scholarly journals A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

1997 ◽  
Vol 17 (12) ◽  
pp. 7362-7374 ◽  
Author(s):  
J A Diehl ◽  
C J Sherr
Keyword(s):  
Cyclin D1 ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Cyclin Dependent Kinase ◽  
Linker Peptide ◽  
Cdk Activating Kinase

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex

Development ◽  
1999 ◽  
Vol 126 (22) ◽  
pp. 5137-5148 ◽  
Author(s):  
H.D. Ryoo ◽  
T. Marty ◽  
F. Casares ◽  
M. Affolter ◽  
R.S. Mann
Keyword(s):  
Nuclear Localization ◽  
Target Genes ◽  
Dominant Negative ◽  
Homeodomain Protein ◽  
Hox Proteins ◽  
Trimeric Complex ◽  
Binding Residue ◽  
Dominant Negative Form

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.

Combined Epigenetic and Immunotherapy For Newly Diagnosed Mantle Cell Lymphoma: Correlative Studies Suggest The Importance Of Enhanced ADCC, Mechanisms of Resistance and Cyclin D1 Nuclear Localization Genotype

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3063-3063
Author(s):  
Kamal Sharma ◽  
Violetta V. Leshchenko ◽  
Zainul Hasanali ◽  
August Stuart ◽  
Sara Shimko ◽  
...  
Keyword(s):  
Complete Remission ◽  
Cyclin D1 ◽  
Nuclear Localization ◽  
Cell Lymphoma ◽  
Newly Diagnosed ◽  
Mantle Cell ◽  
Correlative Studies

Abstract Previously, we reported that epigenetic therapy with cladribine, SAHA, and rituximab (SCR) for newly diagnosed mantle cell lymphoma was remarkably effective, with 100% overall response rate, 85-90% CR rate, and durable responses( Hasanali, AACR,2013 LBA140). Over 40 patients now have been enrolled with all patients completing therapy. Final response and CR rates will be reported at the meeting. This abstract will focus on the correlative studies performed as part of this trial. Cladribine, a purine analog with reported epigenetic activity was shown here by HELP assays to inhibit DNA methylation in vivo in 6 patients with leukemic MCL. Similar activity was also observed in two MCL and two CLL patients treated with cladribine without vorinostat off trial, suggesting cladribine is a DNA hypomethylating agent. Due to cladribine's ability to inhibit the enzyme SAH hydrolase and thus inhibit the donation of methyl groups by S-adenosyl methionine (SAM), we assayed the ability of cladibine to inhibit histone methylation in vitro by Western blot analysis and in vitro assays of histone methyltransferase (HMT) activity on H3lys9 and H3lys27. Both assays demonstrated inhibition of methylated histones (Western) and HMT activity using MCL cell cells and nuclear extract at concentrations of cladribine in the 10-20 um range, higher than the in vivo concentration of 10-20 nm. These observations could be due to the lack of sensitivity of these assays, and more sensitive assays are in development. Studies to help elucidate the mechanism of action of synergy of epigenetic drugs with the monoclonal antibody rituximab were performed. Using cells from patients with leukemic MCL treated with SCR, we assayed for characteristic changes of apoptosis using Western blotting and TUNEL assays. None were detected. We were unable to observe complement mediated cytoxicity in vitro using human serum and rituximab with added cladribine or vorinostat. With ADCC being the primary mechanism of presumed combined epigenetic and rituximab synergy, we investigated ADCC further. CD137 transcriptional upregulation was seen in several but not all treated patients, and some patients showed up regulation of perforin and granzyme mRNA by QRTPCR. An NK cell line, NKL, showed transcriptional upregulation of CD137 after treatment with cladribine and vorinostat. A polymorphism at an intron-exon junction effects the nuclear localization of cyclin D1 by removing a nuclear export signal. Although there is published evidence supporting the role of nuclear cyclin D1 in increased oncogenesis, the role of this polymorphism in MCL remains controversial. Samples from peripheral blood of patients on trial were genotyped at the cyclin D1 locus as AA, AG, or GG, with the A allele being the loss of function allele. The presence of the A allele strongly correlated with the blastic phenotype and the lack of complete remission after SCR therapy, with both being statistically significant (table 1). Immunofluorescent studies with cyclin D1 antibodies showed nuclear and cytoplasmic localization as predicted in patients with the AA and GG genotypes (Fig 1). The heterozygotes are under investigation and will be reported. The mechanism of resistance to SCR was studied in a patient with blastic, leukemic MCL. A 63 male achieved complete remission after two cycles of SCR. Subsequently, he developed neurologic symptoms and was found to have CNS disease. At autopsy, CD20+ disease was found in his CNS and CD20- disease was found systemically. A cell line was established from his peripheral blood that showed significantly reduced levels of CD20 mRNA. Treatment of these cells with a variety of epigenetic drugs was unable to upregulate CD20 mRNA. These cells have been in continuous culture for over 1 year and continue to show diminished levels of CD20 mRNA and protein. Epigenetic changes at the promoter are being studied by chromatin immunoprecipation (ChiP) assays. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cyclin-dependent Kinases Participate in Death of Neurons Evoked by DNA-damaging Agents

1998 ◽  
Vol 143 (2) ◽  
pp. 457-467 ◽  
Author(s):  
David S. Park ◽  
Erick J. Morris ◽  
Jaya Padmanabhan ◽  
Michael L. Shelanski ◽  
Herbert M. Geller ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Sindbis Virus ◽  
Kinase Inhibitors ◽  
Sympathetic Neurons ◽  
Dominant Negative ◽  
Cyclin Dependent Kinase ◽  
Cyclin Dependent Kinases ◽  
Dna Damaging Agents

Previous reports have indicated that DNA-damaging treatments including certain anticancer therapeutics cause death of postmitotic nerve cells both in vitro and in vivo. Accordingly, it has become important to understand the signaling events that control this process. We recently hypothesized that certain cell cycle molecules may play an important role in neuronal death signaling evoked by DNA damage. Consequently, we examined whether cyclin-dependent kinase inhibitors (CKIs) and dominant-negative (DN) cyclin-dependent kinases (CDK) protect sympathetic and cortical neurons against DNA-damaging conditions. We show that Sindbis virus–induced expression of CKIs p16ink4, p21waf/cip1, and p27kip1, as well as DN-Cdk4 and 6, but not DN-Cdk2 or 3, protect sympathetic neurons against UV irradiation– and AraC-induced death. We also demonstrate that the CKIs p16 and p27 as well as DN-Cdk4 and 6 but not DN-Cdk2 or 3 protect cortical neurons from the DNA damaging agent camptothecin. Finally, in consonance with our hypothesis and these results, cyclin D1–associated kinase activity is rapidly and highly elevated in cortical neurons upon camptothecin treatment. These results suggest that postmitotic neurons may utilize Cdk4 and 6, signals that normally control proliferation, to mediate death signaling resulting from DNA-damaging conditions.

scholarly journals Activation of Pre-mRNA Splicing by Human RNPS1 Is Regulated by CK2 Phosphorylation

2005 ◽  
Vol 25 (4) ◽  
pp. 1446-1457 ◽  
Author(s):  
Janeen H. Trembley ◽  
Sawako Tatsumi ◽  
Eiji Sakashita ◽  
Pascal Loyer ◽  
Clive A. Slaughter ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Phosphorylation Site ◽  
Mrna Splicing ◽  
Exon Junction Complex ◽  
Exon Junction ◽  
In Vivo Experiments ◽  
Splicing Regulator ◽  
Major Phosphorylation Site

ABSTRACT Human RNPS1 was originally characterized as a pre-mRNA splicing activator in vitro and was shown to regulate alternative splicing in vivo. RNPS1 was also identified as a protein component of the splicing-dependent mRNP complex, or exon-exon junction complex (EJC), and a role for RNPS1 in postsplicing processes has been proposed. Here we demonstrate that RNPS1 incorporates into active spliceosomes, enhances the formation of the ATP-dependent A complex, and promotes the generation of both intermediate and final spliced products. RNPS1 is phosphorylated in vivo and interacts with the CK2 (casein kinase II) protein kinase. Serine 53 (Ser-53) of RNPS1 was identified as the major phosphorylation site for CK2 in vitro, and the same site is also phosphorylated in vivo. The phosphorylation status of Ser-53 significantly affects splicing activation in vitro, but it does not perturb the nuclear localization of RNPS1. In vivo experiments indicated that the phosphorylation of RNPS1 at Ser-53 influences the efficiencies of both splicing and translation. We propose that RNPS1 is a splicing regulator whose activator function is controlled in part by CK2 phosphorylation.

scholarly journals Stanniocalcin 1 and 2 are secreted as phosphoproteins from human fibrosarcoma cells

2000 ◽  
Vol 350 (2) ◽  
pp. 453-461 ◽  
Author(s):  
Derek A. JELLINEK ◽  
Andy C. CHANG ◽  
Martin R. LARSEN ◽  
Xin WANG ◽  
Phillip J. ROBINSON ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Mrna Levels ◽  
Intact Cells ◽  
Fibrosarcoma Cells ◽  
Ht1080 Cells ◽  
Human Fibrosarcoma Cells

Stanniocalcin 1 (STC1) and stanniocalcin 2 (STC2) are two recently identified mammalian peptide hormones. STC1 plays a role in calcium and phosphate homoeostasis, while the role of STC2 is unknown. We examined a human fibrosarcoma cell line, HT1080, that has high steady-state STC1 and STC2 mRNA levels, to determine whether these proteins are secreted. Following incubation of HT1080 cells with 32P, labelled STC1 and STC2 were found to be secreted into the medium. STC1 was phosphorylated in vitro by protein kinase C (PKC). In vitro and in vivo phosphorylation both occurred exclusively on serine and the phosphopeptide maps were similar, suggesting that PKC might be the in vivo kinase. STC2 was phosphorylated in vitro by casein kinase II (CK2), in vitro and in vivo phosphorylation were exclusively on serine and the phosphopeptide maps were indistinguishable. Phosphorylation of STC2 in intact cells resulted from the action of an ecto-protein kinase, since exogenous STC2 was phosphorylated by HT1080 cells and no phosphorylated STC2 was detectable inside the cells. The ectokinase activity was abolished by heparin and GTP could substitute for ATP as the phosphate donor, indicative of an ecto-CK2-like activity. The in vitro CK2 phosphorylation site was shown by matrix-assisted laser-desorption ionization–time-of-flight MS to be a single serine located between Ser-285 and Ser-298 in the C-terminal region of STC2. This is the first report of the secretion of STC1 or STC2 from mammalian cells. We conclude that these human fibrosarcoma cells express both STC1 and STC2 as secreted phosphoproteins in vivo, with STC2 being phosphorylated by an ecto-CK2-like enzyme.

scholarly journals Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

1993 ◽  
Vol 4 (1) ◽  
pp. 79-92 ◽  
Author(s):  
L Connell-Crowley ◽  
M J Solomon ◽  
N Wei ◽  
J W Harper
Keyword(s):  
Mammalian Cells ◽  
Specific Activity ◽  
Fold Increase ◽  
Cyclin A ◽  
Cyclin B ◽  
Activation Process ◽  
Cyclin Dependent Kinase ◽  
Cell Extracts

p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.

scholarly journals Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5

2004 ◽  
Vol 381 (2) ◽  
pp. 471-481 ◽  
Author(s):  
Mark E. GRAHAM ◽  
Patricia RUMA-HAYNES ◽  
Amanda G. CAPES-DAVIS ◽  
Joanne M. DUNN ◽  
Timothy C. TAN ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Single Mutation ◽  
Cyclin Dependent Kinase ◽  
Major Site ◽  
Multisite Phosphorylation ◽  
Cyclin Dependent Kinase 5

Doublecortin (DCX) is a 40 kDa microtubule-associated protein required for normal neural migration and cortical layering during development. Mutations in the human DCX gene cause a disruption of cortical neuronal migration. Defects in cdk5 (cyclin-dependent kinase 5) also cause defects in neural migration and cortical layering. DCX is a substrate for cdk5 in vitro and in vivo and the major site of in vitro phosphorylation is Ser-297. We used a highly developed MS strategy to identify the cdk5 phosphorylation sites and determine the major and minor sites. Several phosphopeptides were identified from a tryptic digest of 32P-labelled, cdk5-phosphorylated DCX using a combination of off-line HPLC and matrix-assisted laser-desorption ionization-MS with alkaline phosphatase treatment. Tandem MS/MS enabled the identification of seven phosphorylation sites for cdk5. Monitoring of 32P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine/proline-rich domain at the C-terminus. Five other sites, Ser-287, Thr-289, Ser-297, Thr-326 and Ser-332, were also found in the tail. Site-directed mutagenesis largely supported these findings. Single mutation of Ser-28 reduced but did not abolish phosphorylation. Double, rather than single, mutation for Ser-332 and Ser-339 was required to reduce overall phosphorylation, suggesting an interaction between these sites. Truncations of the tail produced a significant reduction in cdk5 phosphorylation of DCX. These results do not support Ser-297 as the major cdk5 phosphorylation site in DCX, but indicate that DCX is subject to complex multisite phosphorylation. This illustrates the importance of a well-developed MS strategy to identify phosphorylation sites.

scholarly journals A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1.

1995 ◽  
Vol 15 (3) ◽  
pp. 1318-1323 ◽  
Author(s):  
L Han ◽  
J Colicelli
Keyword(s):  
Mammalian Cells ◽  
Signal Transduction Pathway ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activating Mutations ◽  
Human Proteins ◽  
Ras Signal Transduction

The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP. Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.

Dominant negative mutants of transforming growth factor-beta 1 inhibit the secretion of different transforming growth factor-beta isoforms

1992 ◽  
Vol 12 (4) ◽  
pp. 1674-1679
Author(s):  
A R Lopez ◽  
J Cook ◽  
P L Deininger ◽  
R Derynck
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Mammalian Cells ◽  
Transforming Growth Factor ◽  
Dominant Negative ◽  
Tgf Beta ◽  
Growth Factor Beta ◽  
Beta 2

Transforming growth factor-beta (TGF-beta) is a secreted polypeptide factor that is thought to play a major role in the regulation of proliferation of many cell types and various differentiation processes. Several related isoforms have been structurally characterized, three of which, TGF-beta 1, -beta 2, and -beta 3, have been detected in mammalian cells and tissues. Each TGF-beta form is a homodimer of a 112-amino-acid polypeptide which is encoded as a larger polypeptide precursor. We have introduced several mutations in the TGF-beta 1 precursor domain, resulting in an inhibition of TGF-beta 1 secretion. Coexpression of these mutants with wild-type TGF-beta 1, -beta 2, and -beta 3 results in a competitive and specific inhibition of the secretion of different TFG-beta forms, indicating that these mutated versions act as dominant negative mutants for TGF-beta secretion. Overexpression of dominant negative mutants can thus be used to abolish endogenous secretion of TGF-beta and structurally related family members, both in vitro and in vivo, and to probe in this way the physiological functions of the members of the TGF-beta superfamily.

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