scholarly journals Human herpesvirus 6B induces phosphorylation of p53 in its regulatory domain by a CK2- and p38-independent pathway

2008 ◽  
Vol 89 (1) ◽  
pp. 87-96 ◽  
Author(s):  
B. Øster ◽  
B. Bundgaard ◽  
T. R. Hupp ◽  
P. Höllsberg
Keyword(s):  
Kinase Inhibitors ◽  
Human Herpesvirus ◽  
Signalling Pathways ◽  
Cyclin Dependent Kinase ◽  
Protein Map ◽  
Mitogen Activated Protein ◽  
Equivalent Site ◽  
Interfering Rna ◽  
Tumour Suppressor P53

Here, we demonstrate that human herpesvirus 6B (HHV-6B) infection upregulates the tumour suppressor p53 and induces phosphorylation of p53 at Ser392. Interestingly, phosphorylation at the equivalent site has previously been shown to correlate with p53 tumour suppression in murine models. Although the signalling pathways leading to Ser392 phosphorylation are poorly understood, they seem to include casein kinase 2 (CK2), double-stranded RNA-activated protein kinase (PKR), p38 or cyclin-dependent kinase 9 (Cdk9). By using column chromatography and in vitro kinase assays, CK2 and p38, but not PKR or Cdk9, eluted in column fractions that phosphorylated p53 at Ser392. However, treatment of cells with neither the CK2 and Cdk9 inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) nor p38 kinase inhibitors reduced HHV-6B-induced Ser392 phosphorylation significantly. Knockdown of the CK2β subunit or p38α by small interfering RNA had no effect on HHV-6B-induced phosphorylation of p53 at Ser392. Thus, HHV-6B induces p53 Ser392 phosphorylation by an atypical pathway independent of CK2 and p38 kinases, whereas mitogen-activated protein (MAP) kinase signalling pathways are involved in viral replication.

scholarly journals Phosphorylation by Cyclin C/Cyclin-Dependent Kinase 2 following Mitogenic Stimulation of Murine Fibroblasts Inhibits Transcriptional Activity of LSF during G1 Progression

2009 ◽  
Vol 29 (9) ◽  
pp. 2335-2345 ◽  
Author(s):  
Utsav H. Saxena ◽  
Christina M. H. Powell ◽  
Jill K. Fecko ◽  
Roxanne Cacioppo ◽  
Hubert S. Chou ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Target Genes ◽  
Cyclin Dependent Kinase ◽  
Mitogenic Stimulation ◽  
Mouse Fibroblasts ◽  
Cyclin C ◽  
Murine Fibroblasts ◽  
Interfering Rna ◽  
Stimulation Of

ABSTRACT Transcription factor LSF is required for progression from quiescence through the cell cycle, regulating thymidylate synthase (Tyms) expression at the G1/S boundary. Given the constant level of LSF protein from G0 through S, we investigated whether LSF is regulated by phosphorylation in G1. In vitro, LSF is phosphorylated by cyclin E/cyclin-dependent kinase 2 (CDK2), cyclin C/CDK2, and cyclin C/CDK3, predominantly on S309. Phosphorylation of LSF on S309 is maximal 1 to 2 h after mitogenic stimulation of quiescent mouse fibroblasts. This phosphorylation is mediated by cyclin C-dependent kinases, as shown by coimmunoprecipitation of LSF and cyclin C in early G1 and by abrogation of LSF S309 phosphorylation upon suppression of cyclin C with short interfering RNA. Although mouse fibroblasts lack functional CDK3 (the partner of cyclin C in early G1 in human cells), CDK2 compensates for this absence. By transient transfection assays, phosphorylation at S309, mediated by cyclin C overexpression, inhibits LSF transactivation. Moreover, overexpression of cyclin C and CDK3 inhibits induction of endogenous Tyms expression at the G1/S transition. These results identify LSF as only the second known target (in addition to pRb) of cyclin C/CDK activity during progression from quiescence to early G1. Unexpectedly, this phosphorylation prevents induction of LSF target genes until late G1.

scholarly journals Histone H3 as a novel substrate for MAP kinase phosphatase-1

2009 ◽  
Vol 296 (2) ◽  
pp. C242-C249 ◽  
Author(s):  
Corttrell M. Kinney ◽  
Unni M. Chandrasekharan ◽  
Lin Yang ◽  
Jianzhong Shen ◽  
Michael Kinter ◽  
...  
Keyword(s):  
Map Kinase ◽  
Map Kinases ◽  
Histone H3 ◽  
Dual Specificity ◽  
Map Kinase Phosphatase ◽  
Mitogen Activated Protein ◽  
And Control ◽  
Interfering Rna ◽  
Endothelial Growth Factor

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a nuclear, dual-specificity phosphatase that has been shown to dephosphorylate MAP kinases. We used a “substrate-trap” technique involving a mutation in MKP-1 of the catalytically critical cysteine to a serine residue (“CS” mutant) to capture novel MKP-1 substrates. We transfected the MKP-1 (CS) mutant and control (wild-type, WT) constructs into phorbol 12-myristate 13-acetate (PMA)-activated COS-1 cells. MKP-1-substrate complexes were immunoprecipitated, which yielded four bands of 17, 15, 14, and 10 kDa with the CS MKP-1 mutant but not the WT MKP-1. The bands were identified by mass spectrometry as histones H3, H2B, H2A, and H4, respectively. Histone H3 was phosphorylated, and purified MKP-1 dephosphorylated histone H3 (phospho-Ser-10) in vitro; whereas, histone H3 (phospho-Thr-3) was unaffected. We have previously shown that thrombin and vascular endothelial growth factor (VEGF) upregulated MKP-1 in human endothelial cells (EC). We now show that both thrombin and VEGF caused dephosphorylation of histone H3 (phospho-Ser-10) and histone H3 (phospho-Thr-3) in EC with kinetics consistent with MKP-1 induction. Furthermore, MKP-1-specific small interfering RNA (siRNA) prevented VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation but had no effect on H3 (phospho-Thr-3 or Thr-11) dephosphorylation. In summary, histone H3 is a novel substrate of MKP-1, and VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation requires MKP-1. We propose that MKP-1-mediated H3 (phospho-Ser-10) dephosphorylation is a key regulatory step in EC activation by VEGF and thrombin.

scholarly journals Regulation of PDGFR-α in rat pulmonary myofibroblasts by staurosporine

2001 ◽  
Vol 280 (2) ◽  
pp. L354-L362 ◽  
Author(s):  
Pamela M. Lindroos ◽  
Yi-Zhe Wang ◽  
Annette B. Rice ◽  
James C. Bonner
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Protein A ◽  
Nuclear Factor Κb ◽  
Mitogen Activated Protein Kinase ◽  
Specific Gene ◽  
Autocrine Loop ◽  
P38 Inhibitor ◽  
Mitogen Activated Protein

Upregulation of the platelet-derived growth factor (PDGF) receptor-α (PDGFR-α) is a mechanism of myofibroblast hyperplasia during pulmonary fibrosis. We previously identified interleukin (IL)-1β as a major inducer of the PDGFR-α in rat pulmonary myofibroblasts in vitro. In this study, we report that staurosporine, a broad-spectrum kinase inhibitor, upregulates PDGFR-α gene expression and protein. A variety of other kinase inhibitors did not induce PDGFR-α expression. Staurosporine did not act via an IL-1β autocrine loop because the IL-1 receptor antagonist protein did not block staurosporine-induced PDGFR-α expression. Furthermore, staurosporine did not activate a variety of signaling molecules that were activated by IL-1β, including nuclear factor-κB, extracellular signal-regulated kinase, and c-Jun NH2-terminal kinase. However, both staurosporine- and IL-1β-induced phosphorylation of p38 mitogen-activated protein kinase and upregulation of PDGFR-α by these two agents was inhibited by the p38 inhibitor SB-203580. Finally, staurosporine inhibited basal and PDGF-stimulated mitogenesis over the same concentration range that induced PDGFR-α expression. Collectively, these data demonstrate that staurosporine is a useful tool for elucidating the signaling mechanisms that regulate PDGFR expression in lung connective tissue cells and possibly for evaluating the role of the PDGFR-α as a growth arrest-specific gene.

Clinical efficacy with dabrafenib and trametinib in a T599_V600insT poorly differentiated metastatic thyroid carcinoma

2021 ◽  
Vol 14 (8) ◽  
pp. e243264
Author(s):  
Chung-Shien Lee ◽  
Emily Miao ◽  
Kasturi Das ◽  
Nagashree Seetharamu
Keyword(s):  
Thyroid Carcinoma ◽  
Kinase Inhibitors ◽  
Mek Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Braf Mutations ◽  
Effective Treatment Option ◽  
Mitogen Activated Protein ◽  
Treatment Data ◽  
Poorly Differentiated

BRAF (v-raf murine sarcoma viral oncogene homolog B1) and MEK (mitogen-activated protein kinase kinase) inhibitors have been shown to improve clinical outcomes in tumours presenting with mutations in the BRAF gene. The most common form of BRAF mutation is V600E/K and has been shown to occur in thyroid cancers. Treatment data for patients harbouring less frequent BRAF mutations are limited. In vitro studies have shown that mutations in codons 599–601 increase kinase activity similar to that in V600E mutations, which suggests that BRAF and MEK inhibitors could be an effective treatment option. Here, we report a case of a patient with thyroid carcinoma harbouring a rare amino acid insertion in codon 599 of the BRAF gene (T599_V600insT) treated with a BRAF and MEK inhibitor.

scholarly journals Expression and modulation of p42/p44 MAPKs and cell cycle regulatory proteins in rat pancreas regeneration

1999 ◽  
Vol 277 (5) ◽  
pp. G953-G959 ◽  
Author(s):  
Jean Morisset ◽  
JoséCristobal Aliaga ◽  
Ezéquiel L. Calvo ◽  
Judith Bourassa ◽  
Nathalie Rivard
Keyword(s):  
Cell Cycle ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Regulatory Proteins ◽  
Mitogen Activated Protein Kinase ◽  
Cyclin Dependent Kinase ◽  
Mapk Activation ◽  
Pancreas Regeneration ◽  
Mitogen Activated Protein ◽  
Cell Cycle Regulatory Proteins

Pancreatic growth occurs after CCK, CCK-induced pancreatitis, and pancreatectomy; the mechanisms involved remain unknown. This study evaluates mitogen-activated protein kinase (MAPK) activation and expression of cell cycle regulatory proteins after pancreatectomy to understand the cellular and molecular mechanisms involved in pancreas regeneration. Rats were killed 1–12 days after pancreatectomy, and p42/p44 MAPK activation, expression of the cyclins D and E, cyclin-dependent kinase (Cdk)-2 activity, retinoblastoma protein (pRb) hyperphosphorylation, and expression of the cyclin kinase inhibitors p15, p21, and p27 were examined. Pancreatic remnants exhibited sustained p42/p44 MAPK activation within 8 h. Cyclins D1 and E showed maximal expression after 2 and 6 days, coinciding with maximal hyperphosphorylation of pRb and Cdk2 activity. The expression of p15 vanished after 12 h, p27 disappeared gradually, and p21 increased early. The p27 complexed with Cdk2 dissociated after 2 days, whereas p21 associated in a reverse fashion. In conclusion, sustained activation of p42/p44 MAPKs and Cdk2 along with overexpression of cyclins D1 and E and reduction of p15 and p27 cyclin inhibitors occurred early after pancreatectomy and are active factors involved in signaling that leads to pancreas regeneration.

scholarly journals Virtual Screening Guided Design, Synthesis and Bioactivity Study of Benzisoselenazolones (BISAs) on Inhibition of c-Met and Its Downstream Signalling Pathways

2019 ◽  
Vol 20 (10) ◽  
pp. 2489 ◽  
Author(s):  
Siqi Zhang ◽  
Qiaoling Song ◽  
Xueting Wang ◽  
Zhiqiang Wei ◽  
Rilei Yu ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Cancer Cell ◽  
Kinase Inhibitors ◽  
Antitumour Activity ◽  
Cancer Cell Line ◽  
Lung Cancer Cell Line ◽  
Signalling Pathways ◽  
Design Synthesis ◽  
Ic50 Values

c-Met is a transmembrane receptor tyrosine kinase and an important therapeutic target for anticancer drugs. In this study, we designed a small library containing 300 BISAs molecules that consisted of carbohydrates, amino acids, isothiourea, tetramethylthiourea, guanidine and heterocyclic groups and screened c-Met targeting compounds using docking and MM/GBSA. Guided by virtual screening, we synthesised a series of novel compounds and their activity on inhibition of the autophosphorylation of c-Met and its downstream signalling pathway proteins were evaluated. We found a panel of benzisoselenazolones (BISAs) obtained by introducing isothiourea, tetramethylthiourea and heterocyclic groups into the C-ring of Ebselen, including 7a, 7b, 8a, 8b and 12c (with IC50 values of less than 20 μM in MET gene amplified lung cancer cell line EBC-1), exhibited more potent antitumour activity than Ebselen by cell growth assay combined with in vitro biochemical assays. In addition, we also tested the antitumour activity of three cancer cell lines without MET gene amplification/activation, including DLD1, MDA-MB-231 and A549. The neuroblastoma SK-N-SH cells with HGF overexpression which activates MET signalling are sensitive to MET inhibitors. The results reveal that our compounds may be nonspecific multitarget kinase inhibitors, just like type-II small molecule inhibitors. Western blot analysis showed that these inhibitors inhibited autophosphorylation of c-MET, and its downstream signalling pathways, such as PI3K/AKT and MARK/ERK. Results suggest that bensoisoselenones can be used as a scaffold for the design of c-Met inhibiting drug leads, and this study opens up new possibilities for future antitumour drug design.

scholarly journals PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum

2000 ◽  
Vol 347 (1) ◽  
pp. 255-263 ◽  
Author(s):  
Valerie BRACCHI-RICARD ◽  
Sailen BARIK ◽  
Cherie DELVECCHIO ◽  
Christian DOERIG ◽  
Ratna CHAKRABARTI ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Sequence Similarity ◽  
Small Subunit ◽  
Mitogen Activated Protein Kinase ◽  
Cdna Clones ◽  
Cyclin Dependent Kinase ◽  
Mitogen Activated Protein

We have isolated a novel protein kinase cDNA, PfPK6, by differential display RT-PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic stages of Plasmodium falciparum, which shows sequence similarity to both cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) family members. The 915 bp open reading frame (ORF) is interrupted by seven introns and encodes a 305-residue polypeptide with a predicted molecular mass of 35848 Da. Several cDNA clones with some of the intron sequences were isolated, indicating alternate or defective splicing of PfPK6 transcripts because the gene seems to be a single copy located on chromosome 13. The similarity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3% and 49.6%, respectively. The signature PSTAIRE (single-letter amino acid codes) CDK motif is changed to SKCILRE in PfPK6. The TXY residues that are phosphorylated in MAPKs for their activation are T173PT in PfPK6. Three size classes of PfPK6 transcripts of 6.5, 2.0 and 1.1 kb are up-regulated during the transition of P. falciparum from ring to trophozoite. Western blot analysis suggested the expression of a 35 kDa polypeptide in trophozoites and schizonts. Immunofluorescence studies indicated both nuclear and cytoplasmic localization of PfPK6 in trophozoite, schizont and segmenter stages. In vitro, recombinant PfPK6 phosphorylated itself and also exogenous substrates, histone and the small subunit of the malarial ribonucleotide reductase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors such as olomoucine and roscovitine. PfPK6 showed a preference for Mn2+ over Mg2+ ions as a cofactor. The Lys38 → Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in catalytic rate for R2 phosphorylation.

scholarly journals Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors.

1996 ◽  
Vol 16 (12) ◽  
pp. 6623-6633 ◽  
Author(s):  
P D Adams ◽  
W R Sellers ◽  
S K Sharma ◽  
A D Wu ◽  
C M Nalin ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Cyclin A ◽  
Cdk Inhibitors ◽  
Cyclin Dependent Kinase ◽  
Binding Motifs ◽  
Recognition Motif ◽  
Binding Inhibition ◽  
In Vivo Growth

Understanding how cyclin-cdk complexes recognize their substrates is a central problem in cell cycle biology. We identified an E2F1-derived eight-residue peptide which blocked the binding of cyclin A and E-cdk2 complexes to E2F1 and p21. Short peptides spanning similar sequences in p107, p130, and p21-like cdk inhibitors likewise bound to cyclin A-cdk2 and cyclin E-cdk2. In addition, these peptides promoted formation of stable cyclin A-cdk2 complexes in vitro but inhibited the phosphorylation of the retinoblastoma protein by cyclin A- but not cyclin B-associated kinases. Mutation of the cyclin-cdk2 binding motifs in p107 and E2F1 likewise prevented their phosphorylation by cyclin A-associated kinases in vitro. The cdk inhibitor p21 was found to contain two functional copies of this recognition motif, as determined by in vitro kinase binding/inhibition assays and in vivo growth suppression assays. Thus, these studies have identified a cyclin A- and E-cdk2 substrate recognition motif. Furthermore, these data suggest that p21-like cdk inhibitors function, at least in part, by blocking the interaction of substrates with cyclin-cdk2 complexes.

scholarly journals Viral Cyclin–Cyclin-Dependent Kinase 6 Complexes Initiate Nuclear DNA Replication

2001 ◽  
Vol 21 (2) ◽  
pp. 624-635 ◽  
Author(s):  
Heike Laman ◽  
Dawn Coverley ◽  
Torsten Krude ◽  
Ronald Laskey ◽  
Nic Jones
Keyword(s):  
Kinase Inhibitors ◽  
Nuclear Dna ◽  
Kaposi Sarcoma ◽  
S Phase ◽  
Replication Initiation ◽  
Herpesvirus Saimiri ◽  
Cyclin Dependent Kinase ◽  
Viral Cyclin ◽  
Replication Systems

ABSTRACT The cyclins encoded by Kaposi sarcoma-associated herpesvirus and herpesvirus saimiri are homologs of human D-type cyclins. However, when complexed to cdk6, they have several activities that distinguish them from D-type cyclin-cdk6 complexes, including resistance to cyclin-dependent kinase inhibitors and an enhanced substrate range. We find that viral cyclins interact with and phosphorylate proteins involved in replication initiation. Using mammalian in vitro replication systems, we show that viral cyclin-cdk6 complexes can directly trigger the initiation of DNA synthesis in isolated late-G1-phase nuclei. Viral cyclin-cdk6 complexes share this capacity with cyclin A-cdk2, demonstrating that in addition to functioning as G1-phase cyclin-cdk complexes, they function as S-phase cyclin-cdk complexes.

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