating co-activator/ co- repressor mediated gene regulation. Earlier studies have demonstrated that Pak1 could be localized in the nuclear compartment and nuclear Pak1 associates with chromatin, thus, raising the possibility that it may be involved in gene transcription which was addressed here. Also, the activation of various protein kinases such as ATM, DNA-PK, ATR and ABL is well documented during DNA damage. However, the role of Pak1 kinase and its targets during DNA damage remains largely unknown. In order to address these questions we carried out genome-wide microarray expression profiling with or without ionization radiation (IR) induced DNA damage. Methodology: Murine Embryonic Fibroblasts were used to generate four sample sets wild-type (WT), Pak1-knockout (Pak1-KO) and wild-type (WT-IR), Pak1-knockout (Pak1-KO-IR) irradiated with ionizing radiation (IR) to induce DNA damage. Genome wide microarray analysis using Affymetrix Mouse Exon 1.0 ST arrays was performed. WT and Pak1-KO samples were analyzed to identify genes whose transcription process might be affected after loss of Pak1. The same samples were also used as controls to compare with the IR treated samples to understand the alterations in the genome after genotoxic insults (IR in our study). Using various comparisons between these sets differentially regulated genes with FDR < 0.05 and fold change of at least ± 2.0 were obtained and hierarchical clustering analysis was performed. Gene Ontology (GO) terms for the target genes were identified to obtain biological significance of the study, followed by the identification of specific functions and canonical pathways. Finally, novel gene targets thus identified were validated using qPCR.Results: We identified 732 significant differentially regulated genes between the WT and Pak1-KO MEFs and 180 `bona fide' Pak1 regulated DNA damage response genes, post IR treatment. 32.61% of the Pak1 regulated genes were found to be involved in molecular functions, 32.08 % are involved in biological processes and the rest 35.31 % are found as the cellular components. Similarly, 15.53% of the Pak1 regulated genes during DNA damage were found to participate in molecular functioning of the cell where as 45.96% were involved in the biological processes and 38.51% are the cellular components. Genes such as Cox7a2l, Pmaip1, Ptgs2, Tmsb4x and Wnt10b whose transcriptional process was regulated by Pak1 and genes such as Ddit4, Eda2r and Gtse1 that were regulated by Pak1 during DNA damage response are few of the targets that were validated using qPCR. These studies reveal important gene targets of pak1 kinase that were not reported so far and hence provide an insight into novel functions of Pak1.
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