No abstract

In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G1 and becomes hyper-phosphorylated in late G1. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F in vivo and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15(INK4b) by transforming growth factor beta treatment of keratinocytes results in G1 arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15(INK4b)-independent transforming growth factor beta-mediated G1 arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16(INK4a) protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G1 allowing continued E2F binding.

The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G 1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G 1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G 0 quiescent cells and becomes hypophosphorylated (ϳ2 mol of PO 4 to 1 mol of pRB) in early G 1 and hyperphosphorylated (ϳ10 mol of PO 4 to 1 mol of pRB) in late G 1 phase. Here, we report that hypophosphorylated pRB, present in early G 1 , represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G 0 and hyperphosphorylated pRB in late G 1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TATdominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G 1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G 1 . Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G 1 and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G 1 cyclin-Cdk complexes.Stimulation by growth factors of resting G 0 quiescent cells to enter the early-G 1 phase of the cell cycle and to transit across the restriction point into late G 1 phase requires the concerted activities of multiple cyclin-dependent kinases (Cdks) that phosphorylate substrates in a cell cycle-specific fashion (for reviews, see references 13, 42, 56, and 64). Activation of cyclin E-Cdk2 at the late G 1 restriction point and activation of cyclin A-Cdk2 at the transition from late G 1 to S phase suggest the involvement of these cyclin-Cdk complexes at specific cell cycle regulatory checkpoints (42,56,57). In contrast, cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) complexes are inactive in G 0 quiescent cells but become activated by growth factor addition in early G 1 phase (36, 38). In addition, whereas cyclin Eand A-associated kinase activities remain cell cycle regulated in cycling cells, cyclin D-Cdk4/6 activity is constitutive in cycling cells (5,17,35,44). Importantly, these observations indicate a distinct role for cyclin D-Cdk4/6 complexes in regulating G 1 cell cycle progression from that of cyclin E-Cdk2 and cyclin A-Cdk2. Indeed, Datar et al. (7) and Meyer et al. (37) using genetic models of Drosophila melanogaster have recently demonstrated that cyclin D-Cdk4 complexes regulate cellular growth (accumulation of mass) and not c...

TNF is a pleiotropic cytokine required for normal development and function of the immune system; however, TNF overexpression also induces inflammation and is associated with autoimmune diseases. TNF exists as both a soluble and a transmembrane protein. Genetic studies in mice have suggested that inflammation in disease models involves soluble TNF (solTNF) and that maintenance of innate immune function involves transmembrane TNF (tmTNF). These findings imply that selective pharmacologic inhibition of solTNF may be anti-inflammatory and yet preserve innate immunity to infection. To address this hypothesis, we now describe dominant-negative inhibitors of TNF (DN-TNFs) as a new class of biologics that selectively inhibits solTNF. DN-TNFs blocked solTNF activity in human and mouse cells, a human blood cytokine release assay, and two mouse arthritis models. In contrast, DN-TNFs neither inhibited the activity of human or mouse tmTNF nor suppressed innate immunity to Listeria infection in mice. These results establish DN-TNFs as the first selective inhibitors of solTNF, demonstrate that inflammation in mouse arthritis models is primarily driven by solTNF, and suggest that the maintenance of tmTNF activity may improve the therapeutic index of future anti-inflammatory agents.