Kinase Deletion Mutant Research Articles

Systematic analysis of the Candida albicans kinome reveals environmentally contingent protein kinase-mediated regulation of filamentation and biofilm formation in vitro and in vivo.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans. An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCECandida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.

Host-derived reactive oxygen species trigger activation of the Candida albicans transcription regulator Rtg1/3.

The signals that denote mammalian host environments and dictate the activation of signaling pathways in human-associated microorganisms are often unknown. The transcription regulator Rtg1/3 in the human fungal pathogen Candida albicans is a crucial determinant of host colonization and pathogenicity. Rtg1/3's activity is controlled, in part, by shuttling the regulator between the cytoplasm and nucleus of the fungus. The host signal(s) that Rtg1/3 respond(s) to, however, have remained unclear. Here we report that neutrophil-derived reactive oxygen species (ROS) direct the subcellular localization of this C. albicans transcription regulator. Upon engulfment of Candida cells by human or mouse neutrophils, the regulator shuttles to the fungal nucleus. Using genetic and chemical approaches to disrupt the neutrophils' oxidative burst, we establish that the oxidants produced by the NOX2 complex-but not the oxidants generated by myeloperoxidase-trigger Rtg1/3's migration to the nucleus. Furthermore, screening a collection of C. albicans kinase deletion mutants, we implicate the MKC1 signaling pathway in the ROS-dependent regulation of Rtg1/3 in this fungus. Finally, we show that Rtg1/3 contributes to C. albicans virulence in the nematode Caenorhabditis elegans in an ROS-dependent manner as the rtg1 and rtg3 mutants display virulence defects in wild-type but not in ROS deficient worms. Our findings establish NOX2-derived ROS as a key signal that directs the activity of the pleiotropic fungal regulator Rtg1/3.

The protein kinase Ire1 has a Hac1-independent essential role in iron uptake and virulence of Candida albicans.

Protein kinases play central roles in virtually all signaling pathways that enable organisms to adapt to their environment. Microbial pathogens must cope with severely restricted iron availability in mammalian hosts to invade and establish themselves within infected tissues. To uncover protein kinase signaling pathways that are involved in the adaptation of the pathogenic yeast Candida albicans to iron limitation, we generated a comprehensive protein kinase deletion mutant library of a wild-type strain. Screening of this library revealed that the protein kinase Ire1, which has a conserved role in the response of eukaryotic cells to endoplasmic reticulum stress, is essential for growth of C. albicans under iron-limiting conditions. Ire1 was not necessary for the activity of the transcription factor Sef1, which regulates the response of the fungus to iron limitation, and Sef1 target genes that are induced by iron depletion were normally upregulated in ire1Δ mutants. Instead, Ire1 was required for proper localization of the high-affinity iron permease Ftr1 to the cell membrane. Intriguingly, iron limitation did not cause increased endoplasmic reticulum stress, and the transcription factor Hac1, which is activated by Ire1-mediated removal of the non-canonical intron in the HAC1 mRNA, was dispensable for Ftr1 localization to the cell membrane and growth under iron-limiting conditions. Nevertheless, expression of a pre-spliced HAC1 copy in ire1Δ mutants restored Ftr1 localization and rescued the growth defects of the mutants. Both ire1Δ and hac1Δ mutants were avirulent in a mouse model of systemic candidiasis, indicating that an appropriate response to endoplasmic reticulum stress is important for the virulence of C. albicans. However, the specific requirement of Ire1 for the functionality of the high-affinity iron permease Ftr1, a well-established virulence factor, even in the absence of endoplasmic reticulum stress uncovers a novel Hac1-independent essential role of Ire1 in iron acquisition and virulence of C. albicans.

The zinc cluster transcription factor Czf1 regulates cell wall architecture and integrity in Candida albicans.

The fungal cell wall is essential for the maintenance of cellular integrity and mediates interactions of the cells with the environment. It is a highly flexible organelle whose composition and organization is modulated in response to changing growth conditions. In the pathogenic yeast Candida albicans, a network of signaling pathways regulates the structure of the cell wall, and mutants with defects in these pathways are hypersensitive to cell wall stress. By harnessing a library of genetically activated forms of all C.albicans zinc cluster transcription factors, we found that a hyperactive Czf1 rescued the hypersensitivity to cell wall stress of different protein kinase deletion mutants. The hyperactive Czf1 induced the expression of many genes with cell wall-related functions and caused visible changes in the cell wall structure. C.albicans czf1Δ mutants were hypersensitive to the antifungal drug caspofungin, which inhibits cell wall biosynthesis. The changes in cell wall architecture caused by hyperactivity or absence of Czf1 resulted in an increased recognition of C.albicans by human neutrophils. Our results show that Czf1, which is known as a regulator of filamentous growth and white-opaque switching, controls the expression of cell wall genes and modulates the architecture of the cell wall.

Dynamic Transcriptomic and Phosphoproteomic Analysis During Cell Wall Stress in Aspergillus nidulans

The fungal cell-wall integrity signaling (CWIS) pathway regulates cellular response to environmental stress to enable wall repair and resumption of normal growth. This complex, interconnected, pathway has been only partially characterized in filamentous fungi. To better understand the dynamic cellular response to wall perturbation, a β-glucan synthase inhibitor (micafungin) was added to a growing A. nidulans shake-flask culture. From this flask, transcriptomic and phosphoproteomic data were acquired over 10 and 120 min, respectively. To differentiate statistically-significant dynamic behavior from noise, a multivariate adaptive regression splines (MARS) model was applied to both data sets. Over 1800 genes were dynamically expressed and over 700 phosphorylation sites had changing phosphorylation levels upon micafungin exposure. Twelve kinases had altered phosphorylation and phenotypic profiling of all non-essential kinase deletion mutants revealed putative connections between PrkA, Hk-8-4, and Stk19 and the CWIS pathway. Our collective data implicate actin regulation, endocytosis, and septum formation as critical cellular processes responding to activation of the CWIS pathway, and connections between CWIS and calcium, HOG, and SIN signaling pathways.

Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes.

Sexual and asexual development in filamentous ascomycetes is controlled by components of conserved signaling pathways. Here, we investigated the development of mutant strains lacking genes for kinases MAK2, MEK2, and MIK2, as well as the scaffold protein HAM5 of the pheromone response (PR) pathway. All had a defect in fruiting body development and hyphal fusion. Another phenotype was a defect in melanin-dependent ascospore germination. However, this deficiency was observed only in kinase deletion mutants, but not in strains lacking HAM5. Notably, the same developmental phenotypes were previously described for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) mutants, but the germination defect was only seen in NOX2 mutants. These data suggest a molecular link between the pheromone signaling pathway and both NOX complexes. Using data from yeast two-hybrid (Y2H) analysis, we found that the scaffolding protein HAM5 interacts with NOR1, the regulator of NOX1 and NOX2 complexes. This interaction was further confirmed using differently fluorescent-labeled proteins to demonstrate that NOR1 and HAM5 co-localize at cytoplasmic spots and tips of mature hyphae. This observation was supported by phenotypic characterization of single and double mutants. The oxidative stress response and the initiation of fruiting bodies were similar in Δham5Δnor1 and Δham5, but distinctly reduced in Δnor1, indicating that the double deletion leads to a partial suppression of the Δnor1 phenotype. We conclude that the PR and NOX1 complexes are connected by direct interaction between HAM5 and NOR1. In contrast, PR kinases are linked to the NOX2 complex without participation of HAM5.

N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism.

Tannerella forsythia is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of the Escherichia coli etherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described for T. forsythia. In between the respective genes on the T. forsythia genome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 104-fold higher catalytic efficiency (kcat/Km) for MurNAc than for N-acetylglucosamine (GlcNAc) with kcat values of 10.5 s-1 and 0.1 s-1 and Km values of 200 μM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition (Ki[S] = 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism of T. forsythia, a kinase deletion mutant (ΔTf_murK::erm) was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, the ΔTf_murK::erm mutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 μg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 μg/ml MurNAc for optimal growth. These data reveal that T. forsythia readily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion of Tf_murK blocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of the T. forsythia cell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis.

Genome Sequences of the Mycobacterium tuberculosis H37Rv-ptkA Deletion Mutant and Its Parental Strain.

ABSTRACTMycobacterium tuberculosis, the etiological agent of tuberculosis, is one of the most devastating infectious agents in the world. Here, we report the draft genome sequences of the M. tuberculosis protein tyrosine kinase (ptkA) deletion mutant and its parental strain H37Rv, which are used in genetic studies and for drug discovery.

The Snf1-activating kinase Sak1 is a key regulator of metabolic adaptation and in vivo fitness of Candida albicans.

The metabolic flexibility of the opportunistic fungal pathogen Candida albicans is important for colonisation and infection of different host niches. Complex regulatory networks, in which protein kinases play central roles, link metabolism and other virulence-associated traits, such as filamentous growth and stress resistance, and thereby control commensalism and pathogenicity. By screening a protein kinase deletion mutant library that was generated in the present work using an improved SAT1 flipper cassette, we found that the previously uncharacterised kinase Sak1 is a key upstream activator of the protein kinase Snf1, a highly conserved regulator of nutrient stress responses that is essential for viability in C. albicans. The sak1Δ mutants failed to grow on many alternative carbon sources and were hypersensitive to cell wall/membrane stress. These phenotypes were mirrored in mutants lacking other subunits of the SNF1 complex and partially compensated by a hyperactive form of Snf1. Transcriptional profiling of sak1Δ mutants showed that Sak1 ensures basal expression of glyoxylate cycle and gluconeogenesis genes even in glucose-rich media and thereby contributes to the metabolic plasticity of C. albicans. In a mouse model of gastrointestinal colonisation, sak1Δ mutants were rapidly outcompeted by wild-type cells, demonstrating that Sak1 is essential for the in vivo fitness of C. albicans.

Dihydroxyacetone metabolism in Haloferax volcanii

Dihydroxyacetone (DHA) is a ketose sugar that can be produced by oxidizing glycerol. DHA in the environment is taken up and phosphorylated to DHA-phosphate by glycerol kinase or DHA kinase. In hypersaline environments, it is hypothesized that DHA is produced as an overflow product from glycerol utilization by organisms such as Salinibacter ruber. Previous research has demonstrated that the halobacterial species Haloquadratum walsbyi can use DHA as a carbon source, and putative DHA kinase genes were hypothesized to be involved in this process. However, DHA metabolism has not been demonstrated in other halobacterial species, and the role of the DHA kinase genes was not confirmed. In this study, we examined the metabolism of DHA in Haloferax volcanii because putative DHA kinase genes were annotated in its genome, and it has an established genetic system to assay growth of mutant knockouts. Experiments in which Hfx. volcanii was grown on DHA as the sole carbon source demonstrated growth, and that it is concentration dependent. Three annotated DHA kinase genes (HVO_1544, HVO_1545, and HVO_1546), which are homologous to the putative DHA kinase genes present in Hqm. walsbyi, as well as the glycerol kinase gene (HVO_1541), were deleted to examine the effect of these genes on the growth of Hfx. volcanii on DHA. Experiments demonstrated that the DHA kinase deletion mutant exhibited diminished, but not absence of growth on DHA compared to the parent strain. Deletion of the glycerol kinase gene also reduced growth on DHA, and did so more than deletion of the DHA kinase. The results indicate that Hfx. volcanii can metabolize DHA and that DHA kinase plays a role in this metabolism. However, the glycerol kinase appears to be the primary enzyme involved in this process. BLASTp analyses demonstrate that the DHA kinase genes are patchily distributed among the Halobacteria, whereas the glycerol kinase gene is widely distributed, suggesting a widespread capability for DHA metabolism.

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6')-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6')-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k(cat)/K(m) = 10(5)-10(7) M(-1) s(-1)). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.

Extracellular sugar phosphates are assimilated by Streptomyces in a PhoP-dependent manner

Filamentous microorganisms of the bacterial genus Streptomyces have a complex life cycle that includes physiological and morphological differentiations. It is now fairly well accepted that lysis of Streptomyces vegetative mycelium induced by programmed cell death (PCD) provides the required nutritive sources for the bacterium to erect spore-forming aerial hyphae. However, little is known regarding cellular compounds released during PCD and the contribution of these molecules to the feeding of surviving cells in order to allow them to reach the late stages of the developmental program. In this work we assessed the effect of extracellular sugar phosphates (that are likely to be released in the environment upon cell lysis) on the differentiation processes. We demonstrated that the supply of phosphorylated sugars, under inorganic phosphate limitation, delays the occurrence of the second round of PCD, blocks streptomycetes life cycle at the vegetative state and inhibits antibiotic production. The mechanism by which sugar phosphates affect development was shown to involve genes of the Pho regulon that are under the positive control of the two component system PhoR/PhoP. Indeed, the inactivation of the response regulator phoP of Streptomyces lividans prevented the 'sugar phosphate effect' whereas the S. lividans ppk (polyphosphate kinase) deletion mutant, known to overexpress the Pho regulon, presented an enhanced response to phosphorylated sugars.

Suppression of Etk/Bmx Protects against Ischemic Brain Injury

Etk/Bmx (epithelial and endothelial tyrosine kinase, also known as BMX), a member of the Tec (tyrosine kinase expressed in hepatocellular carcinoma) family of protein-tyrosine kinases, is an important regulator of signal transduction for the activation of cell growth, differentiation, and development. We have previously reported that activation of Etk leads to apoptosis in MDA-MB-468 cells. The purpose of this study was to examine the role of Etk in neuronal injury induced by H(2)O(2) or ischemia. Using Western blot analysis and immunohistochemistry, we found that treatment with H(2)O(2) significantly enhanced phosphorylation of Etk and its downstream signaling molecule Stat1 in primary cortical neurons. Inhibiting Etk activity by LFM-A13 or knocking down Etk expression by a specific shRNA increased the survival of primary cortical neurons. Similarly, at 1 day after a 60-min middle cerebral artery occlusion (MCAo) in adult rats, both phosphorylated Etk and Stat1 were coexpressed with apoptotic markers in neurons in the penumbra. Pretreatment with LFM-A13 or an adenoviral vector encoding the kinase deletion mutant Etkk attenuated caspase-3 activity and infarct volume in ischemic brain. All together, our data suggest that Etk is activated after neuronal injury. Suppressing Etk activity protects against neurodegeneration in ischemic brain.

Mis17 Is a Regulatory Module of the Mis6-Mal2-Sim4 Centromere Complex That Is Required for the Recruitment of CenH3/CENP-A in Fission Yeast

BackgroundThe centromere is the chromosome domain on which the mitotic kinetochore forms for proper segregation. Deposition of the centromeric histone H3 (CenH3, CENP-A) is vital for the formation of centromere-specific chromatin. The Mis6-Mal2-Sim4 complex of the fission yeast S. pombe is required for the recruitment of CenH3 (Cnp1), but its function remains obscure.Methodology/Principal FindingsMass spectrometry was performed on the proteins precipitated with Mis6- and Mis17-FLAG. The results together with the previously identified Sim4- and Mal2-TAP precipitated proteins indicated that the complex contains 12 subunits, Mis6, Sim4, Mal2, Mis15, Mis17, Cnl2, Fta1-4, Fta6-7, nine of which have human centromeric protein (CENP) counterparts. Domain dissection indicated that the carboxy-half of Mis17 is functional, while its amino-half is regulatory. Overproduction of the amino-half caused strong negative dominance, which led to massive chromosome missegregation and hypersensitivity to the histone deacetylase inhibitor TSA. Mis17 was hyperphosphorylated and overproduction-induced negative dominance was abolished in six kinase-deletion mutants, ssp2 (AMPK), ppk9 (AMPK), ppk15 (Yak1), ppk30 (Ark1), wis4 (Ssk2), and lsk1 (P-TEFb).ConclusionsMis17 may be a regulatory module of the Mis6 complex. Negative dominance of the Mis17 fragment is exerted while the complex and CenH3 remain at the centromere, a result that differs from the mislocalization seen in the mis17-362 mutant. The known functions of the kinases suggest an unexpected link between Mis17 and control of the cortex actin, nutrition, and signal/transcription. Possible interpretations are discussed.

Interleukin-2-inducible T Cell Kinase (Itk) Network Edge Dependence for the Maturation of iNKT Cell

Invariant natural killer T (iNKT) cells are a unique subset of innate T lymphocytes that are selected by CD1d. They have diverse immune regulatory functions via the rapid production of interferon-γ (IFN-γ) and interleukin-4 (IL-4). In the absence of signaling nodes Itk and Txk, Tec family non-receptor tyrosine kinases, mice exhibit a significant block in iNKT cell development. We now show here that although the Itk node is required for iNKT cell maturation, the kinase domain edge of Itk is not required for continued maturation iNKT cells in the thymus compared with Itk-null mice. This rescue is dependent on the expression of the Txk node. Furthermore, this kinase domain independent edge rescue correlates with the increased expression of the transcription factors T-bet, the IL-2/IL-15 receptor β chain CD122, and suppression of eomesodermin expression. By contrast, α-galactosyl ceramide induced cytokine secretion is dependent on the kinase domain edge of Itk. These findings indicate that the Itk node uses a kinase domain independent edge, a scaffolding function, in the signaling pathway leading to the maturation of iNKT cells. Furthermore, the findings indicate that phosphorylation of substrates by the Itk node is only partially required for maturation of iNKT cells, while functional activation of iNKT cells is dependent on the kinase domain/activity edge of Itk.

A Metabolomic View of Staphylococcus aureus and Its Ser/Thr Kinase and Phosphatase Deletion Mutants: Involvement in Cell Wall Biosynthesis

Little is known about intracellular metabolite pools in pathogens such as Staphylococcus aureus. We have studied a particular metabolome by means of the presented LC-MS method. By investigating the central carbon metabolism which includes most of the energy transfer molecules like nucleotides, sugar mono- and biphosphates, and cofactors, a conclusion about phenotypes and stress answers in microorganisms is possible. Quantitative metabolite levels of S. aureus grown in complex lysogeny broth and in minimal medium were compared in the wild-type S. aureus strain 8325 and the isogenic eukaryotic-like protein serine/threonine kinase (DeltapknB) and phosphatase (Deltastp) deletion mutants. Detection of several remarkable differences, e.g., in nucleotide metabolism and especially cell wall precursor metabolites, indicates a previously unreported importance of serine/threonine kinase/phosphatase on peptidoglycan and wall teichoic acid biosynthesis. These findings may lead to new insights into the regulation of staphylococcal cell wall metabolism.

Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant (ΔUL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the ΔUL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or ΔUL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites.

A kinase independent function for Tec kinase ITK in regulating antigen receptor induced serum response factor activation

The Tec family kinases are critical downstream regulators of antigen receptor signals in lymphocytes. As kinases, they act on critical substrates to regulate signals such as calcium increase leading to activation of transcription factors such as NFAT, NFκB and SRF. We now show here that ITK, a member of the Tec family of tyrosine kinases, has a kinase independent function. Mutants of ITK that lack kinase activity or a kinase domain can rescue cells lacking Tec family kinases for antigen receptor induced SRF activation, but not for NFAT, AP-1 or NFκB activation. Furthermore, expression of these mutants in WT cells enhanced SRF activation. This kinase independent function required the SH2 domain since a mutant lacking both the kinase and SH2 domains was much less effective at rescuing SRF activation. This kinase-deleted mutant could partially rescue ERK activation, and interact with multiple tyrosine phosphorylated proteins during antigen receptor signaling, suggesting that ITK uses a scaffolding function that regulates signals leading to specific regulation of SRF activation.

A VicRK Signal Transduction System in Streptococcus mutans Affects gtfBCD , gbpB , and ftf Expression, Biofilm Formation, and Genetic Competence Development

Bacteria exposed to transient host environments can elicit adaptive responses by triggering the differential expression of genes via two-component signal transduction systems. This study describes the vicRK signal transduction system in Streptococcus mutans. A vicK (putative histidine kinase) deletion mutant (SmuvicK) was isolated. However, a vicR (putative response regulator) null mutation was apparently lethal, since the only transformants isolated after attempted mutagenesis overexpressed all three genes in the vicRKX operon (Smuvic+). Compared with the wild-type UA159 strain, both mutants formed aberrant biofilms. Moreover, the vicK mutant biofilm formed in sucrose-supplemented medium was easily detachable relative to that of the parent. The rate of total dextran formation by this mutant was remarkably reduced compared to the wild type, whereas it was increased in Smuvic+. Based on real-time PCR, Smuvic+ showed increased gtfBCD, gbpB, and ftf expression, while a recombinant VicR fusion protein was shown to bind the promoter regions of the gtfB, gtfC, and ftf genes. Also, transformation efficiency in the presence or absence of the S. mutans competence-stimulating peptide was altered for the vic mutants. In vivo studies conducted using SmuvicK in a specific-pathogen-free rat model resulted in significantly increased smooth-surface dental plaque (Pearson-Filon statistic [PF], <0.001). While the absence of vicK did not alter the incidence of caries, a significant reduction in SmuvicK CFU counts was observed in plaque samples relative to that of the parent (PF, <0.001). Taken together, these findings support involvement of the vicRK signal transduction system in regulating several important physiological processes in S. mutans.

Channel Function Is Dissociated from the Intrinsic Kinase Activity and Autophosphorylation of TRPM7/ChaK1

TRPM7/ChaK1 is a unique channel/kinase that contains a TRPM channel domain with 6 transmembrane segments fused to a novel serine-threonine kinase domain at its C terminus. The goal of this study was to investigate a possible role of kinase activity and autophosphorylation in regulation of channel activity of TRPM7/ChaK1. Residues essential for kinase activity were identified by site-directed mutagenesis. Two major sites of autophosphorylation were identified in vitro by mass spectrometry at Ser(1511) and Ser(1567), and these sites were found to be phosphorylated in intact cells. TRPM7/ChaK1 is a cation-selective channel that exhibits strong outward rectification and inhibition by millimolar levels of internal [Mg(2+)]. Mutation of the two autophosphorylation sites or of a key catalytic site that abolished kinase activity did not alter channel activity measured by whole-cell recording or Ca(2+) influx. Inhibition by internal Mg(2+) was also unaffected in the autophosphorylation site or "kinase-dead" mutants. Moreover, kinase activity was enhanced by Mg(2+), was decreased by Zn(2+), and was unaffected by Ca(2+). In contrast, channel activity was inhibited by all three of these divalent cations. However, deletion of much of C-terminal kinase domain resulted in expression of an apparently inactive channel. We conclude that neither current activity nor regulation by internal Mg(2+) is affected by kinase activity or autophosphorylation but that the kinase domain may play a structural role in channel assembly or subcellular localization.