Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity.

To elucidate the regulator-versus-target relationship in the cyclin D1/cdk4/retinoblastoma protein (pRB) pathway, we examined fibroblasts from RB-1 gene-deficient and RB-1 wild-type littermate mouse embryos (ME) and in human tumor cell lines that differed in the status of the RB-1 gene. The RB+/+ and RB-/- ME fibroblasts expressed similar protein levels of D-type cyclins, cdk4, and cdk6, showed analogous spectra and abundance of cellular proteins complexed with cdk4 and/or cyclins D1 and D2, and exhibited comparable associated kinase activities. Of the two human cell lines established from the same sarcoma biopsy, the RB-positive SKUT1B cells contained cdk4 that was mainly associated with D-type cyclins, contrary to a predominant cdk4-p16INK4 complex in the RB-deficient SKUT1A cells. Antibody-mediated neutralization of cyclin D1 arrested the RB-positive ME and SKUT1B cells in G1, whereas this cyclin appeared dispensable in the RB-deficient ME and SKUT1A cells. Lack of requirement for cyclin D1 therefore correlated with absence of functional pRB, regardless of whether active cyclin D1/cdk4 holoenzyme was present in the cells under study. Consistent with a potential role of cyclin D/cdk4 in phosphorylation of pRB, monoclonal anti-cyclin D1 antibodies supporting the associated kinase activity failed to significantly affect proliferation of RB-positive cells, whereas the antibody DCS-6, unable to coprecipitate cdk4, efficiently inhibited G1 progression and prevented pRB phosphorylation in vivo. These data provide evidence for an upstream control function of cyclin D1/cdk4, and a downstream role for pRB, in the order of events regulating transition through late G1 phase of the mammalian cell division cycle.     

Distinct sub-populations of the retinoblastoma protein show a distinct pattern of phosphorylation.

Phosphorylation of the retinoblastoma protein (pRB) is assumed to regulate its growth-controlling function. Moreover, hypophosphorylated and hyperphosphorylated forms of pRB can be distinguished by virtue of the distinct affinities with which they bind to the cell nucleus. This property allows the identification of individual cell nuclei that contain pRB in one or the other form. We show here that after cells emerge from a quiescent (G0) state, conversion of their complement of pRB into a hyperphosphorylated form occurs in late G1, preceding entry into S phase by several hours. Thus, contrary to earlier reports, pRB phosphorylation is not co-ordinated with the G1-S transition and may not directly regulate it. A distinct set of phosphopeptides is found exclusively in those forms of pRB that show the loose nuclear association characteristic of the hyperphosphorylated form of pRB. Another set of phosphopeptides is found with both hypophosphorylated and hyperphosphorylated forms. This suggests the existence of distinct patterns of phosphorylation that are associated with different subsets of pRB molecules. We conclude that substantial phosphorylation of pRB exists in G1 even prior to the hyperphosphorylation point. Cyclin-dependent kinases can cause a liberation of pRB from cell nuclei in vitro. Phosphorylation by members of this kinase family is therefore likely to be directly involved in the change in nuclear affinity in vivo and the associated changes in pRB functioning.     

E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products.

We have studied the initial effects of adenovirus E1A expression on the retinoblastoma (RB) gene product in normal quiescent cells. Although binding of the E1A products to pRB could, in theory, make pRB phosphorylation unnecessary for cell cycle progression, we have found that the 12S wild-type E1A product is capable of inducing phosphorylation of pRB in normal quiescent cells. The induction of pRB phosphorylation correlates with E1A-mediated induction of p34cdc2 expression and kinase activity, consistent with the possibility that p34cdc2 is a pRB kinase. Expression of simian virus 40 T antigen induces similar effects. Induction of pRB phosphorylation is independent of the pRB binding activity of the E1A products; E1A domain 2 mutants do not bind detectable levels of pRB but remain competent to induce pRB phosphorylation and to activate cdc2 protein kinase expression and activity. Although the kinetics of induction are slower, domain 2 mutants induce wild-type levels of pRB phosphorylation and host cell DNA synthesis and yet fail to induce cell proliferation. These results imply that direct physical interaction between the RB and E1A products does not play a required role in the early stages of E1A-mediated cell cycle induction and that pRB phosphorylation is not, of itself, sufficient to allow quiescent cells to divide. These results suggest that the E1A products do not need to bind pRB in order to stimulate resting cells to enter the cell cycle. Indeed, a more important role of the RB binding activity of the E1A products may be to prevent dividing cells from returning to G0.     

Adenovirus E1A is associated with a serine/threonine protein kinase.

The adenovirus E1A proteins form stable protein complexes with a number of cellular proteins, including cyclin A and the product of the retinoblastoma susceptibility gene. We have been interested in learning about the function of proteins associated with E1A and therefore looked for an enzymatic activity present in E1A complexes. We found a serine/threonine kinase activity that phosphorylates two proteins bound to E1A, the 107- and 130-kDa (107K and 130K) proteins. The kinase also phosphorylates histone H1 added as an exogenous substrate. The kinase activity is cell cycle regulated, being most active in S and G2/M-phase cells. The timing of phosphorylation of the 107K protein in vitro correlates with the phosphorylation pattern of the 107K protein in vivo. A variety of genetic and immunochemical approaches indicate that the activity is probably not due to the E1A-associated 300K, 130K, 107K, or pRB protein. Although we have not established the identity of the kinase, we present evidence that the kinase activity is consistent with phosphorylation by p34cdc2 or a related kinase.     

Distinct roles for PP1 and PP2A in phosphorylation of the retinoblastoma protein. PP2a regulates the activities of G(1) cyclin-dependent kinases.

The function of the retinoblastoma protein (pRB) in controlling the G(1) to S transition is regulated by phosphorylation and dephosphorylation on serine and threonine residues. While the roles of cyclin-dependent kinases in phosphorylating and inactivating pRB have been characterized in detail, the roles of protein phosphatases in regulating the G(1)/S transition are not as well understood. We used cell-permeable inhibitors of protein phosphatases 1 and 2A to assess the contributions of these phosphatases in regulating cyclin-dependent kinase activity and pRB phosphorylation. Treating asynchronously growing Balb/c 3T3 cells with PP2A-selective concentrations of either okadaic acid or calyculin A caused a time- and dose-dependent decrease in pRB phosphorylation. Okadaic acid and calyculin A had no effect on pRB phosphatase activity even though PP2A was completely inhibited. The decrease in pRB phosphorylation correlated with inhibitor-induced suppression of G(1) cyclin-dependent kinases including CDK2, CDK4, and CDK6. The inhibitors also caused decreases in the levels of cyclin D2 and cyclin E, and induction of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). The decrease in cyclin-dependent kinase activities were not dependent on induction of cyclin-dependent kinase inhibitors since CDK inhibition still occurred in the presence of actinomycin D or cycloheximide. In contrast, selective inhibition of protein phosphatase 1 with tautomycin inhibited pRB phosphatase activity and maintained pRB in a highly phosphorylated state. The results show that protein phosphatase 1 and protein phosphatase 2A, or 2A-like phosphatases, play distinct roles in regulating pRB function. Protein phosphatase 1 is associated with the direct dephosphorylation of pRB while protein phosphatase 2A is involved in pathways regulating G(1) cyclin-dependent kinase activity.     

The retinoblastoma protein is phosphorylated on multiple sites by human cdc2.

The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase.     

Identification of a cellular protein substrate phosphorylated by the avian sarcoma virus-transforming gene product

The avian sarcoma virus-transforming gene product (pp60src) appears potentially able to mediate cell transformation via phosphorylation since it is tightly associated with a protein kinase activity. We have searched for and have been able to identify a normal cellular protein that appears to be a substrate of pp60src. The phosphorylation of this protein (34K) in transformation-specific in ASV-transformed cells of both avian and mammalian origin. Moreover, the 34K polypeptide serves as a substrate for the pp60src phosphotransferase activity in vitro and is phosphorylated at a site identical to the major site of phosphorylation in vivo. These data suggest that upon transformation the 34,000-dalton protein is phosphorylated directly as a result of pp60src activity.     

Cell cycle arrest by monochloramine through the oxidation of retinoblastoma protein

Impairment of cell cycle control has serious effects on inflammation, tissue repair, and carcinogenesis. We report here the G1 cell cycle arrest by monochloramine (NH2Cl), a physiological oxidant derived from activated neutrophils, and its mechanism. When Jurkat cells were treated with NH2Cl (70 microM, 10 min) and incubated for 24 h, the S phase population decreased significantly with a slight increase in the hypodiploid cell population. The G0/ G1 phase and G2/M phase populations did not show marked changes. Three hours after NH2Cl treatment, the retinoblastoma protein (pRB) was dephosphorylated especially at Ser780 and Ser795, both of which are important phosphorylation sites for the G1 checkpoint function. The phosphorylation at Ser807/811 showed no apparent change. The expression of cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors showed no apparent change. Moreover, the kinase activity that phosphorylates pRB remained constant even after NH2Cl treatment. The protein phosphatase activity that dephosphorylates pRB showed a marginal increase. Notably, when the recombinant pRB was oxidized by NH2Cl in vitro, the oxidized pRB became difficult to be phosphorylated by kinases, especially at Ser780 and Ser795, but not at Ser807/811. Amino acid analysis of oxidized pRB showed methionine oxidation to methionine sulfoxide. The NH2Cl-treated Jurkat cell proteins also showed a decrease in methionine. These observations suggested that direct pRB oxidation was the major cause of NH2Cl-induced cell cycle arrest. In the presence of 2 mM NH4+, NaOCl (200 microM) or activated neutrophils also induced a G1 cell cycle arrest. As protein methionine oxidation has been reported in inflammation and aging, cell cycle modulation by pRB oxidation may occur in various pathological conditions.     

Inhibitory phosphorylation of PP1alpha catalytic subunit during the G(1)/S transition.

We have shown earlier that, in cells expressing the retinoblastoma protein (pRB), a protein phosphatase (PP) 1alpha mutant (T320A) resistant to inhibitory phosphorylation by cyclin-dependent kinases (Cdks) causes G(1) arrest. In this study, we examined the cell cycle-dependent phosphorylation of PP1alpha in vivo using three different antibodies. PP1alpha was phosphorylated at Thr-320 during M-phase and again in late G(1)- through early S-phase. Inhibition of Cdk2 led to a small increase in PP1 activity and also prevented PP1alpha phosphorylation. In vitro, PP1alpha was a substrate for Cdk2 but not Cdk4. In pRB-deficient cells, phosphorylation of PP1alpha occurred in M-phase but not at G(1)/S. G(1)/S phosphorylation was at least partially restored after reintroduction of pRB into these cells. Consistent with this result, PP1alpha phosphorylated at Thr-320 co-precipitated with pRB during G(1)/S but was found in extracts immunodepleted of pRB in M-phase. In conjunction with earlier studies, these results indicate that PP1alpha may control pRB function throughout the cell cycle. In addition, our new results suggest that different subpopulations of PP1alpha regulate the G(1)/S and G(2)/M transitions and that PP1alpha complexed to pRB requires inhibitory phosphorylation by G(1)-specific Cdks in order to prevent untimely reactivation of pRB and permit transition from G(1)- to S-phase and/or complete S-phase.     

Collective inhibition of pRB family proteins by phosphorylation in cells with p16INK4a loss or cyclin E overexpression

The activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G(1) cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin-CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16(INK4a)-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-2 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants, was abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of the RB gene.     

Transforming growth factor beta 1 (TGF beta 1) reduces cellular levels of p34cdc2, and this effect is abrogated by adenovirus independently of the E1A-associated pRB binding activity.

We have used E1A probes to study the roles of the p34cdc2 kinase and the retinoblastoma tumor susceptibility gene product (pRB) in transforming growth factor beta 1 (TGF beta 1)-mediated growth suppression in mink lung epithelial (Mv1Lu) cells. In agreement with previous reports, we see a decline in p34cdc2 kinase activity and a loss of pRB phosphorylation after TGF beta 1 treatment. We report here that TGF beta 1 induces not only a change in p34cdc2 kinase activity but a strong repression of p34cdc2 synthesis. Loss of p34cdc2 kinase activity is not seen until the steady-state level of p34cdc2 declines, suggesting that the intra-cellular signals induced by TGF beta 1 affect p34cdc2 at the level of expression, rather than by altering the posttranslational modifications of p34cdc2 that regulate its kinase activity. Infection with adenovirus expressing either wild-type E1A or a mutant E1A (pm928) defective for pRB binding alleviated TGF beta 1-mediated suppression of DNA synthesis, indicating that E1A does not need to bind pRB physically to keep cell growth-suppressing functions from being activated by TGF beta 1. The E1A.928 mutant virus is able to maintain p34cdc2 expression and kinase activity, as well as pRB phosphorylation in the presence of TGF beta 1, which may account for its ability to maintain cell cycle activity without directly sequestering pRB. Overall our results suggest that TGF beta 1 acts by signaling changes at the level of control of G1 gene expression, not at the level of posttranslational modification of p34cdc2 or its substrates.     

Perturbation of the p53 response by human papillomavirus type 16 E7.

The p53 tumor suppressor protein can induce both cell cycle arrest and apoptosis in DNA-damaged cells. In human carcinoma cell lines expressing wild-type p53, expression of E7 allowed the continuation of full cell cycle progression following DNA damage, indicating that E7 can overcome both G1 and G2 blocks imposed by p53. E7 does not interfere with the initial steps of the p53 response, however, and E7 expressing cells showed enhanced expression of p21(waf1/cip1) and reductions in cyclin E- and A-associated kinase activities following DNA damage. One function of cyclin-dependent kinases is to phosphorylate pRB and activate E2F, thus allowing entry into DNA synthesis. Although E7 may substitute for this activity during cell division by directly targeting pRB, continued cell cycle progression in E7-expressing cells was associated with phosphorylation of pRB, suggesting that E7 permits the retention of some cyclin-dependent kinase activity. One source of this activity may be the E7-associated kinase, which was not inhibited following DNA damage. Despite allowing cell cycle progression, E7 was unable to protect cells from p53-induced apoptosis, and the elevated apoptotic response seen in these cells correlated with the reduction of cyclin A-associated kinase activity. It is possible that inefficient cyclin A-dependent inactivation of E2F at the end of DNA synthesis contributes to the enhanced apoptosis displayed by E7-expressing cells.     

Ras induces anchorage-independent growth by subverting multiple adhesion-regulated cell cycle events.

Anchorage-independent growth is a hallmark of transformed cells, but little is known of the molecular mechanisms that underlie this phenomenon. We describe here studies of cell cycle control of anchorage-independent growth induced by the ras oncogene, with the use of a somatic cell mutant fibroblast line (ER-1-2) that is specifically defective in oncogene-mediated, anchorage-independent growth. Control, nontransformed PKC3-F4 cells and ER-1-2 cells cannot proliferate in semisolid medium. Three important cell cycle events are dependent on adhesion of these cells to a substratum: phosphorylation of the retinoblastoma protein, pRB; cyclin E-dependent kinase activity; and cyclin A expression. PKC3-F4 cells that express ras (PKC3-F4/ras cells) proliferate in nonadherent cultures, and each of these three events occurs in the absence of adhesion in PKC3-F4/ras cells. Thus, ras can override the adhesion requirement of cellular functions that are necessary for cell cycle progression. ER-1-2 cells that express ras (ER-1-2/ras cells) possess hyperphosphorylated forms of pRB and cyclin E-dependent kinase activity in the absence of adhesion but remain adhesion dependent for expression of cyclin A. The adhesion dependence of pRB phosphorylation and cyclin E-dependent kinase activity is therefore dissociable from the adhesion dependence of cyclin A expression. Furthermore, ectopic expression of cyclin A is sufficient to rescue anchorage-independent growth of ER-1-2/ras cells but does not induce anchorage-independent growth of PKC3-F4 or ER-1-2 cells. However, like pRB phosphorylation and cyclin E-dependent kinase activity, the kinase activity associated with ectopically expressed cyclin A is dependent on cell adhesion, and this dependence is overcome by ras. Thus, the induction of anchorage-independent growth by ras may involve multiple signals that lead to both expression of cyclin A and activation of G1 cyclin-dependent kinase activities in the absence of cell adhesion.     

Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B: identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation.

The non-transmembrane protein tyrosine phosphatase, PTP1B, comprises 435 amino acids, of which the C-terminal 114 residues have been implicated in controlling both localization and function of this enzyme. Inspection of the sequence of the C-terminal segment reveals a number of potential sites of phosphorylation. We show that PTP1B is phosphorylated on seryl residues in vivo. Increased phosphorylation of PTP1B is seen to accompany the transition from G2 to M phase of the cell cycle. Two major tryptic phosphopeptides appear in two-dimensional maps of PTP1B from mitotic cells. One of these comigrates with the peptide generated following phosphorylation of PTP1B in vitro at Ser386 by the mitotic protein Ser/Thr kinase p34cdc2:cyclin B. The site of phosphorylation that is responsible for the pronounced retardation in the electrophoretic mobility of PTP1B from mitotic cells has been identified by site directed mutagenesis as Ser352. The identify of the kinase responsible for this modification is presently unknown. We also show that stimulation of HeLa cells with the phorbol ester TPA enhances phosphorylation of PTP1B. Two dimensional phosphopeptide mapping reveals that the bulk of the phosphate is in a single tryptic peptide. The site, identified as Ser378, is also the site of phosphorylation by protein kinase C (PKC) in vitro. Thus the TPA-stimulated phosphorylation of PTP1B in vivo appears to result directly from phosphorylation by PKC. The effect of phosphorylation on the activity of PTP1B has been examined in immunoprecipitates from TPA-treated and nocodazole-arrested cells. TPA treatment does not appear to affect activity directly, whereas the activity of PTP1B from nocodazole-arrested cells is only 70% of that from asynchronous populations.     

Evidence for autocrine activation of a tyrosine kinase in a human gastric carcinoma cell line

Phosphotyrosine (P-Tyr) antibodies have been used to identify the phosphorylated forms of growth factor receptors and oncogene-coded tyrosine kinases. Western blot analysis of a gastric carcinoma cell line with P-Tyr antibodies revealed a tyrosine-phosphorylated protein of Mr 145,000 (P145). In addition, in vitro phosphorylation with (gamma-32P)ATP or P-Tyr immunoprecipitates of the same cells resulted in labelling of this protein on tyrosine. P145 appears to be a transmembrane glycoprotein, with features suggestive of a growth factor receptor. However, the in vivo or in vitro addition of known growth factors did not affect P145 tyrosine phosphorylation. We now report that P145 is rapidly dephosphorylated in vivo when cells are exposed to low pH, a condition known to dissociate ligands from their receptors. The addition of serum-free medium, conditioned by the gastric carcinoma cells, fully restores the tyrosine phosphorylation lost with acid treatment. These data suggest that the activity responsible for P145 phosphorylation on tyrosine, whether intrinsic to P145 itself or due to an associated kinase, is stimulated by a factor secreted by the tumor cells themselves.