RESUMEN
Restitution of lost tumor-suppressor activities may be a promising strategy to target specifically cancer cells. However, the action of ectopically expressed tumor-suppressor genes depends on genetic background of tumoral cells. Ectopic expression of p16(INK4a) induces either cell cycle arrest or apoptosis in different pancreatic cancer cell lines. We examined the molecular mechanisms mediating these two different cellular responses to p16 overexpression. Ectopic expression of p16 leads to G1 arrest in NP-9 cells by redistributing p21/p27 CKIs and inhibiting cyclin-dependent kinase CDK2 activity. In contrast, in NP-18 cells cyclin E (CycE)/CDK2 activity is significantly higher and is not downregulated by p16-mediated redistribution of p21/p27. Moreover, inhibition of CDK4 activity with fascaplysine, which does not affect CycE/CDK2 activity, reduces pocket protein phosphorylation in both cell lines, but fails to induce growth arrest. Like overexpression of p16, fascaplysine induces apoptosis in NP-18 cells, suggesting that inhibition of D-type cyclin/CDK activity in cells with high levels of CycE/CDK2 activity activates an apoptotic pathway. Inhibition of CycE/CDK2 activity via ectopic expression of p21 in NP-18 cells overexpressing p16 induces growth arrest and prevents p16-mediated apoptosis. Accordingly, silencing of p21 expression by using small interfering RNA switches the fate of p16-expressing NP-9 cells from cell cycle arrest to apoptosis. Our data suggest that, after CDK4/6 inactivation, the fate of pancreatic tumor cells depends on the ability to modulate CDK2 activity.
Asunto(s)
Quinasas CDC2-CDC28/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Proteínas E2 de Adenovirus/metabolismo , Apoptosis , Quinasas CDC2-CDC28/antagonistas & inhibidores , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Inhibidores Enzimáticos/farmacología , Expresión Génica , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARNRESUMEN
We have previously shown that the adenoviral 12S E1A protein modulates the phosphorylation status of p130 and p107 without apparent changes in the cell cycle dependent phosphorylation of the retinoblastoma protein. Here we report on the mechanisms by which E1A modifies differentially the phosphorylation status of pocket proteins. In human U-2 OS osteosarcoma cells transiently expressing E1A, ectopic expression of D-type cyclins alone or combined, but not cyclins E and/or A, fully rescues E1A-mediated block in hyperphosphorylation of p130 to form 3. However, cyclins E and A, individually or together, induce hyperphosphorylation of p130 to species with intermediate mobility. Phosphopeptide maps indicate that E1A inhibits phosphorylation of sites phosphorylatable by CDKs. One of these sites is Ser-1044. The effects of blocking the activities of endogenous and exogenous cyclins with p16 and dominant negative CDK2 in E1A expressing cells further indicate that p130 is phosphorylated by both D-type cyclin and cyclin E/CDK complexes and that E1A modulates the activity of these G1/S CDKs by independent mechanisms. Stable expression of E1A in MC3T3-E1 cells leads to downregulation of D-type cyclins, and upregulation of cyclins E and A. This is accompanied by increased CDK2 kinase activity. Downregulation of D-type cyclins in these cells correlates with a block on both p130 hyperphosphorylation to form 3 and hyperphosphorylation of p107. This is rescued by D-type cyclins but not by cyclin E. In addition, we show that the upregulation of cyclins E and A is at least partially dependent on an intact pocket protein/E2F pathway, but downregulation of D-type cyclins is not. Moreover, we provide evidence that while the lack of a functional pRB pathway also results in a block on hyperphosphorylation of p130 to form 3, this is not sufficient to induce constitutive expression of p130 form 2b.
Asunto(s)
Proteínas E1A de Adenovirus/fisiología , Quinasas CDC2-CDC28 , Ciclina A/fisiología , Ciclina E/fisiología , Quinasas Ciclina-Dependientes/fisiología , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas , Quinasa 2 Dependiente de la Ciclina , Fase G1 , Humanos , Fosforilación , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Fase S , Células Tumorales CultivadasRESUMEN
The phosphorylation status of the pRB family of growth suppressor proteins is regulated in a cell cycle entry-, progression-, and exit-dependent manner in normal cells. We have shown previously that p130, a member of this family, exhibits patterns of phosphorylated forms associated with various cell growth and differentiation stages. However, human 293 cells, which are transformed cells that express the adenoviral oncoproteins E1A and E1B, exhibit an abnormal pattern of p130 phosphorylated forms. Here we report that, unlike pRB, the phosphorylation status of both p130 and p107 is not modulated during the cell cycle in 293 cells as it is in other cells. Conditional overexpression of individual G(1)/S cyclins in 293 cells does not alter the phosphorylation status of p130, suggesting that the expression of E1A and/or E1B blocks hyperphosphorylation of p130. In agreement with these observations, transient cotransfection of vectors expressing E1A 12S, but not E1B, in combination with pocket proteins into U-2 OS cells blocks hyperphosphorylation of both p130 and p107. However, the phosphorylation status of pRB is not altered by cotransfection of E1A 12S vectors. Moreover, MC3T3-E1 preosteoblasts stably expressing E1A 12S also exhibit a block in hyperphosphorylation of endogenous p130 and p107. Direct binding of E1A to p130 and p107 is not required for the phosphorylation block since E1A 12S mutants defective in binding to the pRB family also block hyperphosphorylation of p130 and p107. Our data reported here identify a novel function of E1A, which affects p130 and p107 but does not affect pRB. Since E1A does not bind the hyperphosphorylated forms of p130, this function of E1A might prevent the existence of "free" hyperphosphorylated p130, which could act as a CDK inhibitor.
Asunto(s)
Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas , Proteína de Retinoblastoma/metabolismo , Línea Celular , Humanos , Fosforilación , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del RetinoblastomaRESUMEN
By binding to the transactivation response element (TAR) RNA, the transcriptional transactivator (Tat) from the human immunodeficiency virus increases rates of elongation rather than initiation of viral transcription. Two cyclin-dependent serine/threonine kinases, CDK7 and CDK9, which phosphorylate the C-terminal domain of RNA polymerase II, have been implicated in Tat transactivation in vivo and in vitro. In this report, we demonstrate that CDK9, which is the kinase component of the positive transcription elongation factor b (P-TEFb) complex, can activate viral transcription when tethered to the heterologous Rev response element RNA via the regulator of expression of virion proteins (Rev). The kinase activity of CDK9 and cyclin T1 is essential for these effects. Moreover, P-TEFb binds to TAR only in the presence of Tat. We conclude that Tat-P-TEFb complexes bind to TAR, where CDK9 modifies RNA polymerase II for the efficient copying of the viral genome.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Productos del Gen tat/metabolismo , VIH-1/genética , Factores de Elongación de Péptidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Activación Transcripcional , Línea Celular Transformada , Ciclina T , Quinasas Ciclina-Dependientes/genética , Productos del Gen rev/genética , Duplicado del Terminal Largo de VIH , Células HeLa , Humanos , Factor B de Elongación Transcripcional Positiva , Transcripción Genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
In the present study we have analysed the regulation of pocket protein expression and post-transcriptional modifications on cell proliferation and differentiation, both in vivo and in vitro. There are marked changes in pocket protein levels during these transitions, the most striking differences being observed between p130 and p107. The mechanisms responsible for regulating pocket protein levels seem to be dependent on both cell type and pocket protein, in addition to their dependence on the cell growth status. Changes in retinoblastoma protein and p107 levels are independent of their state of phosphorylation. However, whereas p130 phosphorylation to forms characteristic of quiescent/differentiated cells results in the accumulation of p130 protein, phosphorylation of p130 to one or more forms characteristic of cycling cells is accompanied by down-regulation of its protein levels. We also show here that the phosphorylation status and protein levels of p130 and p107 are regulated in vivo as in cultured cells. In vivo, changes in p130 forms are correlated with changes in E2F complexes. Moreover, the modulation of p130 and p107 status during cell differentiation in vitro is consistent with the patterns of protein expression and phosphorylation status found in mouse tissues. Thus in addition to the direct disruption of pocket protein/E2F complexes induced by cyclin/cyclin-dependent kinase, the results we report here indicate that the differential modulation of pocket protein levels constitutes a major mechanism that regulates the pool of each pocket protein that is accessible to E2F and/or other transcription factors.
Asunto(s)
Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Proteínas , Proteína de Retinoblastoma/fisiología , Células 3T3/metabolismo , Animales , Diferenciación Celular/fisiología , División Celular/fisiología , Glioblastoma/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Granulocitos/citología , Granulocitos/efectos de los fármacos , Humanos , Regeneración Hepática/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/metabolismo , Fosfoproteínas/biosíntesis , Fosfoproteínas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Ratas , Ratas Sprague-Dawley , Proteína de Retinoblastoma/biosíntesis , Proteína de Retinoblastoma/metabolismo , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Células Tumorales CultivadasRESUMEN
Pocket proteins, including the retinoblastoma susceptibility gene product (pRB) and the related proteins p107 and p130, function at cell cycle regulatory steps that link cyclin/CDK-integrated positive and negative growth signals with E2F transcription factor activity on genes required for cell cycle progression. Protein complex formation between pocket proteins and members of the E2F family of transcription factors determines whether E2F complexes act as transcriptional activators or repressors. Experimental work over the last few years indicates that individual pocket proteins interact with specific E2F members to regulate the transcription of certain genes under diverse cell growth conditions. Among these protein associations, p130-containing E2F complexes seem to be of particular importance in controlling gene transcription in quiescent and differentiating cells by repressing the transcription of a set of E2F-responsive genes. Once the cells are progressing through the G1 phase of the cell cycle, pocket protein-mediated regulation of E2F activity is assumed by pRB and p107. p130-mediated transcriptional regulation thus seems to prevent a gene expression program characteristic of dividing cells at the cell cycle exit and re-entrance transitions and in quiescent cells.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Factor de Transcripción E2F4/fisiología , Proteína p130 Similar a la del Retinoblastoma/fisiología , Animales , Diferenciación Celular/fisiología , Fase G1/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Fosforilación , Fase de Descanso del Ciclo Celular/fisiología , Proteína p107 Similar a la del Retinoblastoma/fisiología , Transcripción Genética/efectos de los fármacosRESUMEN
The retinoblastoma family of proteins, also known as pocket proteins, includes the product of the retinoblastoma susceptibility gene and the functionally and structurally related proteins p107 and p130. Pocket proteins control growth processes in many cell types, and this has been linked to the ability of pocket proteins to interact with a multitude of cellular proteins that regulate gene expression at various levels. By regulating gene expression, pocket proteins control cell cycle progression, cell cycle entry and exit, cell differentiation and apoptosis. This review will focus on the mechanisms of regulation of pocket proteins and how modulation of pocket protein levels and phosphorylation status regulate association with their cellular targets. The coordinated regulation of pocket proteins provides the cells with a competence mechanism for passage through certain cell growth and differentiation transitions.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Proteínas , Proteína de Retinoblastoma/fisiología , Animales , Diferenciación Celular/genética , División Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/fisiología , Ciclinas/metabolismo , Ciclinas/fisiología , Factores de Transcripción E2F , Activación Enzimática , Histona Desacetilasas/fisiología , Humanos , Ratones , Ratones Noqueados , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , ARN Polimerasa I/biosíntesis , ARN Polimerasa III/biosíntesis , Proteína de Retinoblastoma/metabolismo , Proteína 1 de Unión a Retinoblastoma , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
Cyclin T1 has been identified recently as a regulatory subunit of CDK9 and as a component of the transcription elongation factor P-TEFb. Cyclin T1/CDK9 complexes phosphorylate the carboxy terminal domain (CTD) of RNA polymerase II (RNAP II) in vitro. Here we report that the levels of cyclin T1 are dramatically upregulated by two independent signaling pathways triggered respectively by PMA and PHA in primary human peripheral blood lymphocytes (PBLs). Activation of these two pathways in tandem is sufficient for PBLs to enter and progress through the cell cycle. However, the expression of cyclin T1 is not growth and/or cell cycle regulated in other cell types, indicating that regulation of cyclin T1 expression is dependent on tissue-specific signaling pathways. Upregulation of cyclin T1 in stimulated PBLs results in induction of the CTD kinase activity of the cyclin T1/CDK9 complex, which in turn correlates directly with phosphorylation of RNAP II in vivo, linking for the first time activation of the cyclin T1/ CDK9 pair with phosphorylation of RNAP II in vivo. In addition, we report here that endogenous CDK9 and cyclin T1 complexes associate with HIV-1 generated Tat in relevant cells and under physiological conditions (HIV-1 infected T cells). This, together with our results showing that HIV-1 replication in stimulated PBLs correlates with the levels of cyclin T1 protein and associated CTD kinase activity, suggests that the cyclin T1/CDK9 pair is one of the HIV-1 required host cellular cofactors generated during T cell activation.
Asunto(s)
Ciclinas/metabolismo , Activación de Linfocitos , Proteínas Quinasas/metabolismo , Linfocitos T/metabolismo , Regulación hacia Arriba , Ciclo Celular , Células Cultivadas , Ciclina T , Quinasa 9 Dependiente de la Ciclina , VIH-1 , Células HeLa , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Mitógenos/farmacología , Fosforilación , Fitohemaglutininas/farmacología , ARN Polimerasa II/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The mammalian cell cycle engine, which is composed of cyclin/CDK holoenzymes, controls the progression throughout the cell cycle by regulating, at least in part, the transcription of two types of genes: genes whose protein products are required for DNA metabolism and genes whose protein products are involved in cell cycle control. Among the targets of cyclin/CDKs, there is a family of negative growth regulators collectively known as pocket proteins. This family of pocket proteins includes the product of the retinoblastoma tumor suppressor gene, pRB and the functionally and structurally related proteins p107 and p130. In this review, the mechanisms by which pocket proteins are thought to regulate cell growth and differentiation are discussed.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Diferenciación Celular/fisiología , División Celular/fisiología , Proteínas de Unión al ADN , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas , Proteína de Retinoblastoma/metabolismo , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Diferenciación Celular/genética , División Celular/genética , Factores de Transcripción E2F , Humanos , Proteína 1 de Unión a Retinoblastoma , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
We report here experimental evidence indicating that the p21CIP, the universal inhibitor of cyclin-dependent protein kinases, general inhibitor CDKs is a substrate oof cyclin A-cdk2. The evidence comes from phosphorylation experiments in which the endogenous p21CIP present in using the original cyclin A-cdk2 complexes immunoprecipitated from HeLa cells extracts can be phosphorylated by the cdk2 of the same complexes. In vitro experiments showing that reconstituted GSTcyclin A-GSTcdk2 complexes from phosphorylate recombinant GSTp21CIP confirms that p21CIP is a cyclin A-cdk2 substrate.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclina A/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidores Enzimáticos/metabolismo , Células HeLa , Humanos , Fosforilación , Especificidad por SustratoRESUMEN
PITALRE is a human protein kinase belonging to the cell division cycle 2 (CDC2) kinase family, and is the catalytic subunit of a multimeric complex that contains several cellular proteins. PITALRE complexes from several cell lines and tissues phosphorylate retinoblastoma protein and myelin basic protein (MBP). In the present work, we have found that MBP is phosphorylated by PITALRE complexes on both Ser and Thr residues. Two different antibodies raised to PITALRE purified virtually identical kinase activities, as analysed by MBP phosphopeptide mapping and phosphoamino acid analysis. We have identified the proline-directed residue Ser-162 of MBP as a major phosphorylation site for PITALRE. In addition, our results suggest that one of the two MBP proline-directed threonine residues, Thr-97, is also selectively phosphorylated by PITALRE. These data, together with analysis of different peptide substrates derived from sites on MBP that are phosphorylated by PITALRE, indicate that PITALRE is a Ser/Thr proline-directed kinase. In addition, our results show that PITALRE has a substrate site specificity distinguishable from those of the CDC2 and cyclin-dependent kinase 2 (CDK2).
Asunto(s)
Proteína Básica de Mielina/metabolismo , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Quinasa 9 Dependiente de la Ciclina , Electroforesis en Gel Bidimensional , Células HeLa , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Fosfopéptidos/química , Fosforilación , Análisis de Secuencia , Tripsina/metabolismoRESUMEN
PITALRE is a human protein kinase identified by means of its partial sequence identity to the cell division cycle regulatory kinase CDC2. Immunopurified PITALRE protein complexes exhibit an in vitro kinase activity that phosphorylates the retinoblastoma protein, suggesting that PITALRE catalyses this phosphorylation reaction. However, the presence of other kinases in the immunopurified complex could not be ruled out. In the present work, an inactive mutant of the PITALRE kinase has been used to demonstrate that PITALRE is the catalytic subunit responsible for the PITALRE-complex-associated kinase activity, Ectopic overexpression of PITALRE did not increase the total PITALRE kinase activity in the cell, suggesting that PITALRE is regulated by limiting cellular factor(s). Characterization of the PITALRE-containing protein complexes indicated that most of the cellular PITALRE protein exists as a subunit in at least two different active multimeric complexes. Although monomeric PITALRE is also active in vitro, PITALRE present in multimeric complexes exhibits several-fold higher activity than monomeric PITALRE. In addition, overexpression of PITALRE demonstrated the existence of two new associated proteins of approx. 48 and 98 kDa. Altogether these results suggest that, in contrast to the situation with cyclin-dependent kinases, monomeric PITALRE is active, and that association with other proteins modulates its activity and/or its ability to recognize substrates in vivo.
Asunto(s)
Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Catálisis , Línea Celular Transformada , Cromatografía Líquida de Alta Presión , Quinasa 9 Dependiente de la Ciclina , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Hemaglutininas , Humanos , Riñón/enzimología , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutación Puntual , Unión Proteica , TransfecciónRESUMEN
During the transition from G1 to G0, p130 undergoes a specific phosphorylation event-leading to p130-form 2- that is mediated by a kinase/s other than the known G1, S and G2/M cyclin/CDKs. Changes in the phosphorylation status of p130 during this transition are responsible, at least in part, for the concomitant formation of p130/E2F-4 complexes, which are characteristic of G0. These complexes remain abundant during early G1 upon restimulation, but not after mitosis, and are dissociated in mid G1 when p130 is abruptly hyperphosphorylated to form 3. Subsequently, p130 forms 1 and 2 are no longer detected during the remainder of the cell cycle. Consistently, phosphorylation to form 3 and dissociation from E2F-4 complexes is reproduced by a cyclin/CDK holoenzyme in vitro. TGF-beta-induced G1 arrest abrogates cyclin/CDK phosphorylation of p130 but not phosphorylation to form 2. The cell cycle-dependent phosphorylation pattern of p130 is thus shown to comprise two distinct steps that are catalyzed by different kinases. The differential regulation of p130 and pRB phosphorylation during the transition from G1 to G0 may explain the fact that p130 and E2F-4 are the major components of E2F complexes in quiescent cells. Moreover, the newly described phosphorylation of p130 at the transition from G1 to G0 defines a novel mechanism of cell cycle exit regulation.
Asunto(s)
Células 3T3/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Fase G1/fisiología , Fosfoproteínas/metabolismo , Proteínas , Fase de Descanso del Ciclo Celular/fisiología , Factores de Transcripción/metabolismo , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Factor de Transcripción E2F4 , Humanos , Ratones , Fosforilación , Proteína p130 Similar a la del Retinoblastoma , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
To evaluate transcriptional mechanisms during cytokine induction of myeloid progenitor cell proliferation, we examined the expression and activity of transcription factors that control cell cycle-dependent histone genes in interleukin-3 (IL-3)-dependent FDC-P1 cells. Histone genes are transcriptionally upregulated in response to a series of cellular regulatory signals that mediate competency for cell cycle progression of the G1/S-phase transition. We therefore focused on factors that are functionally related to activity of the principal cell cycle regulatory element of the histone H4 promoter: CDC2, cyclin A, as well as RB- and IRF-related proteins. Comparisons were made with activities of ubiquitous transcription factors that influence a broad spectrum of promoters independent of proliferation or expression of tissue-specific phenotypic properties. Northern blot analysis indicates that cellular levels of cyclin A and CDC2 mRNAs increase when DNA synthesis and H4 gene expression are initiated, supporting involvement in cell cycle progression. Using gel-shift assays, incorporating factor-specific antibody and oligonucleotide competition controls, we define three sequential period following cytokine stimulation of FDC-P1 cells when selective upregulation of a subset of transcription factors is observed. In the initial period, the levels of SP1 and HiNF-P are moderately elevated; ATF, AP-1, and HiNF-M/IRF-2 are maximal during the second period; while E2F and HiNF-D, which contain cyclin A as a component, predominate during the third period, coinciding with maximal H4 gene expression and DNA synthesis. Differential regulation of H4 gene transcription factors following growth stimulation is consistent with a principal role of histone gene promoter elements in integrating cues from multiple signaling pathways that control cell cycle induction and progression. Regulation of transcription factors controlling histone gene promoter activity within the context of a staged cascade of responsiveness to cyclins and other physiological mediators of proliferation in FDC-P1 cells provides a paradigm for experimentally addressing interdependent cell cycle and cell growth parameters that are operative in hematopoietic stem cells.
Asunto(s)
Ciclinas/genética , Citocinas/farmacología , Regulación de la Expresión Génica , Granulocitos , Células Madre Hematopoyéticas/citología , Histonas/genética , Factores de Transcripción/genética , Secuencia de Bases , Células de la Médula Ósea , Proteína Quinasa CDC2/genética , Ciclo Celular/genética , División Celular , Línea Celular , ADN/biosíntesis , Células Madre Hematopoyéticas/metabolismo , Cinética , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/biosíntesisRESUMEN
We have previously demonstrated that maize (Zea mays) 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (PGAM-i) is not related to 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase. With the aid of specific anti-maize PGAM-i antibodies, we demonstrate here the presence of a closely related PGAM-i in other plants. We also describe the isolation and sequencing of a cDNA-encoding almond (Prunus amygdalus) PGAM-i that further demonstrates this relationship among plant PGAM-i. A search of the major databases for related sequences allowed us to identify some novel PGAM-i from different sources: plants (Arabidopsis thaliana, Oryza sativa and Antithamniom sp.), monera (Escherichia coli, Bacillus subtilis and Bacillus megaterium) and animals (Caenorhabditis elegans). All of these amino acid sequences share a high degree of homology with plant PGAM-i. These observations suggest that the PGAM-i from several biological kingdoms constitute a family of protein different from other proteins with related enzymatic function and arose from a common ancestral gene that has diverged throughout its evolution.
Asunto(s)
Fosfoglicerato Mutasa/genética , Secuencia de Aminoácidos , Animales , Bacterias , Evolución Biológica , Secuencia Conservada , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Datos de Secuencia Molecular , Plantas , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
The retinoblastoma-related protein p130 is a putative negative regulator of cell proliferation in mammalian cells. In this study, p130 is shown to exist in multiple phosphorylated forms in human cells. In glioblastoma T98G cells synchronized by serum deprivation, specific phosphorylated forms of p130 are found at different times after serum re-stimulation. Two phosphorylated forms of p130 only found in serum-arrested T98G cells and in early G1 phase associate with the adenovirus oncoprotein E1A in vitro. One of these two forms corresponds to the in vivo E1A-associated p130 in 293 cells, which express endogenous E1A protein. Moreover, p130 undergoes an abrupt shift to a unique phosphorylated form in mid G1 which is the only p130 form found during the remaining phases of the cell cycle. This phosphorylated form possesses an associated histone H1 kinase activity that is most active in late S phase and G2/M. The cell cycle-dependent expression pattern of cyclins in T98G cells is compatible with cyclin D1/CDK complexes driving the shift to this phosphorylated p130 form in mid G1. These results suggest that the putative growth inhibitory function of p130 is regulated by phosphorylation of this protein. They also suggest that differential phosphorylation of p130 during the cell cycle plays distinct roles in the regulation of p130 function.
Asunto(s)
Ciclo Celular , Fosfoproteínas/metabolismo , Proteínas , Proteína de Retinoblastoma/metabolismo , Proteínas E1A de Adenovirus/biosíntesis , Proteínas E1A de Adenovirus/metabolismo , Neoplasias Encefálicas , Línea Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Fase G1 , Glioblastoma , Humanos , Cinética , Fosforilación , Proteína p130 Similar a la del Retinoblastoma , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
All eukaryotic cells possess similar mechanisms to regulate the progression of the cell cycle. However, higher eukaryotes have evolved to respond to a large array of positive and negative signals with an intracellular or extracellular origin. These signals are eventually integrated by a conserved protein engine consisting of holoenzymes with kinase activity, which trigger crucial transitions during the cell cycle. In this review, the mechanisms by which the mammalian cell cycle engine integrates intracellular and extracellular signals of different nature are discussed.
Asunto(s)
Ciclo Celular/fisiología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/fisiología , Ciclinas/fisiología , Inhibidores Enzimáticos/farmacología , Genes Supresores de Tumor , Animales , HumanosRESUMEN
Employing the myeloblastic leukemia M1 cell line, which does not express endogenous p53, and genetically engineered variants, it was recently shown that activation of p53, using a p53 temperature-sensitive mutant transgene (p53ts), resulted in rapid apoptosis that was delayed by high level ectopic expression of bcl-2. In this report, advantage has been taken of these M1 variants to investigate the relationship between p53-mediated G1 arrest and apoptosis. Flow cytometric cell cycle analysis has provided evidence that activation of wild-type (wt) p53 function in M1 cells resulted in the induction of G1 growth arrest; this was clearly seen in the M1p53/bcl-2 cells because of the delay in apoptosis that unmasked p53-induced G1 growth arrest. This finding was further corroborated at the molecular level by analysis of the expression and function of key cell cycle regulatory genes in M1p53 versus M1p53/bcl-2 cells after the activation of wt p53 function; events that take place at early times during the p53-induced G1 arrest occur in both the M1p53 and the M1p53/bcl-2 cells, whereas later events occur only in the M1p53/bcl-2 cells, which undergo delayed apoptosis, thereby allowing the cells to complete G1 arrest. Finally, it was observed that a spectrum of p53 target genes implicated in p53-induced growth suppression and apoptosis were similarly regulated, either induced (gadd45, waf1, mdm2, and bax) or suppressed (c-myc and bcl-2), after activation of wt p53 function in M1p53 and M1p53/bcl-2 cells. Taken together, these findings show that wt p53 can simultaneously induce the genetic programs of both G1 growth arrest and apoptosis within the same cell type, in which the genetic program of cell death can proceed in either G1-arrested (M1p53/bcl-2) or cycling (M1p53) cells. These findings increase our understanding of the functions of p53 as a tumor suppressor and how alterations in these functions could contribute to malignancy.
Asunto(s)
Apoptosis , Ciclo Celular , Proteínas Nucleares , Proteína p53 Supresora de Tumor/fisiología , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/fisiología , Expresión Génica , Genes myc , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteínas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas c-mdm2 , ARN Mensajero/genética , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2 , Proteinas GADD45RESUMEN
Cell cycle-controlled human histone genes are coordinately expressed during S phase, and transcriptional regulation involves a series of trans-acting factors (HiNFs). The proliferation-specific factor HiNF-D interacts with multiple recognition motifs in histone H4, H3, and H1 promoters. Using gel shift immunoassays, we show that CDC2, cyclin A, and an RB-related protein are ubiquitous subunits of HiNF-D binding activity isolated from several cell types. HiNF-D levels in vivo are sensitive to okadaic acid and staurosporine, indicating that HiNF-D activity and/or assembly is influenced by phosphorylation status. Thus, HiNF-D appears to be a multicomponent phosphoprotein that participates in coordinate control of multiple histone H4, H3, and H1 genes during the cell cycle. The presence of cell cycle mediators in the HiNF-D complex suggests linkage between transcriptional control of histones, enzymes involved in DNA synthesis, and the onset of DNA replication during the G1/S phase transition.
Asunto(s)
Ciclo Celular , Histonas/genética , Regiones Promotoras Genéticas , Factores de Transcripción/química , Transcripción Genética , Alcaloides/farmacología , Secuencia de Bases , Proteína Quinasa CDC2/metabolismo , Ciclinas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Éteres Cíclicos/farmacología , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Datos de Secuencia Molecular , Ácido Ocadaico , Oligodesoxirribonucleótidos/química , Fosforilación , ARN Mensajero/genética , Proteína de Retinoblastoma/química , Estaurosporina , Activación TranscripcionalRESUMEN
Two different fractions of cdk2 and cdc2 have been found in the nucleus of HeLa cells. One, which can be extracted by nuclease treatment, possibly associated with DNA- or RNA-containing structures and another one, which is bound to the nuclear matrix. Nuclear cdk2 forms high molecular weight complexes which migrate at the same position as DNA polymerase alpha and proliferating cell nuclear antigen in sucrose gradient centrifugation experiments. These results suggest that nuclear cdk2 complexes could be associated with the replication factories. Immunoprecipitation experiments reveal that nuclear cdk2 complexes display histone H1-kinase activity and phosphorylate a protein of 18 kDa which is present in these complexes.