scholarly journals Functional Domains of Fused, a Serine-Threonine Kinase Required for Signaling in Drosophila

Genetics ◽  
1996 ◽  
Vol 142 (4) ◽  
pp. 1181-1198
Author(s):  
Pascal Thérond ◽  
Georges Alves ◽  
Bernadette Limbourg-Bouchon ◽  
Hervé Tricoire ◽  
Elizabeth Guillemet ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Kinase Domain ◽  
In Vitro Mutagenesis ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Terminal Domain ◽  
Segment Polarity ◽  
Protein Functions

Abstract fused (fu) is a segment-polarity gene encoding a putative serine-threonine kinase. In a wild-type context, all fu mutations display the same set of phenotypes. Nevertheless, mutations of the Suppressor of fused [Su(fu)] gene define three classes of alleles (fu0, fuI, fuII). Here, we report the molecular analysis of known fu mutations and the generation of new alleles by in vitro mutagenesis. We show that the Fused (Fu) protein functions in vivo as a kinase. The N-terminal kinase and the extreme C-terminal domains are necessary for Fu+ activity while a central region appears to be dispensable. We observe a striking correlation between the molecular lesions of fu mutations and the phenotype displayed in their interaction with Su(fu). Indeed, fuI alleles which are suppressed by Su(fu) mutations are defined by inframe alterations of the N-terminal catalytic domain whereas the C-terminal domain is missing or altered in all fuII alleles. An unregulated FuII protein, which can be limited to the 80 N-terminal amino acids of the kinase domain, would be responsible for the neomorphic costal-2 phenotype displayed by the fuII-Su(fu) interaction. We propose that the Fu C-terminal domain can differentially regulate the Fu catalytic domain according to cell position in the parasegment.

scholarly journals A PERK-Like Receptor Kinase Interacts with the Geminivirus Nuclear Shuttle Protein and Potentiates Viral Infection

2006 ◽  
Vol 80 (13) ◽  
pp. 6648-6656 ◽  
Author(s):  
Lilian H. Florentino ◽  
Anésia A. Santos ◽  
Mariana R. Fontenelle ◽  
Guilherme L. Pinheiro ◽  
Francisco M. Zerbini ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Kinase Domain ◽  
Receptor Protein ◽  
Nuclear Shuttle Protein ◽  
Serine Threonine Kinase ◽  
Viral Dna ◽  
Functional Link ◽  
Threonine Kinase

ABSTRACT The nuclear shuttle protein (NSP) from bipartite geminiviruses facilitates the intracellular transport of viral DNA from the nucleus to the cytoplasm and acts in concert with the movement protein (MP) to promote the cell-to-cell spread of the viral DNA. A proline-rich extensin-like receptor protein kinase (PERK) was found to interact specifically with NSP of Cabbage leaf curl virus (CaLCuV) and of tomato-infecting geminiviruses through a yeast two-hybrid screening. The PERK-like protein, which we designated NsAK (for NSP-associated kinase), is structurally organized into a proline-rich N-terminal domain, followed by a transmembrane segment and a C-terminal serine/threonine kinase domain. The viral protein interacted stably with defective versions of the NsAK kinase domain, but not with the potentially active enzyme, in an in vitro binding assay. In vitro-translated NsAK enhanced the phosphorylation level of NSP, indicating that NSP functions as a substrate for NsAK. These results demonstrate that NsAK is an authentic serine/threonine kinase and suggest a functional link for NSP-NsAK complex formation. This interpretation was corroborated by in vivo infectivity assays showing that loss of NsAK function reduces the efficiency of CaLCuV infection and attenuates symptom development. Our data implicate NsAK as a positive contributor to geminivirus infection and suggest it may regulate NSP function.

scholarly journals A phyB-PIF1-SPA1 kinase regulatory complex promotes photomorphogenesis in Arabidopsis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Inyup Paik ◽  
Fulu Chen ◽  
Vinh Ngoc Pham ◽  
Ling Zhu ◽  
Jeong-Il Kim ◽  
...  
Keyword(s):  
Red Light ◽  
Kinase Domain ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
C Terminus ◽  
Subsequent Degradation ◽  
Regulatory Complex

Abstract CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) is a highly conserved E3 ubiquitin ligase from plants to animals and acts as a central repressor of photomorphogenesis in plants. SUPPRESSOR OF PHYA-105 1 family members (SPA1-SPA4) directly interact with COP1 and enhance COP1 activity. Despite the presence of a kinase domain at the N-terminus, no COP1-independent role of SPA proteins has been reported. Here we show that SPA1 acts as a serine/threonine kinase and directly phosphorylates PIF1 in vitro and in vivo. SPAs are necessary for the light-induced phosphorylation, ubiquitination and subsequent degradation of PIF1. Moreover, the red/far-red light photoreceptor phyB interacts with SPA1 through its C-terminus and enhances the recruitment of PIF1 for phosphorylation. These data provide a mechanistic view on how the COP1-SPA complexes serve as an example of a cognate kinase-E3 ligase complex that selectively triggers rapid phosphorylation and removal of its substrates, and how phyB modulates this process to promote photomorphogenesis.

scholarly journals Regulation of Chk1 by Its C-terminal Domain

2008 ◽  
Vol 19 (11) ◽  
pp. 4546-4553 ◽  
Author(s):  
Ana Kosoy ◽  
Matthew J. O'Connell
Keyword(s):  
Catalytic Domain ◽  
Kinase Domain ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Terminal Domain ◽  
C Terminus ◽  
The Face ◽  
Chk1 Kinase

Chk1 is a protein kinase that is the effector molecule in the G2 DNA damage checkpoint. Chk1 homologues have an N-terminal kinase domain, and a C-terminal domain of ∼200 amino acids that contains activating phosphorylation sites for the ATM/R kinases, though the mechanism of activation remains unknown. Structural studies of the human Chk1 kinase domain show an open conformation; the activity of the kinase domain alone is substantially higher in vitro than full-length Chk1, and coimmunoprecipitation studies suggest the C-terminal domain may contain an autoinhibitory activity. However, we show that truncation of the C-terminal domain inactivates Chk1 in vivo. We identify additional mutations within the C-terminal domain that activate ectopically expressed Chk1 without the need for activating phosphorylation. When expressed from the endogenous locus, activated alleles show a temperature-sensitive loss of function, suggesting these mutations confer a semiactive state to the protein. Intragenic suppressors of these activated alleles cluster to regions in the catalytic domain on the face of the protein that interacts with substrate, suggesting these are the regions that interact with the C-terminal domain. Thus, rather than being an autoinhibitory domain, the C-terminus of Chk1 also contains domains critical for adopting an active configuration.

scholarly journals WNK4 Kinase: from structure to physiology

2021 ◽  
Author(s):  
Adrian Rafael Murillo-de-Ozores ◽  
Alejandro Rodriguez-Gama ◽  
Hector Carbajal-Contreras ◽  
Gerardo Gamba ◽  
Maria Castaneda-Bueno
Keyword(s):  
Oxidative Stress ◽  
Mouse Models ◽  
Serine Threonine Kinase ◽  
Gene Encoding ◽  
Threonine Kinase ◽  
Physiological Conditions ◽  
Different Levels ◽  
Familial Hyperkalemic Hypertension

With No Lysine (K) kinase 4 (WNK4) belongs to a serine-threonine kinase family characterized by the atypical positioning of its catalytic lysine. Despite the fact that WNK4 has been found in many tissues, the majority of its study has revolved around its function in the kidney, specifically as a positive regulator of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the nephron. This is explained by the description of gain-of-function mutations in the gene encoding WNK4 that cause Familial Hyperkalemic Hypertension (FHHt). This disease is mainly driven by increased downstream activation of the Ste20-related Proline Alanine Rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1)-NCC pathway, which increases salt reabsorption in the DCT and indirectly impairs renal K+ secretion. Here, we review the large volume of information that has accumulated about different aspects of WNK4 function. We first review the knowledge on WNK4 structure and enumerate the functional domains and motifs that have been characterized. Then, we discuss WNK4 physiological functions based on the information obtained from in vitro studies and from a diverse set of genetically modified mouse models with altered WNK4 function. We then review in vitro and in vivo evidence on the different levels of regulation of WNK4. Finally, we go through the evidence that has suggested how different physiological conditions act through WNK4 to modulate NCC activity.

scholarly journals A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette.

1993 ◽  
Vol 13 (12) ◽  
pp. 7625-7635 ◽  
Author(s):  
P D Walden ◽  
N J Cowan
Keyword(s):  
Protein Complex ◽  
Catalytic Domain ◽  
Signal Transduction Pathway ◽  
Kinase Domain ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Serine Threonine Kinase ◽  
Expression Clone

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.

scholarly journals Point Mutations in Yeast CBF5 Can Abolish In Vivo Pseudouridylation of rRNA

1999 ◽  
Vol 19 (11) ◽  
pp. 7461-7472 ◽  
Author(s):  
Yeganeh Zebarjadian ◽  
Tom King ◽  
Maurille J. Fournier ◽  
Louise Clarke ◽  
John Carbon
Keyword(s):  
Catalytic Domain ◽  
Sequence Similarity ◽  
Point Mutations ◽  
Rrna Synthesis ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Sequence Motifs ◽  
Conserved Sequence

ABSTRACT In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Ψ) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known Ψ synthase, and shares the “KP” and “XLD” conserved sequence motifs found in the catalytic domains of three distinct families of known and putative Ψ synthases. To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative Ψ synthase domain of Cbf5p. Yeast strains expressing these mutatedcbf5 genes in a cbf5Δ null background are viable at 25°C but display pronounced cold- and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of Ψ in rRNA at extreme temperatures. Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutantcbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some of the mutants are temperature sensitive both for growth and for formation of Ψ in the rRNA. In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects. There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutantcbf5D95A, which lacks Ψ in rRNA. A subset of mutations in the Ψ synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction. Our results provide additional evidence that Cbf5p is the Ψ synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes.

scholarly journals Protein Serine/Threonine Kinase StkP Positively Controls Virulence and Competence in Streptococcus pneumoniae

2004 ◽  
Vol 72 (4) ◽  
pp. 2434-2437 ◽  
Author(s):  
Jose Echenique ◽  
Aras Kadioglu ◽  
Susana Romao ◽  
Peter W. Andrew ◽  
Marie-Claude Trombe
Keyword(s):  
Streptococcus Pneumoniae ◽  
Lung Infection ◽  
Signaling Network ◽  
Serine Threonine Kinase ◽  
Positive Regulation ◽  
Threonine Kinase

ABSTRACT In the Streptococcus pneumoniae genome, stkP, encoding a membrane-associated serine/threonine kinase, is not redundant (L. Novakova, S. Romao, J. Echenique, P. Branny, and M.-C. Trombe, unpublished results). The data presented here demonstrate that StkP belongs to the signaling network involved in competence triggering in vitro and lung infection and bloodstream invasion in vivo. In competence, functional StkP is required for activation of comCDE upstream of the autoregulated ring orchestrated by the competence-stimulating peptide. This is the first description of positive regulation of comCDE transcription in balance with its repression by CiaRH.

scholarly journals Regulation of Yeast Actin Cytoskeleton-Regulatory Complex Pan1p/Sla1p/End3p by Serine/Threonine Kinase Prk1p

2001 ◽  
Vol 12 (12) ◽  
pp. 3759-3772 ◽  
Author(s):  
Guisheng Zeng ◽  
Xianwen Yu ◽  
Mingjie Cai
Keyword(s):  
Actin Cytoskeleton ◽  
Regulatory Protein ◽  
Strong Support ◽  
Stable Complex ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization

The serine/threonine kinase Prk1p is known to be involved in the regulation of the actin cytoskeleton organization in budding yeast. One possible function of Prk1p is the negative regulation of Pan1p, an actin patch regulatory protein that forms a complex in vivo with at least two other proteins, Sla1p and End3p. In this report, we identified Sla1p as another substrate for Prk1p. The phosphorylation of Sla1p by Prk1p was established in vitro with the use of immunoprecipitated Prk1p and in vivo with the use ofPRK1 overexpression, and was further supported by the finding that immunoprecipitated Sla1p contained PRK1- and ARK1-dependent kinase activities. Stable complex formation between Prk1p and Sla1p/Pan1p in vivo could be observed once the phosphorylation reaction was blocked by mutation in the catalytic site of Prk1p. Elevation of Prk1p activities in wild-type cells resulted in a number of deficiencies, including those in colocalization of Pan1p and Sla1p, endocytosis, and cell wall morphogenesis, likely attributable to a disintegration of the Pan1p/Sla1p/End3p complex. These results lend a strong support to the model that the phosphorylation of the Pan1p/Sla1p/End3p complex by Prk1p is one of the important mechanisms by which the organization and functions of the actin cytoskeleton are regulated.

scholarly journals The Metallo-β-Lactamase/β-CASP Domain of Artemis Constitutes the Catalytic Core for V(D)J Recombination

2004 ◽  
Vol 199 (3) ◽  
pp. 315-321 ◽  
Author(s):  
Catherine Poinsignon ◽  
Despina Moshous ◽  
Isabelle Callebaut ◽  
Régina de Chasseval ◽  
Isabelle Villey ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Catalytic Domain ◽  
Single Amino Acid ◽  
In Vitro Mutagenesis ◽  
Recombination Activity ◽  
Catalytic Core ◽  
Class B ◽  
Repair Factor

The V(D)J recombination/DNA repair factor Artemis belongs to the metallo-β-lactamase (β-Lact) superfamily of enzymes. Three regions can be defined within the Artemis protein sequence: (a) the β-Lact homology domain, to which is appended (b) the β-CASP region, specific of members of the β-Lact superfamily acting on nucleic acids, and (c) the COOH-terminal domain. Using in vitro mutagenesis, here we show that the association of the β-Lact and the β-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates. Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity. The results presented here define the β-Lact/β-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B β-Lact enzymes hydrolyzing β-lactam compounds.

scholarly journals Inhibition of Bcr serine kinase by tyrosine phosphorylation.

1996 ◽  
Vol 16 (3) ◽  
pp. 998-1005 ◽  
Author(s):  
J Liu ◽  
Y Wu ◽  
G Z Ma ◽  
D Lu ◽  
L Haataja ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Philadelphia Chromosome ◽  
Wild Type ◽  
Serine Kinase ◽  
Serine Threonine Kinase ◽  
Threonine Kinase

The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.

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