Sqd interacts with the Drosophila retinoblastoma tumor suppressor Rbf

The retinoblastoma tumor suppressor (RB) serves as a scaffold to coordinate binding of numerous proteins, including E2F and histone deacetylases, through its C-terminal domain. The amino-terminal half of RB has few known binding partners and its function is not well understood. We used the amino-terminal domain of the Drosophila retinoblastoma tumor suppressor Rbf (RbfN) to identify novel binding partners by immunoprecipitation coupled with mass spectrometry. Our experiment revealed that the RNA-binding protein Squid (Sqd) is a putative interacting partner of RbfN. Western blot confirmed that Sqd interacts with the amino-terminal domain of Rbf. We observed that Sqd colocalizes with RbfN in Drosophila salivary gland cells. We also show that double RNAi knockdown of Rbf and Sqd in the eye results in an extensive loss of eye bristles, suggesting that Rbf and Sqd function in a common pathway. We conclude from our studies that Rbf physically and genetically interacts with Sqd. We propose that the retinoblastoma tumor suppressor may play a novel role in RNA processing through interaction with RNA-binding proteins.     

p55, the Drosophila ortholog of RbAp46/RbAp48, is required for the repression of dE2F2/RBF-regulated genes

Many proteins have been proposed to be involved in retinoblastoma protein (pRB)-mediated repression, but it is largely uncertain which cofactors are essential for pRB to repress endogenous E2F-regulated promoters. Here we have taken advantage of the stream-lined Drosophila dE2F/RBF pathway, which has only two E2Fs (dE2F1 and dE2F2), and two pRB family members (RBF1 and RBF2). With RNA interference (RNAi), we depleted potential corepressors and looked for the elevated expression of groups of E2F target genes that are known to be directly regulated by RBF1 and RBF2. Previous studies have implicated histone deacetylase (HDAC) and SWI/SNF chromatin-modifying complexes in pRB-mediated repression. However, our results fail to support the idea that the SWI/SNF proteins are required for RBF-mediated repression and suggest that a requirement for HDAC activities is likely to be limited to a subset of targets. We found that the chromatin assembly factor p55/dCAF-1 is essential for the repression of dE2F2-regulated targets. The removal of p55 deregulated the expression of E2F targets that are normally repressed by dE2F2/RBF1 and dE2F2/RBF2 complexes in a cell cycle-independent manner but had no effect on the expression of E2F targets that are normally coupled with cell proliferation. The results indicate that the mechanisms of RBF regulation at these two types of E2F targets are different and suggest that p55, and perhaps p55's mammalian orthologs RbAp46 and RbAp48, have a conserved function in repression by pRB-related proteins.     

Nucleolar GTP-binding Protein-1 (NGP-1) Promotes G1 to S Phase Transition by Activating Cyclin-dependent Kinase Inhibitor p21Cip1/Waf1

Nucleolar GTP-binding protein (NGP-1) is overexpressed in various cancers and proliferating cells, but the functional significance remains unknown. In this study, we show that NGP-1 promotes G₁ to S phase transition of cells by enhancing CDK inhibitor p21^Cip-1/Waf1 expression through p53. In addition, our results suggest that activation of the cyclin D1-CDK4 complex by NGP-1 via maintaining the stoichiometry between cyclin D1-CDK4 complex and p21 resulted in hyperphosphorylation of retinoblastoma protein at serine 780 (p-RB^Ser-780) followed by the up-regulation of E2F1 target genes required to promote G₁ to S phase transition. Furthermore, our data suggest that ribosomal protein RPL23A interacts with NGP-1 and abolishes NGP-1-induced p53 activity by enhancing Mdm2-mediated p53 polyubiquitination. Finally, reduction of p-RB^Ser-780 levels and E2F1 target gene expression upon ectopic expression of RPL23a resulted in arrest at the G₁ phase of the cell cycle. Collectively, this investigation provides evidence that NGP-1 promotes cell cycle progression through the activation of the p53/p21^Cip-1/Waf1 pathway.