Kinase Mutant (kinase + mutant)

Distribution by Scientific Domains

Selected Abstracts

DAP kinase activity is critical for C2 -ceramide-induced apoptosis in PC12 cells

FEBS JOURNAL, Issue 1 2002
Mutsuya Yamamoto
Exposure of PC12 cells to C2 -ceramide results in dose-dependent apoptosis. Here, we investigate the involvement of death-associated protein (DAP) kinase, initially identified as a positive mediator of the interferon-,-induced apoptosis of HeLa cells, in the C2 -ceramide-induced apoptosis of PC12 cells. DAP kinase is endogenously expressed in these cells. On exposure of PC12 cells to 30 µm C2 -ceramide, both the total (assayed in the presence of Ca2+/calmodulin) and Ca2+/calmodulin-independent (assayed in the presence of EGTA) DAP kinase activities were transiently increased 5.0- and 12.2-fold, respectively, at 10 min, and then decreased to 1.7- and 3.4-fold at 90 min. After 10 min exposure to 30 µm C2 -ceramide, the Ca2+/calmodulin independent activity/ total activity ratio increased from 0.22 to 0.60. These effects were dependent on the C2 -ceramide concentration. C8 -ceramide, another active ceramide analog, also induced apoptosis and activated DAP kinase, while C2 -dihydroceramide, an inactive ceramide analog, failed to induce apoptosis and increase DAP kinase activity. Furthermore, transfection studies revealed that overexpression of wild-type DAP kinase enhanced the sensitivity to C2 - and C8 -ceramide, while a catalytically inactive DAP kinase mutant and a construct containing the death domain and C-terminal tail of DAP kinase, which act in a dominant-negative manner, rescued cells from C2 -, and C8 -ceramide-induced apoptosis. These findings demonstrate that DAP kinase is an important component of the apoptotic machinery involved in ceramide-induced apoptosis, and that the intrinsic DAP kinase activity is critical for ceramide-induced apoptosis. [source]

A novel gene, ecl1+, extends the chronological lifespan in fission yeast

Hokuto Ohtsuka
Abstract We have identified a novel gene from Schizosaccharomyces pombe that we have named ecl1+ (extender of the chronological lifespan). When ecl1+ is provided on a high-copy number plasmid, it extends the viability of both the ,sty1 MAP kinase mutant and the wild-type cells after entry into the stationary phase. ecl1+ encodes an 80-amino acid polypeptide that had not been annotated in the current database. The ecl1+ -mRNA increases transiently when the growth phase is changed from the log phase to the stationary phase. The Ecl1 protein is localized in the nucleus. Calorie restriction extends the chronological lifespan of wild-type and ,ecl1 cells but not ecl1+ -overproducing cells. The ,pka1 mutant shows little, if any, additional extension of viability when Ecl1 is overproduced. The ste11+ gene that is negatively controlled by Pka1 is up regulated when Ecl1 is overproduced. From these results we propose that the effect of Ecl1 overproduction may be mainly linked to and negatively affects the Pka1-dependent pathway. [source]

Catalytic activity of Cdk9 is required for nuclear co-localization of the Cdk9/cyclin T1 (P-TEFb) complex

Giuliana Napolitano
Cdk9 and its binding partner cyclin T1 comprise the positive elongation factor b (P-TEFb). P-TEFb phosphorylates the RNA polymerase II carboxyl-terminal-domain (CTD) allowing efficient transcription elongation. Recent studies showed that Cdk9 is a predominant nuclear protein, and here we investigated the functional requirement for nuclear localization of Cdk9. We found that the catalytic inactive kinase mutant (Cdk9dn) fails to accumulate in the nucleus showing a diffuse sub-cellular localization. In addition to the catalityc activity, nuclear localization of Cdk9 protein requires the presence of the phospho-acceptor sites at the C-terminus tail. Finally, enforced expression of wild-type cyclinT1, which enhances nuclear localization of Cdk9wt, fails to direct the Cdk9 mutants to the nucleus. Collectively, these findings implicate that nuclear localization of Cdk9 requires auto-phosphorylation of the kinase, and highlight the presence of a regulatory mechanism underlying the nuclear localization of the P-TEFb complex. J. Cell. Physiol. 197: 1,7, 2003© 2003 Wiley-Liss, Inc. [source]

The role of mitogen-activated protein (MAP) kinase signalling components and the Ste12 transcription factor in germination and pathogenicity of Botrytis cinerea

SUMMARY In all fungi studied so far, mitogen-activated protein (MAP) kinase cascades serve as central signalling complexes that are involved in various aspects of growth, stress response and infection. In this work, putative components of the yeast Fus3/Kss1-type MAP kinase cascade and the putative downstream transcription factor Ste12 were analysed in the grey mould fungus Botrytis cinerea. Deletion mutants of the MAP triple kinase Ste11, the MAP kinase kinase Ste7 and the MAP kinase adaptor protein Ste50 all resulted in phenotypes similar to that of the previously described BMP1 MAP kinase mutant, namely defects in germination, delayed vegetative growth, reduced size of conidia, lack of sclerotia formation and loss of pathogenicity. Mutants lacking Ste12 showed normal germination, but delayed infection as a result of low penetration efficiency. Two differently spliced ste12 transcripts were detected, and both were able to complement the ste12 mutant, except for a defect in sclerotium formation, which was only corrected by the full-sized transcript. Overexpression of the smaller ste12 transcript resulted in delayed germination and strongly reduced infection. Bc-Gas2, a homologue of Magnaporthe grisea Gas2 that is required for appressorial function, was found to be non-essential for growth and infection, but its expression was under the control of both Bmp1 and Ste12. In summary, the role and regulatory connections of the Fus3/Kss1-type MAP kinase cascade in B. cinerea revealed both common and unique properties compared with those of other plant pathogenic fungi, and provide evidence for a regulatory link between the BMP1 MAP kinase cascade and Ste12. [source]

Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect,

HUMAN MUTATION, Issue 11 2009
Eng-King Tan
Abstract Mutations in the gene encoding phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) have been associated with the loss of dopaminergic neurons characteristic of familial and sporadic Parkinson disease. We developed an in vitro system of stable human dopaminergic neuronal cell lines coexpressing an equivalent copy of normal and mutant PINK1 to simulate "heterozygous" and "homozygous" states in patients. Mutants in the N-terminus, C-terminus, and kinase domain were generated and cloned into a two-gene mammalian expression vector to generate stable mammalian expression cell lines producing an equivalent copy number of wild-type/mutant PINK1. The cell lines were subjected to oxidative stress and the rate of apoptosis and change in mitochondrial membrane potential (,,m) were assessed. Cell lines expressing kinase and C-terminus mutants exhibited a greater rate of apoptosis and decrease in ,,m, and increased time-dependent cell loss when subjected to oxidative stress compared to the wild-type. Cell lines expressing two copies of kinase mutants exhibited a greater apoptosis rate and ,,m decrease than those expressing one copy of the mutant. In time-dependent experiments, there was a significant difference between "homozygous," "heterozygous," and wild-type cell lines, with decreasing cell survival in cell lines expressing mutant copies of PINK1 compared to the wild-type. We provided the first experimental evidence that clinically reported PINK1 heterozygous mutations exert a gene dosage effect, suggesting that haploinsufficiency of PINK1 is the most likely mechanism that increased the susceptibility to dopaminergic cellular loss. Hum Mutat 30:1551,1557, 2009. © 2009 Wiley-Liss, Inc. [source]

Characterization of Herpes Simplex Virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity

Mark S. Kokoris
Abstract Herpes Simplex Virus type 1 (HSV-1) thymidine kinase (TK) is currently the most widely used suicide agent for gene therapy of cancer. Tumor cells that express HSV-1 thymidine kinase are rendered sensitive to prodrugs due to preferential phosphorylation by this enzyme. Although ganciclovir (GCV) is the prodrug of choice for use with TK, this approach is limited in part by the toxicity of this prodrug. From a random mutagenesis library, seven thymidine kinase variants containing multiple amino acid substitutions were identified on the basis of activity towards ganciclovir and acyclovir based on negative selection in Escherichia coli. Using a novel affinity chromatography column, three mutant enzymes and the wild-type TK were purified to homogeneity and their kinetic parameters for thymidine, ganciclovir, and acyclovir determined. With ganciclovir as the substrate, one mutant (mutant SR39) demonstrated a 14-fold decrease in Km compared to the wild-type enzyme. The most dramatic change is displayed by mutant SR26, with a 124-fold decrease in Km with acyclovir as the substrate. Such new "prodrug kinases" could provide benefit to ablative gene therapy by now making it feasible to use the relatively nontoxic acyclovir at nanomolar concentrations or ganciclovir at lower, less immunosuppressive doses. [source]

Inhibition of protein kinase CK2 leads to a modulation of androgen receptor dependent transcription in prostate cancer cells

THE PROSTATE, Issue 2 2007
Claudia Götz
Abstract BACKGROUND The androgen receptor (AR) mediates the biological responses of androgens in the prostate gland. In prostate cancer, this pathway is often deregulated and causes an uncontrolled proliferation. METHODS The current study focuses on the effects of an inhibition of protein kinase CK2 on the AR-mediated transcription in LNCaP prostate cancer cells. We used chemical inhibitors of CK2 as well as dominant-negative kinase mutants to downregulate the CK2 activity. We determined the effects of the inhibition by Western blot analysis of endogenous target genes of the AR as well as by reporter assays. RESULTS We found that inhibition of CK2 led to a downregulation of the AR-dependent transcription. Moreover, the amount of the AR protein decreased significantly. CONCLUSION According to the fact that AR pathways are involved in the development and progression of prostate cancer, the ability to modulate AR function should provide an alternative basis for the development of new cancer therapies. Prostate © 2006 Wiley-Liss, Inc. [source]

Activation of NF-,B and IL-8 by Yersinia enterocolitica invasin protein is conferred by engagement of Rac1 and MAP kinase cascades

Guntram A. Grassl
Summary Yersinia enterocolitica triggers activation of the nuclear factor (NF)-,B and production of the proinflammatory chemokine interleukin (IL)-8 in intestinal epithelial cells. This activation is due to adhesion of the bacteria via their outer membrane protein invasin to the host cells. Using Clostridium difficile toxins that specifically inactivate small GTPases, and transfection of inhibitory proteins of the Rho-GTPases, we demonstrate that Rac1, but not Cdc42 or Rho, is required for activation of NF-,B by invasin. Invasin activated the mitogen activated protein kinases (MAPK) p38 and c-Jun N-terminal protein kinase (JNK) but not extracellular signal regulated kinase (ERK). The functional relevance of these pathways for invasin-mediated IL-8 expression was assessed by protein kinase inhibitors and dominant-negative kinase mutants. While NF-,B and JNK contribute to IL-8 transcription, p38 MAPK also acts through stabilization of IL-8 mRNA, as confirmed by quantitative RT-PCR and electrophoretic mobility shift assays. Transfection experiments with I-,B kinase (IKK)1 and IKK2 mutants indicate that the release of NF-,B from its cytoplasmic inhibitor I-,B and its translocation into the nucleus is mediated by these kinases. Our data identify Rac1 as a key intermediate in invasin-triggered IL-8 synthesis and demonstrate that maximum IL-8 induction involves several MAP kinase cascades. [source]