The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

Abstract

We previously characterized the retinoblastoma tumor suppressor protein (Rb) as a regulator of adherens junction assembly and cell-to-cell adhesion in osteoblasts. This is a novel function since Rb is predominantly known as a cell cycle repressor. Herein, we characterized the molecular mechanisms by which Rb performs this function, hypothesizing that Rb controls the activity of known regulators of adherens junction assembly. We found that Rb represses the expression of the p21-activated protein kinase (Pak1), an effector of the small Rho GTPase Rac1. Rac1 is a well-known regulator of adherens junction assembly whose increased activity in cancer is linked to perturbations of intercellular adhesion. Using nuclear run-on and luciferase reporter transcription assays, we found that Pak1 repression by Rb is transcriptional, without affecting Pak1 mRNA and protein stability. Pak1 promoter bioinformatics showed multiple E2F1 binding sites within 155 base pairs of the transcriptional start site, and a Pak1-promoter region containing these E2F sites is susceptible to transcriptional inhibition by Rb. Chromatin immunoprecipitations showed that an Rb-E2F complex binds to the region of the Pak1 promoter containing the E2F1 binding sites, suggesting that Pak1 is an E2F target and that the repressive effect of Rb on Pak1 involves blocking the trans-activating capacity of E2F. A bioinformatics analysis showed elevated Pak1 expression in several solid tumors relative to adjacent normal tissue, with both Pak1 and E2F increased relative to normal tissue in breast cancer, supporting a cancer etiology for Pak1 up-regulation. Therefore, we propose that by repressing Pak1 expression, Rb prevents Rac1 hyperactivity usually associated with cancer and related to cytoskeletal derangements that disrupt cell adhesion, consequently enhancing cancer cell migratory capacity. This de-regulation of cell adhesion due to Rb loss could be part of the molecular events associated with cancer progression and metastasis.

The molecular basis of p21-activated kinase-associated neurodevelopmental disorders: From genotype to phenotype

“…Transcriptional and post-transcriptional regulations of each PAK present differences that may also change the way mutations affect brain development and functions. PAK1 expression is activated via FOXO and E2F2 transcription factors and is inhibited by Rb (de la Torre-Ubieta et al, 2010;Sosa-García et al, 2015). PAK2 transcriptional regulation is still an understudied subject, however, PAK2 was found as differentially methylated in children with ASD (Jasoliya et al, 2022).…”

CDK4/6 inhibition synergizes with inhibition of P21-Activated Kinases (PAKs) in lung cancer cell lines

“…Another ATP competitive inhibitor in our custom compound library, FRAX486, has strong activity against PAK1-3, and although some activity was detected against PAK4 it was much less potent [ 28 , 29 ]. Our previous work has shown that PAK1 (p21-activated kinase 1) is regulated by Rb [ 30 ]. Analysis of the PAK6 promotor sequence shows binding sites for E2F suggesting regulation by Rb as well [ 31 ].…”

E2F site in the essential promoter region does not confer S phase-specific transcription of the ABCC10 gene in human prostate cancer cells

“…E2Fs are also known to control transcription of genes referred to as non-classical targets whose functions are not directly involved in cell cycle progression (e.g. p21activated protein kinase, prolyl isomerase Pin1, neo-genin), (Andrusiak et al, 2011;Julian & Blais, 2015;Julian et al, 2016;Ryo et al, 2002;Sosa-Garcia et al, 2015). According to a classical cell cycle regulation model by retinoblastoma RB/E2F pathway, E2F1-3 factors released from pocket protein pRB upon its phosphorylation by cyclin G 1 -dependent kinase complexes (cyclin D/cdk4 and cyclin E/cdk2), transactivate transcription of target genes that mediate S phase entry (e.g.…”