scholarly journals FLR-4, a Novel Serine/Threonine Protein Kinase, Regulates Defecation Rhythm in Caenorhabditis elegans

2005 ◽  
Vol 16 (3) ◽  
pp. 1355-1365 ◽  
Author(s):  
Masaya Take-uchi ◽  
Yuri Kobayashi ◽  
Koutarou D. Kimura ◽  
Takeshi Ishihara ◽  
Isao Katsura
Keyword(s):  
Caenorhabditis Elegans ◽  
Protein Kinase ◽  
Temperature Shift ◽  
Kinase Domain ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Pharyngeal Muscles ◽  
Unique Structural Feature

The defecation behavior of the nematode Caenorhabditis elegans is controlled by a 45-s ultradian rhythm. An essential component of the clock that regulates the rhythm is the inositol trisphosphate receptor in the intestine, but other components remain to be discovered. Here, we show that the flr-4 gene, whose mutants exhibit very short defecation cycle periods, encodes a novel serine/threonine protein kinase with a carboxyl terminal hydrophobic region. The expression of functional flr-4::GFP was detected in the intestine, part of pharyngeal muscles and a pair of neurons, but expression of flr-4 in the intestine was sufficient for the wild-type phenotype. Furthermore, laser killing of the flr-4–expressing neurons did not change the defecation phenotypes of wild-type and flr-4 mutant animals. Temperature-shift experiments with a temperature-sensitive flr-4 mutant suggested that FLR-4 acts in a cell-functional rather than developmental aspect in the regulation of defecation rhythms. The function of FLR-4 was impaired by missense mutations in the kinase domain and near the hydrophobic region, where the latter allele seemed to be a weak antimorph. Thus, a novel protein kinase with a unique structural feature acts in the intestine to increase the length of defecation cycle periods.

scholarly journals Molecular evidence for the direct involvement of a protein kinase C in developmental and behavioural susceptibility to tumour-promoting phorbol esters in Caenorhabditis elegans

1995 ◽  
Vol 312 (1) ◽  
pp. 69-74 ◽  
Author(s):  
Y Tabuse ◽  
T Sano ◽  
K Nishiwaki ◽  
J Miwa
Keyword(s):  
Protein Kinase C ◽  
Caenorhabditis Elegans ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Kinase Domain ◽  
Molecular Evidence ◽  
Wild Type ◽  
C Elegans ◽  
Kinase C ◽  
Tumour Promoters

The nematode Caenorhabditis elegans displays developmental and behavioural sensitivity to tumour-promoting phorbol esters. This sensitivity involves the gene tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B. Here we report the molecular nature of the sensitivity in this animal. Characterization of transposon Tc1-induced phorbol ester-resistant mutants has revealed that Tc1 was inserted in a region encoding the kinase domain, resulting in the loss of tpa-1 products. Introduction of a genomic DNA containing the entire wild-type tpa-1 locus into a Tc1-inserted mutant restored the sensitivity to tumour promoters, and tpa-1 products were also produced. These results suggest that the function of wild-type TPA-1 is necessary and sufficient for tumour promoters to cause developmental and behavioural sensitivity in C. elegans.

scholarly journals Caenorhabditis elegans lin-45 raf Is Essential for Larval Viability, Fertility and the Induction of Vulval Cell Fates

Genetics ◽  
2002 ◽  
Vol 160 (2) ◽  
pp. 481-492
Author(s):  
Virginia Hsu ◽  
Cheri L Zobel ◽  
Eric J Lambie ◽  
Tim Schedl ◽  
Kerry Kornfeld
Keyword(s):  
Caenorhabditis Elegans ◽  
Protein Kinase ◽  
Map Kinase ◽  
Signal Transmission ◽  
Kinase Domain ◽  
Binding Domain ◽  
Protein Kinase Activity ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Cell Fates

Abstract The protein kinase Raf is an important signaling protein. Raf activation is initiated by an interaction with GTP-bound Ras, and Raf functions in signal transmission by phosphorylating and activating a mitogen-activated protein (MAP) kinase kinase named MEK. We identified 13 mutations in the Caenorhabditis elegans lin-45 raf gene by screening for hermaphrodites with abnormal vulval formation or germline function. Weak, intermediate, and strong loss-of-function or null mutations were isolated. The phenotype caused by the most severe mutations demonstrates that lin-45 is essential for larval viability, fertility, and the induction of vulval cell fates. The lin-45(null) phenotype is similar to the mek-2(null) and mpk-1(null) phenotypes, indicating that LIN-45, MEK-2, and MPK-1 ERK MAP kinase function in a predominantly linear signaling pathway. The lin-45 alleles include three missense mutations that affect the Ras-binding domain, three missense mutations that affect the protein kinase domain, two missense mutations that affect the C-terminal 14-3-3 binding domain, three nonsense mutations, and one small deletion. The analysis of the missense mutations indicates that Ras binding, 14-3-3-binding, and protein kinase activity are necessary for full Raf function and suggests that a 14-3-3 protein positively regulates Raf-mediated signaling during C. elegans development.

scholarly journals Distinct roles of the Drosophila ninaC kinase and myosin domains revealed by systematic mutagenesis

1993 ◽  
Vol 122 (3) ◽  
pp. 601-612 ◽  
Author(s):  
JA Porter ◽  
C Montell
Keyword(s):  
Protein Kinase ◽  
Retinal Degeneration ◽  
Kinase Domain ◽  
Specific Protein ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Multiple Functions

The Drosophila ninaC locus encodes a rhabdomere specific protein (p174) with linked protein kinase and myosin domains, required for a wild-type ERG and to prevent retinal degeneration. To investigate the role for linked kinase and myosin domains, we analyzed mutants generated by site-directed mutagenesis. Mutation of the kinase domain resulted in an ERG phenotype but no retinal degeneration. Deletion of the myosin domain caused a change in the subcellular distribution of p174 and resulted in both ERG and retinal degeneration phenotypes. Temperature-sensitive mutations in the myosin domain resulted in retinal degeneration, but no ERG phenotype. These results indicated that the ERG and retinal degeneration phenotypes were not strictly coupled suggesting that the myosin domain has multiple functions. We propose that the role of the kinase domain is to regulate other rhabdomeric proteins important in phototransduction and that the myosin domain has at least two roles: to traffic the kinase into the rhabdomeres and to maintain the rhabdomeres.

In silico profiling and structural insights of missense mutations in RET protein kinase domain by molecular dynamics and docking approach

2014 ◽  
Vol 10 (3) ◽  
pp. 421-436 ◽  
Author(s):  
C. George Priya Doss ◽  
B. Rajith ◽  
Chiranjib Chakraboty ◽  
V. Balaji ◽  
R. Magesh ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Protein Kinase ◽  
In Silico ◽  
Kinase Domain ◽  
Missense Mutations ◽  
Protein Kinase Domain ◽  
Docking Approach ◽  
Structural Insights

Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast.

Genetics ◽  
1992 ◽  
Vol 132 (1) ◽  
pp. 53-62 ◽  
Author(s):  
R E Hollingsworth ◽  
R M Ostroff ◽  
M B Klein ◽  
L A Niswander ◽  
R A Sclafani
Keyword(s):  
Protein Kinase ◽  
Insertional Mutagenesis ◽  
Molecular Genetic ◽  
Dna Lesions ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Wild Type ◽  
Mutant Proteins ◽  
Repair Enzymes ◽  
Induced Mutagenesis

Abstract The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.

scholarly journals Heterozygous mutations affecting the protein kinase domain of CDK13 cause a syndromic form of developmental delay and intellectual disability

2017 ◽  
Vol 55 (1) ◽  
pp. 28-38 ◽  
Author(s):  
Mark J Hamilton ◽  
Richard C Caswell ◽  
Natalie Canham ◽  
Trevor Cole ◽  
Helen V Firth ◽  
...  
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
Intellectual Disability ◽  
Protein Kinase ◽  
Developmental Delay ◽  
Congenital Heart ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Protein Kinase Domain

IntroductionRecent evidence has emerged linking mutations in CDK13 to syndromic congenital heart disease. We present here genetic and phenotypic data pertaining to 16 individuals with CDK13 mutations.MethodsPatients were investigated by exome sequencing, having presented with developmental delay and additional features suggestive of a syndromic cause.ResultsOur cohort comprised 16 individuals aged 4–16 years. All had developmental delay, including six with autism spectrum disorder. Common findings included feeding difficulties (15/16), structural cardiac anomalies (9/16), seizures (4/16) and abnormalities of the corpus callosum (4/11 patients who had undergone MRI). All had craniofacial dysmorphism, with common features including short, upslanting palpebral fissures, hypertelorism or telecanthus, medial epicanthic folds, low-set, posteriorly rotated ears and a small mouth with thin upper lip vermilion. Fifteen patients had predicted missense mutations, including five identical p.(Asn842Ser) substitutions and two p.(Gly717Arg) substitutions. One patient had a canonical splice acceptor site variant (c.2898–1G>A). All mutations were located within the protein kinase domain of CDK13. The affected amino acids are highly conserved, and in silico analyses including comparative protein modelling predict that they will interfere with protein function. The location of the missense mutations in a key catalytic domain suggests that they are likely to cause loss of catalytic activity but retention of cyclin K binding, resulting in a dominant negative mode of action. Although the splice-site mutation was predicted to produce a stable internally deleted protein, this was not supported by expression studies in lymphoblastoid cells. A loss of function contribution to the underlying pathological mechanism therefore cannot be excluded, and the clinical significance of this variant remains uncertain.ConclusionsThese patients demonstrate that heterozygous, likely dominant negative mutations affecting the protein kinase domain of the CDK13 gene result in a recognisable, syndromic form of intellectual disability, with or without congenital heart disease.

scholarly journals Casein kinase II is required for efficient transcription by RNA polymerase III.

1996 ◽  
Vol 16 (3) ◽  
pp. 892-898 ◽  
Author(s):  
D J Hockman ◽  
M C Schultz
Keyword(s):  
Protein Kinase ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Rna Polymerases ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Polymerase Iii

Casein kinase II (CKII) is a ubiquitous and highly conserved serine/threonine protein kinase found in the nucleus and cytoplasm of most cells. Using a combined biochemical and genetic approach in the yeast Saccharomyces cerevisiae, we assessed the role of CKII in specific transcription by RNA polymerases I, II, and III. CKII is not required for basal transcription by RNA polymerases I and II but is important for polymerase III transcription. Polymerase III transcription is high in extracts with normal CKII activity but low in extracts from a temperature-sensitive mutant that has decreased CKII activity due to a lesion in the enzyme's catalytic alpha' subunit. Polymerase III transcription of 5S rRNA and tRNA templates in the temperature-sensitive extract is rescued by purified, wild-type CKII. An inhibitor of CKII represses polymerase III transcription in wild-type extract, and this repression is partly overcome by supplementing reaction mixtures with active CKII. Finally, we show that polymerase III transcription in vivo is impaired when CKII is inactivated. Our results demonstrate that CKII, an oncogenic protein kinase previously implicated in cell cycle and growth control, is required for high-level transcription by RNA polymerase III.

scholarly journals Evidence for an Essential Catalytic Role of the F10 Protein Kinase in Vaccinia Virus Morphogenesis

2004 ◽  
Vol 78 (1) ◽  
pp. 257-265 ◽  
Author(s):  
Patricia Szajner ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Protein Kinase ◽  
Vaccinia Virus ◽  
Early Stage ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Membrane Formation ◽  
Virus Morphogenesis ◽  
Catalytic Role

ABSTRACT Temperature-sensitive mutants of vaccinia virus, with genetic changes that map to the open reading frame encoding the F10 protein kinase, exhibit a defect at an early stage of viral morphogenesis. To further study the role of the enzyme, we constructed recombinant vaccinia virus vF10V5i, which expresses inducible V5 epitope-tagged F10 and is dependent on a chemical inducer for plaque formation and replication. In the absence of inducer, viral membrane formation was delayed and crescents and occasional immature forms were detected only late in infection. When the temperature was raised from 37 to 39°C, the block in membrane formation persisted throughout the infection. The increased stringency may be explained by a mild temperature sensitivity of the wild-type F10 kinase, which reduced the activity of the very small amount expressed in the absence of inducer, or by the thermolability of an unphosphorylated kinase substrate or uncomplexed F10-interacting protein. Further analyses demonstrated that tyrosine and threonine phosphorylation of the A17 membrane component was inhibited in the absence of inducer. The phosphorylation defect could be overcome by transfection of plasmids that express wild-type F10, but not by plasmids that express F10 with single amino acid substitutions that abolished catalytic activity. Although the mutated forms of F10 were stable and concentrated in viral factories, only the wild-type protein complemented the assembly and replication defects of vF10V5i in the absence of inducer. These studies provide evidence for an essential catalytic role of the F10 kinase in vaccinia virus morphogenesis.

scholarly journals Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (10) ◽  
pp. 3637-3645 ◽  
Author(s):  
J Schultz ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Nucleotide Sequence ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Kinase Gene ◽  
Multiple Copies ◽  
Protein Kinase Gene ◽  
Rna Disruption ◽  
High Level

Mutations in the SSN6 gene suppress the invertase derepression defect caused by a lesion in the SNF1 protein kinase gene. We cloned the SSN6 gene of Saccharomyces cerevisiae and identified its 3.3-kilobase poly(A)-containing RNA. Disruption of the gene caused phenotypes similar to, but more severe than, those caused by missense mutations: high-level constitutivity for invertase, clumpiness, temperature-sensitive growth, alpha-specific mating defects, and failure to homozygous diploids to sporulate. In contrast, the presence of multiple copies of SSN6 interfered with derepression of invertase. An ssn6 mutation was also shown to cause glucose-insensitive expression of a GAL10-lacZ fusion and maltase. The mating defects of MAT alpha ssn6 strains were associated with production of two a-specific products, a-factor and barrier, and reduced levels of alpha-factor; no deficiency of MAT alpha 2 RNA was detected. We showed that ssn6 partially restored invertase expression in a cyr1-2 mutant, although ssn6 was clearly not epistatic to cyr1-2. We also determined the nucleotide sequence of SSN6, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). Possible functions of the SSN6 product are discussed.

scholarly journals Functional characterization of Polr3a hypomyelinating leukodystrophy mutations in the S. cerevisiae homolog, RPC160

2020 ◽  
Author(s):  
Robyn D. Moir ◽  
Christian Lavados ◽  
JaeHoon Lee ◽  
Ian M. Willis
Keyword(s):  
Functional Characterization ◽  
Rna Polymerase Iii ◽  
State Level ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Wild Type ◽  
Functional Deficits ◽  
Pol Iii ◽  
Transcriptional Output

AbstractMutations in RNA polymerase III (Pol III) cause hypomeylinating leukodystrophy (HLD) and neurodegeneration in humans. POLR3A and POLR3B, the two largest Pol III subunits, together form the catalytic center and carry the majority of disease alleles. Disease-causing mutations include invariant and highly conserved residues that are predicted to negatively affect Pol III activity and decrease transcriptional output. A subset of HLD missense mutations in POLR3A cluster in the pore region that provides nucleotide access to the Pol III active site. These mutations were engineered at the corresponding positions in the Saccharomyces cerevisiae homolog, Rpc160, to evaluate their functional deficits. None of the mutations caused a growth or transcription phenotype in yeast. Each mutation was combined with a frequently occurring pore mutation, POLR3A G672E, which was also wild-type for growth and transcription. The double mutants showed a spectrum of phenotypes from wild-type to lethal, with only the least fit combinations showing an effect on Pol III transcription. In one slow-growing temperature-sensitive mutant the steady-state level of tRNAs was unaffected, however global tRNA synthesis was compromised, as was the synthesis of RPR1 and SNR52 RNAs. Affinity-purified mutant Pol III was broadly defective in both factor-independent and factor-dependent transcription in vitro across genes that represent the yeast Pol III transcriptome. Thus, the robustness of yeast to Pol III leukodystrophy mutations in RPC160 can be overcome by a combinatorial strategy.

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