The retinoblastoma protein and the regulation of cell cycling.

Increasing attention has been focused on how the retinoblastoma (RB) protein regulates cell growth. Recent evidence indicates that it is a substrate for phosphorylation by cyclin-dependent kinase-cyclin complexes and suggests that this phosphorylation modulates the ability of this protein to regulate transit through the cell cycle, perhaps in its G1 phase.     

Proline-directed protein phosphorylation and cell cycle regulation.

Recent discoveries have converged on the emerging enzymology that governs the G1-S phase transition of the mammalian somatic cell cycle. These discoveries have led to an appreciation of the regulatory role of proline-directed protein phosphorylation in molecular signalling, and have resulted in the identification of a putative proto-oncogene.     

Preimplantation-embryo-specific cell cycle regulation is attributed to the low expression level of retinoblastoma protein

Biosynthesis of heme A, a prosthetic group of cytochrome oxidase (COX), involves an initial farnesylation of heme B. The heme O product formed in this reaction is modified by hydroxylation of the methyl group at carbon C-8 of the porphyrin ring. This reaction was proposed to be catalyzed by Cox15p, ferredoxin, and ferredoxin reductase. Oxidation of the alcohol to the corresponding aldehyde yields heme A. In the present study we have assayed heme A and heme O in yeast COX mutants. The steady state concentrations of the two hemes in the different strains studied indicate that hydroxylation of heme O, catalyzed by Cox15p, is regulated either by a subunit or assembly intermediate of COX. The heme profiles of the mutants also suggest positive regulation of heme B farnesylation by the hydroxylated intermediate formed at the subsequent step or by Cox15p itself.     

The retinoblastoma protein is phosphorylated during specific phases of the cell cycle

p105-RB is the product of the retinoblastoma tumor suppressor gene. It is a nuclear phosphoprotein hypothesized to act as an inhibitor of cellular proliferation, yet surprisingly it is present in actively dividing cells. To look for changes in p105-RB that may regulate its activity during the cell cycle, we generated synchronized cell populations and followed their progression through the cell cycle. p105-RB is synthesized throughout the cycle, but is phosphorylated in a phase-specific manner. In the G0 and G1 phases of the cell cycle, an unphosphorylated species of the protein is the only detectable form, whereas in the S and G2/M phases, multiple phosphorylated species of p105-RB are detected.     

The retinoblastoma pathway in plant cell cycle and development

The activity of cyclin-dependent kinases (CDKs) on specific targets mediates the temporal regulation of plant cell cycle transitions. The sequential activity of CDKs and the spatial regulation of cell proliferation during plant development, however, are still poorly understood. Understanding these aspects depends on the identification of the downstream targets and upstream modulators of CDKs and their regulation in response to mitogenic and/or differentiation signals. Current efforts to elucidate the answers to these questions are very promising; in particular, recent works reveal the essential role that the retinoblastoma pathway plays in controlling cell cycle progression and, presumably, some developmental events.     

Analysis of site-specific phosphorylation of the retinoblastoma protein during cell cycle progression

Differential phosphorylation of the retinoblastoma protein plays a pivotal role in cell cycle regulation. The retinoblastoma protein is specifically phosphorylated during the cell cycle by cyclin-dependent kinase complexes which intersect with many cellular signaling networks. Since the loss of the retinoblastoma signaling pathways occurs in a wide variety of human tumors, understanding the significance of site-specific phosphorylation can clarify the role of selected cyclin-dependent kinase complexes during cell cycle progression. Here we describe the phosphospecificity and cellular characterization of a panel of polyclonal antibodies that recognize unique phosphorylation sites within the retinoblastoma protein. These reagents were used to validate authentic cellular retinoblastoma phosphorylation sites at amino acids 780, 795, and 807/811 correlating with the G1-S transition.     

Nuclear localization and cell cycle regulation of a murine protein tyrosine phosphatase.

MPTP is a murine homolog of the human T-cell protein tyrosine phosphatase (PTPase) and the rat PTP-S enzyme. Enzymatic activity of this ubiquitously expressed protein was demonstrated in immunoprecipitates from NIH 3T3 cells and in recombinant protein overexpressed in bacteria. Expression of beta-galactosidase-MPTP MPTP chimeric proteins in COS1 cells identified a nuclear localization signal at the carboxyl terminus of the MPTP that was sufficient to direct beta-galactosidase as well as a tagged version of the MPTP to the nucleus. Deletion analysis of amino acids within the nuclear targeting signal showed that this sequence does not conform to the bipartite type of nuclear localization signals. Furthermore, it was shown that the steady-state levels of MPTP RNA fluctuate in a cell cycle-specific manner. On the basis of these experiments, we discuss the possible function of MPTP in the cell cycle and other nuclear processes.     

Human papillomavirus type 16 E6 promotes retinoblastoma protein phosphorylation and cell cycle progression

We show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G(1)/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16(INK4a), CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16(INK4a). The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21(WAF1/CIP1) were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21(WAF1/CIP1) gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21(WAF1/CIP1) levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G(1) arrest imposed by oncogenic ras. Immunofluorescence staining of cells coexpressing ras and E6 from either HPV16 or HPV1 revealed that antiproliferative (p16(INK4a)) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.     

The dynamics of cell cycle regulation

Major events of the cell cycle--DNA synthesis, mitosis and cell division-are regulated by a complex network of protein interactions that control the activities of cyclin-dependent kinases. The network can be modeled by a set of nonlinear differential equations and its behavior predicted by numerical simulation. Computer simulations are necessary for detailed quantitative comparisons between theory and experiment, but they give little insight into the qualitative dynamics of the control system and how molecular interactions determine the fundamental physiological properties of cell replication. To that end, bifurcation diagrams are a useful analytical tool, providing new views of the dynamical organization of the cell cycle, the role of checkpoints in assuring the integrity of the genome, and the abnormal regulation of cell cycle events in mutants. These claims are demonstrated by an analysis of cell cycle regulation in fission yeast.     

Phosphorylation of the Oxytricha telomere protein: possible cell cycle regulation.

In the macronucleus of the ciliate Oxytricha nova, telomeres end with single-stranded (T4G4)2 DNA bound to a heterodimeric telomere protein (alpha beta). Both the alpha and beta subunits (alpha-TP and beta-TP) were phosphorylated in asynchronously growing Oxytricha; beta-TP was phosphorylated to a much higher degree. In vitro, mouse cyclin-dependent kinases (Cdks) phosphorylated beta-TP in a lysine-rich domain that is not required for specific DNA binding but is implicated in higher order structure formation of telomeres. Therefore, phosphorylation of beta-TP could modulate a function of the telomere protein that is separate from specific DNA binding. Phosphoamino acid analysis revealed that the mouse Cdks modify predominantly threonine residues in beta-TP, consistent with the observation that beta-TP contains two consensus Cdk recognition sequences containing threonine residues. In Xenopus egg extracts that undergo cell cycling, beta-TP was phosphorylated in M phase and dephosphorylated in interphase. This work provides the first direct evidence of phosphorylation at telomeres in any organism, as well as indirect evidence for cell cycle regulation of telomere phosphorylation. The Cdc2/cyclin A and Cdc2/cyclin B kinases are required for major mitotic events. An attractive model is that phosphorylation of beta-TP by these kinases is required for the breakdown of telomere associations with each other and/or with nuclear structures prior to nuclear division.     

The retinoblastoma protein-binding region of simian virus 40 large T antigen alters cell cycle regulation in lenses of transgenic mice.

Regulation of the cell cycle is a critical aspect of cellular proliferation, differentiation, and transformation. In many cell types, the differentiation process is accompanied by a loss of proliferative capability, so that terminally differentiated cells become postmitotic and no longer progress through the cell cycle. In the experiments described here, the ocular lens has been used as a system to examine the role of the retinoblastoma protein (pRb) family in regulation of the cell cycle during differentiation. The ocular lens is an ideal system for such studies, since it is composed of just two cell types: epithelial cells, which are capable of proliferation, and fiber cells, which are postmitotic. In order to inactivate pRb in viable mice, genes encoding either a truncated version of simian virus 40 large T antigen or the E7 protein of human papillomavirus were expressed in a lens-specific fashion in transgenic mice. Lens fiber cells in the transgenic mice were found to incorporate bromodeoxyuridine, implying inappropriate entry into the cell cycle. Surprisingly, the lens fiber cells did not proliferate as tumor cells but instead underwent programmed cell death, resulting in lens ablation and microphthalmia. Analogous lens alterations did not occur in mice expressing a modified version of the truncated T antigen that was mutated in the binding domain for the pRb family. These experimental results indicate that the retinoblastoma protein family plays a crucial role in blocking cell cycle progression and maintaining terminal differentiation in lens fiber cells. Apoptotic cell death ensues when fiber cells are induced to remain in or reenter the cell cycle.     

Developmental regulation of the cell cycle.

The control of metazoan cell proliferation, a problem long the domain of cell culture studies, is now being examined in developing animals. Surprisingly, developmental regulation is mediated at a variety of cell-cycle stages. Highly conserved cell-cycle control mechanisms provide a focus for studying the regulatory processes involved.     

Phosphorylation-dephosphorylation of retinoblastoma protein not necessary for passage through the mammalian cell division cycle

Phosphorylation of the retinoblastoma protein (Rb) during the G1-phase of the mammalian cell division cycle is currently believed to be a controlling element regulating the passage of cells into S-phase. We find, however, that the suspension-grown cell lines U937, L1210, and MOLT-4 contain exclusively hyperphosphorylated Rb. Furthermore, when adherent NIH3T3 cells are grown at very low densities to avoid overgrowth and contact inhibition, they also contain only hyperphosphorylated Rb. NIH3T3 cells exhibit hypophosphorylation when the cells are grown at moderate to high cell densities. We propose that cultures of adherent cells such as NIH3T3, when grown to moderate cell densities, are made up of two populations of cells: (a) cells that are relatively isolated and therefore growing exponentially without contact inhibition, and (b) cells that are growth-inhibited by local cell density or contact inhibition. The common observation in adherent cell lines, that Rb is both hyper- and hypophosphorylated in the G1-phase and only hyperphosphorylated in the S- and G2-phases, is explained by the effects of cell density and contact inhibition. Thus, phosphorylation-dephosphorylation of Rb protein during the G1 phase is not a necessary process during the NIH3T3, L1210, MOLT-4, and U937 division cycles. We propose that phosphorylation-dephosphorylation of Rb is independent of the division cycle and is primarily determined by growth conditions throughout the division cycle.     

Accessory protein regulation of microtubule dynamics throughout the cell cycle

A number of accessory proteins capable of stabilizing or destabilizing microtubule polymers in dividing cells have been identified recently. Many of these accessory proteins are modified and regulated by cell-cycle-dependent phosphorylation. Through this regulation, microtubule dynamics are modified to generate rapid microtubule turnover during mitosis. In general, although some microtubule-stabilizing proteins are inactivated at entry into mitosis, a critical balance between microtubule stabilizers and destabilizers is necessary for assembly of the mitotic spindle.     

Role of the retinoblastoma protein in cell cycle arrest mediated by a novel cell surface proliferation inhibitor

A novel cell regulatory sialoglycopeptide (CeReS-18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial-like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS-18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS-18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS-18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS-18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS-18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density-dependent growth inhibition. © 1994 Wiley-Liss, Inc.     

Expression and regulation of protein kinase CK2 during the cell cycle

There are indications from genetic, biochemical and cell biological studies that protein kinase CK2 (formerly casein kinase II) has a variety of functions at different stages in the cell cycle. To further characterize CK2 and its potential roles during cell cycle progression, one of the objectives of this study was to systematically examine the expression of all three subunits of CK2 at different stages in the cell cycle. To achieve this objective, we examined levels of CK2, CK2 and CK2 on immunoblots as well as CK2 activity in samples prepared from: (i) elutriated populations of MANCA (Burkitt lymphoma) cells, (ii) serum-stimulated GL30-92/R (primary human fibroblasts) cells and (iii) drug-arrested chicken bursal lymphoma BK3A cells. On immunoblots, we observed a significant and co-ordinate increase in the expression of CK2 and CK2 following serum stimulation of quiescent human fibroblasts. By comparison, no major fluctuations in CK2 activity were detected during any other stages during the cell cycle. Furthermore, we did not observe any dramatic differences between the relative levels of CK2 to CK2 during different stages in the cell cycle. However, we observed a significant increase in the amount of CK2 relative to CK2 in cells arrested with nocodazole. We also examined the activity of CK2 in extracts or in immunoprecipitates prepared from drug-arrested cells. Of particular interest is the observation that the activity of CK2 is not changed in nocodazole-arrested cells. Since CK2 is maximally phosphorylated in these cells, this result suggests that the phosphorylation of CK2 by p34cdc2 does not affect the catalytic activity of CK2. However, the activity of CK2 was increased by incubation with p34cdc2 in vitro. Since this activation was independent of ATP we speculate that p34cdc2 may have an associated factor that stimulates CK2 activity. Collectively, the observations that relative levels of CK2 increase in mitotic cells, that CK2 and CK2 are phosphorylated in mitotic cells and that p34cdc2 affects CK2 activity in vitro suggest that CK2 does have regulatory functions associated with cell division.