Abstract A68: A functional link between the retinoblastoma protein and the small Rho GTPase Rac1

Abstract

Abstract A tumor suppressor commonly targeted in human cancers is the retinoblastoma (pRb) protein, a regulator of the G1/S phase transition of the cell cycle. pRb itself is inactivated with a frequency of over 90% in a subset of human tumors such as retinoblastomas, osteosarcomas, and small cell lung carcinomas. Ours studies suggest a novel role of pRb as a regulator of the activity of the small Rho/GTPase Rac1. Our data show that pRb can regulate Rac1 activity by controlling the levels of the p21-activated protein kinase 1 (PAK1), a main Rac-1 effector. qRT-PCR, immunoblot and immunofluorescence analyses showed that pRb loss in murine osteoblasts results in a dramatic increase of PAK1 mRNA and protein. In order to determine if this increase is due to increased mRNA half-we performed mRNA stability assays using culturing cells with actinomycin D to block new transcription and to assess the half-life of pre-existing PAK1 mRNA molecules. We did not find a significant change in PAK1 mRNA half-life when comparing pRb-expressing vs pRb-deficient osteoblasts, suggesting that pRb regulation of PAK1 is not post-We next tested whether PAK1 regulation by pRb occurs at the trancriptional level, a scenario that would be consistent with pRb's well characterized role as a transcriptional regulator. We used a genomic PCR strategy to amplify several fragments of the PAK1 promoter spanning an area from − 601 to + 159 downstream into the coding region. Fragments were cloned into a luciferase reporter plasmid and the resulting constructs were transfected into pRb-expressing and pRb-osteoblasts, followed by luciferase activity assays. We identified a pRb-responsive element in the promoter fragment spanning from −201 to +159 that seems to be responsible for PAK1 transcriptional repression in pRb-expressing osteoblasts, suggesting the presence of pRb-sensitive elements within this Pak1 promoter fragment. We next performed bioinformatics analyses aimed at identifying binding sites for transcription factor known for their capacity to interact with pRb. We found that this fragment of the PAK1 promoter contains binding sites for several well known transcriptional factors, notable among this being E2F transcription factors. This observation makes it tempting to speculate that pRb represses PAK1 transcription by blocking the E2F mediated transcriptional of the PAK1 gene. Taken together our results implicate pRb in the regulation of the activity of the small Rho/GTPases Rac1. This in turn brings forth the interesting possibility that pRb, acting via small Rho/GTPases, could have additional novel roles as a regulator of processes such as changes in cell shape, migration, and cytoskeletal reorganization, processes in which small Rho GTPases are known to play a role. This project is supported by PSM Institutional Funds, by the U56 Partnership between PSM and the H. Lee Moffitt Cancer Center (Pilot Grant No. 10-14352-02-03), and RISE Program 1R25GMO82406 from NIH-NIGMS. Citation Information: Cancer Res 2009;69(23 Suppl):A68.