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G Draetta

Publications and source records attributed to G Draetta.

At least 19 recordsLinked to original sources
PubMed1992

Distinct forms of human CDC2 identified by novel monoclonal antibodies.

Studies on the functional and structural properties of the cdc2 kinase, a key cell-cycle regulator, have been possible thanks to the availability of cdc2-specific immunoreagents. In an attempt to elucidate the biochemical regulation of the cdc2 kinase in more detail, we have raised a series of novel mouse monoclonal antibodies against human recombinant cdc2 protein. The five Mab reported here can be subclassified into two groups according to their interspecies cross-reactivity and distinct immunoprecipitation patterns. Thus, the target epitopes of Mab POH-1, POH-2 and POH-7 (group 1) appear to be limited to a few mammalian species and the fraction of cdc2 immunoprecipitable by these Mab from cellular extracts is considerably enhanced by denaturation. In contrast, the POH-3 and POH-8 (group 2) Mab recognize a denaturation-sensitive epitope on cdc2 which is present in all tested mammalian species. More importantly, each of the two groups of Mab immunoprecipitate forms of cdc2 associated with a characteristic set of cellular proteins, none of which appears to be cyclin A or cyclin B. None of the antibodies precipitated a histone-H1 or casein-kinase activity, although an activity which phosphorylated some of the coprecipitated proteins was coprecipitated with the group 2 Mab. These novel Mab did not interfere with the association of cdc2 with cyclin A in vitro and efficient immunoprecipitation of a panel of cdc2 mutant proteins suggests that the target epitopes may not involve amino acid residues essential for currently known cdc2 functions. The results of the present study provide evidence for the existence of additional forms of the cdc2 protein in exponentially growing human cells, distinct from both the monomeric and the cyclin-bound cdc2 identified so far.

Animals↗
PubMed1992

The consensus sequences for cdc2 kinase and for casein kinase-2 are mutually incompatible. A study with peptides derived from the beta-subunit of casein kinase-2.

Two series of synthetic peptides that reproduce the amino- and carboxyl-terminal segments of the beta-subunit of casein kinase-2, including the sites phosphorylated by CK2 and cdc2 kinase, respectively, have been used as model substrates for these enzymes. The N-terminal peptide beta(1-9), MSSSEEVSW, is readily phosphorylated by CK2 but not all by cdc2. The opposite is true of the C-terminal peptide beta(206-215), NFKSPVKTIR, whose Ser-4 is a good target for cdc2 while being unaffected by CK2. The individual substitutions of Pro-5 and Lys-7 in the latter peptide with Gly and Ala (or Glu), respectively, prevent its phosphorylation by cdc2, whereas the substitution of Lys-3 with Ala is well tolerated and the substitution of the target Ser with Thr actually improves phosphorylation. Thus the consensus sequence for cdc2 is shown to be X-S-P-X-K. Such a requirement for a basic residue at position +3 is opposite to that of CK2 whose consensus sequence (S-X-X-E/D/Yp/Sp) includes an acidic residue at the same position. Moreover the motif Ser-Pro is detrimental for CK2, preventing the phosphorylation of otherwise suitable peptides. These observations would rule out the possibility that the site specificity of CK2 might overlap with that of cdc2 and possibly of other Pro-directed protein kinases.

Amino Acid Sequence↗
PubMed1992

Association of cdk2 kinase with the transcription factor E2F during S phase.

The transcription factor E2F controls the expression of several proliferation-related genes and is a target of the adenovirus E1A oncogene. In human cells, both cyclin A and the cdk2 protein kinase were found in complexes with E2F. Although the total amounts of cdk2 were constant in the cell cycle, binding to E2F was detected only when cells entered S phase, a time when the cdk2 kinase is activated. These data suggest that the interaction between cdk2 and E2F requires an active kinase that has cyclin A as a targeting component.

Adenovirus Early Proteins↗
PubMed1992

cdc2 protein kinase: structure-function relationships.

Activation of the cdc2 kinase in the cell cycle occurs upon binding to a regulatory subunit called cyclin. Cyclin A associates with both Cdc2 and its homologue Cdk2. The two complexes appear in S phase but cyclin A/Cdk2 is activated earlier than cyclin A/Cdc2. Several regions in Cdc2 are involved in binding cyclins A and B. Phosphorylation of cyclin/Cdk complexes ensures that the kinase activity peaks at a specific time in the cell cycle. Phosphorylation of Thr161 in Cdc2 is required for strong cyclin binding and kinase activity in vitro; its dephosphorylation is necessary for cells to exit mitosis. We have identified a novel 'Activating factor' that stimulates binding between cyclin and Cdc2 by inducing phosphorylation of Cdc2 on Thr161. We propose that Thr161 is targeted by an additional cell cycle regulatory pathway.

CDC2 Protein Kinase↗
PubMed1992

Cyclin A is required at two points in the human cell cycle.

Cyclins play a fundamental role in regulating cell cycle events in all eukaryotic cells. The human cyclin A gene was identified as the site of integration of hepatitis B virus in a hepatocarcinoma cell line; in addition, cyclin A is associated with the E2F transcription factor in a complex which is dissociated by the E1A oncogene product. Such findings suggest that cyclin A is a target for oncogenic signals. We have now found that DNA synthesis and entry into mitosis are inhibited in human cells microinjected with anti-cyclin A antibodies at distinct times. Cyclin A binds both cdk2 and cdc2, giving two distinct cyclin A kinase activities, one appearing in S phase, the other in G2. These results suggest that cyclin A defines novel control points of the human cell cycle.

Adenovirus Early Proteins↗
PubMed1992

Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase.

Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (approximately 15 microM instead of 60 microM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement greater than 6,000 urea-inactivated centrosomes. The material is heat inactivated above 56 degrees C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native centrosomes require approximately 15 microM tubulin to begin nucleating microtubules, whereas centrosomes complemented in interphase extracts begin nucleating microtubules around 7-8 microM tubulin. Therefore, the critical tubulin concentrations for polymer assembly off native centrosomes is higher than that observed for the centrosomes first denatured and then complemented in egg extracts. In vivo, the microtubule nucleating activity of centrosomes seems to be regulated by phosphorylation at the onset of mitosis (Centonze, V. E., and G. G. Borisy. 1990. J. Cell Sci. 95:405-411). Since cyclins are major regulators of mitosis, we tested the effect of adding bacterially produced cyclins to interphase egg extracts. Both cyclin A and B activate an H1 kinase in the extracts. Cyclin A-associated kinase causes an increase in the microtubule nucleating activity of centrosomes complemented in the extract but cyclin B does not. The critical tubulin concentration for polymer assembly off centrosomes complemented in cyclin A-treated extracts is similar to that observed for centrosomes complemented in interphase extracts. However, centrosomes complemented in cyclin A treated extracts nucleate much more microtubules at high tubulin concentration. We define this as the "capacity" of centrosomes to nucleate microtubules. It seems that the microtubule nucleating activity of centrosomes can be defined by two distinct parameters: (a) the critical tubulin concentration at which they begin to nucleate microtubules and (b) their capacity to nucleate microtubules at high tubulin concentrations, the latter being modulated by phosphorylation.

Adenosine Triphosphate↗
PubMed1992

Binding of the human E2F transcription factor to the retinoblastoma protein but not to cyclin A is abolished in HPV-16-immortalized cells.

The adenovirus E1A, SV40 large T and papillomavirus E7 proteins immortalize primary cells by virtue of their ability to bind the retinoblastoma gene product (pRB) and other cellular proteins, including cyclin A and the prRB-related protein, p107. It has been demonstrated that these viral oncogene products will prevent the inhibition of positive growth regulators by pRB, one of them being the E2F transcription factor. Here we show that the interactions of pRB and cyclin A with E2F are present also in normal keratinocytes and in primary human fibroblasts. In human keratinocytes immortalized by human papillomavirus 16 (HPV-16), expressing high levels of HPV-16 E7 protein, complexes between E2F and pRB are disrupted. In this cell line, as well as in HeLa cells which express HPV-18 E7, complexes containing E2F and cyclin A are maintained, indicating that this interaction is not sensitive to the viral oncoprotein and that cyclin A can associate with E2F independently of pRB. In vitro binding experiments suggest that the E7 gene product is able to preferentially abolish the interaction of pRB with E2F, leaving the cyclin A complexes intact. Our findings suggest that E7-dependent immortalization of human cells is associated with modifications of E2F multiprotein complexes.

Base Sequence↗
PubMed1991

cdc2 phosphorylation is required for its interaction with cyclin.

Activation of the cdc2 protein kinase at different stages of the cell cycle is regulated by post-translational modifications and interactions with cyclins. We show that in vitro translated human cdc2 binds very poorly to A and B cyclins, unless it has been preincubated with a Xenopus egg extract. This results in the phosphorylation of cdc2 which allows binding to cyclins. The replacement of Thr161, a residue conserved and phosphorylated in other protein kinases, with valine inhibits cdc2 association with A and B cyclins. In addition, mutations in the amino-terminus of cdc2 and within the conserved 'PSTAIR' region strongly inhibit binding. The Thr161Val mutation causes a lethal phenotype in the fission yeast Schizosaccharomyces pombe, while replacement of Thr161 with glutamic acid, potentially mimicking phosphorylation, causes uncoordination of mitosis and multiple cytokinesis. These results suggest that a threonine phosphorylation/dephosphorylation cycle is involved in regulating cdc2 function.

Amino Acid Sequence↗
PubMed1991

A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena.

Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells.

Animals↗
PubMed1991

Cyclin A, cell cycle control and oncogenesis.

One of the most fundamental questions in biology is how a cell is able to regulate its division cycle. Initially it was thought that in mammalian cells control over entry into the cell cycle is exerted at a restriction point in G1; once past this point the cell would be free to undergo all the steps needed until the following division. Hence, for many years research on tumorigenesis focused on the mitogenic activation of quiescent cells by growth factors, peptide hormones and oncogene products (for reviews see [1, 2]). These studies investigated the initial steps required to induce a quiescent, nondividing cell to proliferate, and led to the identification of many growth factor receptors, of both the tyrosine kinase family and the G-protein coupled family. Receptors bearing protein tyrosine phosphatase or serine kinase catalytic domains were also identified via this route (for reviews see [3, 4, 5]). However more recent studies on the cooperation between different growth factors for mitogenesis have shown that multiple requirements exist for a cell to proceed through the entire division cycle. Indeed studies in several different organisms, pioneered by investigators working with Ascomycetes [6, 7, 8], have now clearly shown that the eukaryotic cell cycle proceeds through multiple check-points. Furthermore, it now appears that many of the regulatory elements and even pathways have been conserved throughout evolution. In this review we discuss the possible involvement of one of the transducing molecules, cyclin A, in abnormal cell proliferation.

Amino Acid Sequence↗
PubMed1991

Mutations at sites involved in Suc1 binding inactivate Cdc2.

suc1+ encodes an essential cell cycle regulator of the fission yeast Schizosaccharomyces pombe. Its product, a 13-kDa protein, interacts with the Cdc2 protein kinase. Both positive and negative effects on cell cycle progression have been attributed to Suc1. To date, the exact mechanisms and the physiological role of the interaction between Suc1 and Cdc2 remain unclear. Here we have studied the molecular basis of this association. We show that Cdc2 can bind Suc1 or its mammalian homolog directly in the absence of any additional protein component. Using an alanine scanning mutagenesis method, we analyzed the interaction between Cdc2 and Suc1. We show that the integrity of several domains on the Cdc2 protein, including sites directly involved in catalytic activity, is required for binding to Suc1. Furthermore, Cdc2 mutant proteins unable to bind Suc1 (but able to bind cyclins) are nonfunctional when overexpressed in S. pombe, indicating that a specific interaction with Suc1 is required for Cdc2 function.

Amino Acid Sequence↗
PubMed1991

Transforming growth factor beta 1 inhibition of p34cdc2 phosphorylation and histone H1 kinase activity is associated with G1/S-phase growth arrest.

Transforming growth factor beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell proliferation. We present data which indicate that epithelial cell proliferation is inhibited when TGF beta 1 is added throughout the prereplicative G1 phase. Cultures become reversibly blocked in late G1 at the G1/S-phase boundary. The inhibitory effects of TGF beta 1 on cell growth occur in the presence of the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Associated with this inhibitory effect is a decrease in the phosphorylation and histone H1 kinase activity of the p34cdc2 protein kinase. These data suggest that TGF beta 1 growth inhibition in epithelial cells involves the regulation of p34cdc2 activity at the G1/S transition.

Animals↗
PubMed1991

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.

Adenoviridae↗
PubMed1990

Fission yeast cdc25 is a cell-cycle regulated protein.

Fission yeast cell division is initiated by the cdc2/cdc13-cyclin protein kinase which in its catalytically active state comprises the mitotic inducer. During interphase the cdc2/cyclin complex is assembled in an inactive state that requires cdc25+ gene function for M-phase activation. The cdc25+ product, a 76 kd phosphoprotein, is shown to oscillate in abundance during the cell cycle, reaching a peak at G2/M, and to be sensitive to nitrogen starvation. The level of cdc25 is subject to feedback regulation involving both cdc25 and cdc2.

Blotting, Western↗
PubMed1990

Cell cycle control in eukaryotes: molecular mechanisms of cdc2 activation.

cdc2 kinase regulates the progression of eukaryotic cells through the division cycle. Events such as cell growth, DNA replication and mitosis are coordinated through the activation of specific forms of this kinase. Here I discuss our present knowledge of the mechanisms that regulate the activity of cdc2 kinase.

Amino Acid Sequence↗
PubMed1989

A 60 kd cdc2-associated polypeptide complexes with the E1A proteins in adenovirus-infected cells.

p60 is a cellular protein that binds to the adenovirus E1A protein complex in virally infected or transformed human cells. In both infected and uninfected cells, p60 was found in a complex with the cdc2 protein kinase. Immune complexes containing p60 and cdc2 display a cell cycle-dependent histone H1 kinase activity that is most active in interphase. The previously described cdc2-p62/cyclin complex also acts as a histone H1 kinase but is maximally active in mitotic metaphase. The shift in the timing of activation of different cdc2-containing complexes suggests that each might play a distinct role in regulation of the cell cycle.

Adenovirus Early Proteins↗