Kinase Deletion Mutant Research Articles

Phosphorylation of Candida glabrata ATP-binding Cassette Transporter Cdr1p Regulates Drug Efflux Activity and ATPase Stability

Fungal ATP-binding cassette transporter regulation was investigated using Candida glabrata Cdr1p and Pdh1p expressed in Saccharomyces cerevisiae. Rephosphorylation of Pdh1p and Cdr1p was protein kinase A inhibitor-sensitive but responded differentially to Tpk isoforms, stressors, and glucose concentration. Cdr1p Ser(307), which borders the nucleotide binding domain 1 ABC signature motif, and Ser(484), near the membrane, were dephosphorylated on glucose depletion and independently rephosphorylated during glucose exposure or under stress. The S484A enzyme retained half the wild type ATPase activity without affecting azole resistance, but the S307A enzyme was unstable to plasma membrane isolation. Studies of pump function suggested conformational interaction between Ser(484) and Ser(307). An S307A/S484A double mutant, which failed to efflux the Cdr1p substrate rhodamine 6G, had a fluconazole susceptibility 4-fold greater than the Cdr1p expressing strain, twice that of the S307A mutant, but 64-fold less than the control null strain. Stable intragenic suppressors indicative of homodimer nucleotide binding domain 1-nucleotide binding domain 1 interactions partially restored rhodamine 6G pumping and increased fluconazole and rhodamine 6G resistance in the S307A/S484A mutant. Nucleotide binding domain 1 of Cdr1p is a sensor of important physiological stimuli.

The Nuclear Kinase Lsk1p Positively Regulates the Septation Initiation Network and Promotes the Successful Completion of Cytokinesis in Response to Perturbation of the Actomyosin Ring inSchizosaccharomyces pombe

Cytokinesis in fission yeast requires the function of an actomyosin-based contractile ring whose constriction is dependent on a signaling module termed the septation initiation network (SIN). In response to minor perturbation of the ring, the duration of SIN signaling is extended concurrently with a delay in nuclear cycle progression. These mechanisms require the conserved phosphatase Clp1p/Flp1p and facilitate the successful completion of cytokinesis, thereby increasing cellular viability. To isolate novel components of this cytokinesis monitoring system, we screened a genome-wide bank of protein kinase deletion mutants and identified Lsk1p, a nuclear-localized protein kinase. Similar to clp1Delta mutants, and in contrast to wild type, lsk1Delta cells are unable to maintain the integrity of the actomyosin ring upon treatment with low doses (0.3 microM) of latrunculin A. However, unlike clp1Delta mutants, lsk1Delta cells are competent to delay nuclear cycle progression after cytokinetic failure. In addition, lsk1Delta mutants suppress the lethal, multiseptate phenotype conferred by hyperactivation of the SIN, demonstrating that Lsk1p is a positive regulator of this module. In this report, we demonstrate that Lsk1p acts in parallel to Clp1p to promote actomyosin ring stability upon checkpoint activation. Our studies also establish that actomyosin ring maintenance and nuclear cycle delay in response to cytokinetic perturbation can be genetically resolved into independent pathways.

Corpus Callosum Deficiency in Transgenic Mice Expressing a Truncated Ephrin-A Receptor

The A-class of the erythropoietin-producing hepatocellular carcinoma cell-derived (EphA) tyrosine kinase receptors and their ligands, the A-ephrins, play critical roles in the specification of topographic axon projection maps during development. In this study, the role of the EphA subfamily in callosal projections was investigated using transgenic mice expressing a kinase deletion mutant of EphA5. In approximately half of these transgenic mice, cerebral cortical neurons in various cortical regions (primary and secondary somatosensory cortices and frontal as well as visual areas) failed to project to the contralateral cortex. When commissural axons were examined with DiI labeling, few callosal fibers were found to traverse the midline in both the adult and neonatal transgenic mice. This defect in callosal development correlates with the expression of the transgene, because neurons in the superficial layers of the motor cortex, where transgene expression is low, show normal contralateral projection through the corpus callosum. In addition, multiple EphA receptors are expressed in callosal neurons and ephrin-A5 stimulates neurite outgrowth of callosal neurons in vitro. The midline glia structures important for callosal axon midline crossing appear normal in the transgenic mice, suggesting that the defects are unrelated to defective guidance structures at the midline. These observations suggest critical functions for EphA receptor in establishing callosal connections during brain development.

IKKβ Is Required for Bcl-2-mediated NF-κB Activation in Ventricular Myocytes

The transcription factor nuclear factor kappa B (NF-kappa B) is regulated by cytoplasmic inhibitor I kappa B alpha. An integral step in the activation of NF-kappa B involves the phosphorylation and degradation of I kappa B alpha. We have previously reported that I kappa B alpha activity is diminished in ventricular myocytes expressing Bcl-2 (de Moissac, D., Zheng, H., and Kirshenbaum, L. A. (1999) J. Biol. Chem. 274, 29505-29509). The underlying mechanism by which Bcl-2 activates NF-kappa B is undefined. In view of growing evidence that the I kappa B kinases (IKKs), notably IKK beta, are involved in signal induced phosphorylation of I kappa B alpha, we ascertained whether IKK beta is necessary and sufficient for Bcl-2 mediated NF-kappa B activation. Here we demonstrate that expression of Bcl-2 in ventricular myocytes resulted in an increase in NF-kappa B-dependent DNA binding, NF-kappa B gene transcription and reduced I kappa B alpha levels. An increase in the IKK beta kinase activity was observed in cells expressing full-length Bcl-2 but not in cells expressing the BH4 deletion mutant of Bcl-2 (Delta BH4; residues 10-30). Catalytically inactive mutants of IKK beta, but not IKK alpha, suppressed Bcl-2-mediated I kappa B alpha phosphorylation and NF-kappa B activation. Transfection of human embryonic 293 cells with a kinase-defective Raf-1 or a kinase-defective mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 (MEKK-1) suppressed Bcl-2-mediated IKK beta activity and NF-kappa B activation. Further, Bcl-2-mediated NF-kappa B activity was impaired in nullizygous mouse embryonic fibroblasts deficient for IKK beta. In this report, we provide the first direct evidence that Bcl-2 activates NF-kappa B by a signaling mechanism that involves Raf-1/MEKK-1 mediated activation of IKK beta.

Selective Inhibition of MAPKK Wis1 in the Stress-activated MAPK Cascade of Schizosaccharomyces pombe by Novel Berberine Derivatives

Intracellular molecular targets of novel berberine derivatives, HWY 289 and HWY 336, were identified by a screen of a variety of mutants in fission yeast Schizosaccharomyces pombe. HWY 289 and HWY 336 completely inhibited the proliferation of wild type as well as various mutant fission yeast cells (minimal inhibitory concentrations were 29.52 microm for HWY 289 and 11.83 microm for HWY 336), but did not affect the proliferation of Wis1 mitogen-activated protein kinase kinase (MAPKK) deletion mutants. In addition, HWY 289 with an IC(50) value of 7.3 microm or HWY 336 with IC(50) of 5.7 microm specifically inhibited in vitro kinase activities of purified Wis1, whereas either compound did not affect the activities of other kinases in the mitogen-activated protein kinase (MAPK) cascades of fission yeast. These genetic and biochemical results demonstrate the high degree of specificity of HWY 289 and HWY 336 to MAPKK Wis1 and suggest that the cytotoxicity of these compounds is not simply due to the inhibition of Wis1 kinase activity. High salt wash experiments have shown that strong noncovalent binding occurs between Wis1 and either HWY 289 or HWY 336. The preincubation of Wis1 kinase with ATP did not affect the inhibition of Wis1 by HWY 289 and HWY 336, but when Wis1 was preincubated with MBP, a protein substrate, Wis1 kinase activity was no longer inhibited. These observations demonstrate that HWY 289/HWY 336 do inhibit Wis1 kinase, not by binding to the ATP-binding site but by disturbing the binding of substrate to the kinase. Target validation of the complex of HWY 289/HWY 336 and Wis1 kinase will provide important clues for the mechanism of specific cytotoxicity of these compounds in S. pombe. On a broader aspect, it would create an initiative to further modify and develop compounds that selectively inhibit kinases and cause cytotoxicity in various MAPK cascades including those of mammals.

Homologues of LMPK, a mitogen-activated protein kinase from Leishmania mexicana, in different Leishmania species.

LMPK, a mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana, is essential for the proliferation of the amastigote, the mammalian stage of the protozoan parasite. This has been demonstrated using deletion mutant promastigotes, the insect stage of the parasite: first, in vitro after differentiation to amastigotes, which subsequently lost their potential to proliferate; second, by infection of peritoneal macrophages, which were able to cope with the infection and cleared the parasites; third, by infection of BALB/c mice, which showed no lesion development. The lmpk deletion mutant promastigotes are a potential live vaccine because they infect macrophages, transform to amastigotes and deliver amastigote antigens to raise an immune response without causing the disease. In addition, inhibition of LMPK in a wild-type infection is likely to resolve the disease and as such, is an ideal target for drug development against leishmaniasis. Here we investigated the presence and copy number of lmpk homologues in Leishmania amazonensis, L. major, L. tropica, L. aethiopica, L. donovani, L. infantum, and L. braziliensis and discuss the results with regard to drug development and vaccination using kinase deletion mutants.

Stimulation of pro-alpha(1)(I) collagen by TGF-beta(1) in mesangial cells: role of the p38 MAPK pathway.

Transforming growth factor-beta(1) (TGF-beta(1)) is a potent inducer of extracellular matrix protein synthesis and a key mediator of renal fibrosis. However, the intracellular signaling mechanisms by which TGF-beta(1) stimulates this process remain incompletely understood. In this report, we examined the role of a major stress-activated intracellular signaling cascade, belonging to the mitogen-activated protein kinase (MAPK) superfamily, in mediating TGF-beta(1) responses in rat glomerular mesangial cells, using dominant-negative inhibition of TGF-beta(1) signaling receptors. We first stably transfected rat glomerular mesangial cells with a kinase-deleted mutant TGF-beta type II receptor (TbetaR-II(M)) designed to inhibit TGF-beta(1) signaling in a dominant-negative fashion. Next, expression of TbetaR-II(M) mRNA was confirmed by Northern analysis. Cell surface expression and ligand binding of TbetaR-II(M) protein were demonstrated by affinity cross-linking with (125)I-labeled-TGF-beta(1). TGF-beta(1) rapidly induced p38 MAPK phosphorylation in wild-type and empty vector (pcDNA3)-transfected control mesangial cells. Interestingly, transfection with dominant-negative TbetaR-II(M) failed to block TGF-beta(1)-induced p38 MAPK phosphorylation. Moreover, dominant-negative TbetaR-II(M) failed to block TGF-beta(1)-stimulated pro-alpha(1)(I) collagen mRNA expression and cellular protein synthesis, whereas TGF-beta(1)-induced extracellular signal-regulated kinase (ERK) 1/ERK2 activation and antiproliferative responses were blocked by TbetaR-II(M). In the presence of a specific inhibitor of p38 MAPK, SB-203580, TGF-beta(1) was unable to stimulate pro-alpha(1)(I) collagen mRNA expression in the control and TbetaR-II(M)-transfected mesangial cells. Finally, we confirmed that both p38 MAPK activation and pro-alpha(1)(I) collagen stimulation were TGF-beta(1) effects that were abrogated by dominant-negative inhibition of TGF-beta type I receptor. Thus we show first demonstration of p38 MAPK activation by TGF-beta(1) in mesangial cells, and, given the rapid kinetics, this TGF-beta(1) effect is likely a direct one. Furthermore, our findings suggest that the p38 MAPK pathway functions as a component in the signaling of pro-alpha(1)(I) collagen induction by TGF-beta(1) in mesangial cells.

Mapping the functional domains of elongation factor-2 kinase.

A new class of eukaryotic protein kinases that are not homologous to members of the serine/threonine/tyrosine protein kinase superfamily was recently identified [Futey, L. M., et al. (1995) J. Biol. Chem. 270, 523-529; Ryazanov, A. G., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4884-4889]. This class includes eukaryotic elongation factor-2 kinase, Dictyostelium myosin heavy chain kinases A, B, and C, and several mammalian putative protein kinases that are not yet fully characterized [Ryazanov, A. G., et al. (1999) Curr. Biol. 9, R43-R45]. eEF-2 kinase is a ubiquitous protein kinase that phosphorylates and inactivates eukaryotic translational elongation factor-2, and thus can modulate the rate of polypeptide chain elongation during translation. eEF-2 was the only known substrate for eEF-2 kinase. We demonstrate here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid peptide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylation site in Dictyostelium myosin heavy chains. This enabled us to develop a rapid assay for eEF-2 kinase activity. To localize the functional domains of eEF-2 kinase, we expressed human eEF-2 kinase in Escherichia coli as a GST-tagged fusion protein, and then performed systematic in vitro deletion mutagenesis. We analyzed eEF-2 kinase deletion mutants for the ability to autophosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, MH-1. Mutants with deletions between amino acids 51 and 335 were unable to autophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1. Mutants with deletions between amino acids 521 and 725 were unable to phosphorylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-1. The kinases with deletions between amino acids 2 and 50 and 336 and 520 were able to catalyze all three reactions. In addition, the C-terminal domain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitation assay. These results suggest that eEF-2 kinase consists of two domains connected by a linker region. The amino-terminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-2 targeting domain. The calmodulin-binding region is located between amino acids 51 and 96. The amino acid sequence of the carboxyl-terminal domain of eEF-2 kinase displays similarity to several proteins, all of which contain repeats of a 36-amino acid motif that we named "motif 36".

Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant.

Tumor-directed gene therapy, such as "suicide gene" therapy, requires high levels of gene expression in a high percentage of tumor cells in vivo to be effective. Current vector strategies have been ineffective in achieving these goals. This report introduces the attenuated (thymidine kinase (TK)-negative) replication-competent vaccinia virus (VV) as a potential vector for tumor-directed gene therapy by studying the biodistribution of VV in animal tumor models. A TK-deleted recombinant VV (Western Reserve strain) expressing luciferase on a synthetic promoter was constructed. Luciferase activity was measured in vitro after transduction of a variety of human and murine tumor cell lines and in vivo after intraperitoneal (i.p.) delivery in C57BL/6 mice with 7-day i.p. tumors (10(6) MC-38 cells). Three other in vivo tumor models were examined for tumor-specific gene expression after intravenous delivery of VV (human melanoma in nude mice, adenocarcinoma liver metastasis in immunocompetent mice, and subcutaneous sarcoma in the rat). In addition, a replication-incompetent vaccinia (1 microg of psoralen and ultraviolet light, 365 nm, 4 minutes) was tested in vitro and in vivo and compared with active virus. Luciferase activity in i.p. tumors at 4 days after i.p. injection of VV was >7000-fold higher than lung, >3000-fold higher than liver, and >250-fold higher than ovary. In addition, intravenous injection of VV resulted in markedly higher tumor luciferase activity compared with any other organ in every model tested (up to 188,000-fold higher than liver and 77,000-fold higher than lung). Inactivation of the virus resulted in negligible gene expression in vivo. In summary, VV has a high transduction efficiency in tumor cells with high levels of gene expression. The results suggest a selective in vivo replication of TK-deleted VV in tumor cells. Replication competent, TK-deleted VV appears to be an ideal vector for testing the in vivo delivery of toxic genes to tumor cells.

Thymidine kinase-deleted vaccinia virus expressing purine nucleoside phosphorylase as a vector for tumor-directed gene therapy.

Tumor-directed gene therapy faces many obstacles. Lack of tissue targeting and low in vivo transduction efficiency represent some of the limitations for a successful therapeutic outcome. A thymidine kinase-deleted mutant vaccinia virus has been shown in marker studies to replicate selectively in tumor tissue in animal models. Purine nucleoside phosphorylase (PNP), from E. coli, converts the nontoxic prodrug 6-methylpurine deoxyriboside (6-MPDR) to the toxic purine 6-methylpurine. In this study, we investigated the cytotoxic properties of PNP, expressed by an optimized synthetic early/late promoter in a vaccinia virus (vMPPNP). In vitro cytotoxicity of psoralen-inactivated vMPPNP (1 microg of psoralen, 4 min of LWUV [365 nm]) at the maximum tolerated dose (MTD) of 6-MPDR (80 microM) reduced cell viability by day 3 to 1.7%. At an MOI of 0.002, replication-competent vMPPNP and 6-MPDR (80 microM) caused reduction of cell viability to 19.8% within 4 days. Furthermore, there was complete abrogation of viral replication after intracellular conversion of prodrug into the active toxin. The potency of such a system was similar among all histologies tested. Finally, the cytotoxic efficacy has been shown to be more rapid and complete than that of cytosine deaminase (CD), a more established enzyme/prodrug system. When virus was delivered intraperitoneally into athymic mice with hepatic metastases, followed by administration of prodrug, there was a significant prolongation of survival and a 30% cure rate. In summary, owing to its tumor-targeting capabilities, high transduction efficiency, and high gene expression, a vaccinia virus expressing PNP could prove to be a potent and valuable vector for tumor-targeted gene therapy.

Regulation of expression of the valine (branched-chain amino acid) dehydrogenase-encoding gene from Streptomyces coelicolor

Expression of the Streptomyces coelicolor (Sc) valine (branched-chain amino acid) dehydrogenase-encoding gene (vdh) is regulated by valine, glucose and NH 4 + at the transcriptional level. The results of assays for the level of accumulated vdh mRNA in the Sc J802 strain by primer extension experiments and for the level of catechol dioxygenase (Xy1E) activity in Sc J802 (vdh::xylE) transformants show that transcription of the vdh gene is induced approx. 2.5-fold by valine, as compared to asparagine as the sole nitrogen source in the presence of glucose as carbon source. Valine induction is repressed by glucose, as compared to glycerol as the carbon source, and by NH 4 +. Glucose catabolite repression is relieved in Sc M480, a glucose kinase ( glkA) deletion mutant. This suggests that glucose repression of vdh and carbohydrate metabolism are due to the same mechanism in Sc, which involves glucose kinase.

Quantitative analysis of herpes simplex virus DNA and transcriptional activity in ganglia of mice latently infected with wild-type and thymidine kinase-deficient viral strains.

The relationship between herpes simplex virus (HSV) DNA replication and establishment of latent infection was examined using an experimental model that makes use of the segmental sensory innervation of mouse flanks (T7 to T12). Ganglia from consecutive thoracic segments of C57BL/10 mice latently infected with a virulent strain of HSV-1 (SC16) were compared with respect to (i) HSV DNA levels, (ii) latency-associated transcripts (LATs) and (iii) numbers of LAT+ neurons. In concordance with previous results, two patterns of virus persistence were detected distinguished by either a low (10 to 23) or high (approx. 200) number of viral genomes/LAT+ neuron. The high copy pattern was associated, anatomically, with ganglia directly innervating inoculated skin (T7/8). Paradoxically, the highest number of LAT+ neurons and the highest concentrations of LATs were detected in spinal segments (e.g. T10) containing the lowest number of viral genomes, implying that most of the latent SC16 DNA detected at T7 and T8 was transcriptionally repressed. When neuronal amplification of HSV DNA during the establishment phase was prevented by infecting mice with a viral thymidine kinase deletion mutant (TKDM21), the high copy pattern was eliminated and each LAT+ neuron contained, on average, 22 TKDM21 genomes. We conclude that input (i.e. unamplified) and progeny (i.e. amplified) DNA sequences persist in the peripheral nervous systems of mice infected with SC16. Structurally, latent TKDM21 DNA lacked free genomic termini, consistent with persistence of input DNA in an integrated or circular episomal configuration.

The trigeminal ganglion is a location for equine herpesvirus 1 latency and reactivation in the horse.

Four specific pathogen-free ponies were infected intranasally with equine herpesvirus 1 (EHV-1) and two were similarly infected with an EHV-1 thymidine kinase deletion mutant. The primary infections were characterized by a transient fever accompanied by virus shedding into nasal mucus and viraemia. No virus was detected in clinical specimens after 15 days post-infection. Two months later a reactivation stimulus was administered to all six ponies and only the four that had been previously inoculated with wild-type EHV-1 shed virus into nasal mucus (for 10 days), proving the presence of a latent infection. No recurrence of viraemia was observed. The animals were monitored for a further 6 weeks and were consistently shown to be free from infectious virus. Tissues were then obtained postmortem. Co-cultivation of explanted trigeminal ganglia from two out of the four ponies that carried the wild-type virus yielded cultures positive for infectious virus. Apart from nasal epithelium, no infectious virus was recovered from any other tissue. PCR confirmed the presence of virus DNA in the ganglia from all six ponies. Lymphoid tissues also yielded positive signals using this technique. The relevance of virus detection by PCR in lymphoid and neural tissues is discussed in relation to the potential for reactivation of latent virus in the host. However, evidence is presented to show that EHV-1 is neurotropic and, in common with other members of the alpha-herpesvirus subfamily, establishes latency in sensory ganglia from which virus can be reactivated.

The Role of Herpes Simplex Virus Type 1 Thymidine Kinase in Pathogenesis

A genetically engineered herpes simplex virus type 1 (HSV-1)thymidine kinase (TK) deletion mutant has been constructed and used to investigate the role of this gene in pathogenesis. Inoculation of mice with the HSV TK deletion mutant resulted in the establishment of latent ganglionic infection as demonstrated by superinfection of explanted ganglia with wild-type (wt) virus but not by routine explant culture suggesting that the virus-encoded TK is not essential for the establishment of latent infection but may be necessary for either reactivation or virus replication following reactivation. In addition, Southern blot hybridization has been used to demonstrate in vivo complementation of this mutant by wt virus in both peripheral and central nervous system tissues of mice during acute infection and to show that such complementation can result in the establishment and reactivation of latent TK- infection.

4824667 Thymidine kinase deletion mutants of bovine herpesvirus-1, vaccines against infectious bovine rhinotracheitis containing same and methods for the production and use of same: Malon Kit, Saul Kit assigned to NovaGene Inc; Baylor College of Medici

4824667 Thymidine kinase deletion mutants of bovine herpesvirus-1, vaccines against infectious bovine rhinotracheitis containing same and methods for the production and use of same: Malon Kit, Saul Kit assigned to NovaGene Inc; Baylor College of Medici

4824667 Thymidine kinase deletion mutants of bovine herpesvirus-1, vaccines against infectious bovine rhinotracheitis containing same and methods for the production and use of same: Malon Kit, Saul Kit assigned to NovaGene Inc; Baylor College of Medici

4824667 Thymidine kinase deletion mutants of bovine herpesvirus-1, vaccines against infectious bovine rhinotracheitis containing same and methods for the production and use of same: Malon Kit, Saul Kit assigned to NovaGene Inc; Baylor College of Medici

4703011 Thymidine kinase deletion mutants of bovine herpesvirus-1: Malon Kit, Saul Kit assigned to NovaGene Inc; Baylor College of Medici

4703011 Thymidine kinase deletion mutants of bovine herpesvirus-1: Malon Kit, Saul Kit assigned to NovaGene Inc; Baylor College of Medici