scholarly journals The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases

2005 ◽  
Vol 391 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Alberto C. Vitari ◽  
Maria Deak ◽  
Nick A. Morrice ◽  
Dario R. Alessi
Keyword(s):  
Protein Kinase ◽  
Glutamic Acid ◽  
Protein Kinases ◽  
Catalytic Domain ◽  
Cellular Processes ◽  
Basal Activity ◽  
Human Genes ◽  
Genes Encoding ◽  
Equivalent Residue ◽  
Phosphopeptide Mapping

Mutations in the human genes encoding WNK1 [with no K (lysine) protein kinase-1] and the related protein kinase WNK4 are the cause of Gordon's hypertension syndrome. Little is known about the molecular mechanism by which WNK isoforms regulate cellular processes. We immunoprecipitated WNK1 from extracts of rat testis and found that it was specifically associated with a protein kinase of the STE20 family termed ‘STE20/SPS1-related proline/alanine-rich kinase’ (SPAK). We demonstrated that WNK1 and WNK4 both interacted with SPAK as well as a closely related kinase, termed ‘oxidative stress response kinase-1’ (OSR1). Wildtype (wt) but not catalytically inactive WNK1 and WNK4 phosphorylated SPAK and OSR1 to a much greater extent than with other substrates utilized previously, such as myelin basic protein and claudin-4. Phosphorylation by WNK1 or WNK4 markedly increased SPAK and OSR1 activity. Phosphopeptide mapping studies demonstrated that WNK1 phosphorylated kinase-inactive SPAK and OSR1 at an equivalent residue located within the T-loop of the catalytic domain (Thr233 in SPAK, Thr185 in OSR1) and a serine residue located within a C-terminal non-catalytic region (Ser373 in SPAK, Ser325 in OSR1). Mutation of Thr185 to alanine prevented the activation of OSR1 by WNK1, whereas mutation of Thr185 to glutamic acid (to mimic phosphorylation) increased the basal activity of OSR1 over 20-fold and prevented further activation by WNK1. Mutation of Ser325 in OSR1 to alanine or glutamic acid did not affect the basal activity of OSR1 or its ability to be activated by WNK1. These findings suggest that WNK isoforms operate as protein kinases that activate SPAK and OSR1 by phosphorylating the T-loops of these enzymes, resulting in their activation. Our analysis also describes the first facile assay that can be employed to quantitatively assess WNK1 and WNK4 activity.

scholarly journals The ubiquitin-associated domain of AMPK-related protein kinases allows LKB1-induced phosphorylation and activation

2006 ◽  
Vol 394 (3) ◽  
Author(s):  
Mark H. Rider
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Related Protein ◽  
Biochemical Journal ◽  
Kinase Catalytic Domain ◽  
Uba Domain ◽  
Amp Activated Protein Kinase

The AMPK (AMP-activated protein kinase)-related protein kinase subfamily of the human kinome comprises 12 members closely related to the catalytic α1/α2 subunits of AMPK. The precise role of the AMPK-related kinases and their in vivo substrates is rather unclear at present, but some are involved in regulating cell polarity, whereas others appear to control cellular differentiation. Of the 12 human AMPK-related protein kinase family members, 11 can be activated following phosphorylation of their T-loop threonine residue by the LKB1 complex. Nine of these AMPK-related kinases activated by LKB1 contain an UBA (ubiquitin-associated) domain immediately C-terminal to the kinase catalytic domain. In this issue of the Biochemical Journal, Jaleel et al. show that the presence of an UBA domain in AMP-related kinases allows LKB1-induced phosphorylation and activation. The findings have implications for understanding the molecular mechanisms of activation of this fascinating family of protein kinases. Also, mutations in the UBA domains of the AMP-related kinase genes might be present in families with Peutz–Jehgers syndrome and in other cancer patients.

scholarly journals Functional Characterization of a Serine/Threonine Protein Kinase of Pseudomonas aeruginosa

1999 ◽  
Vol 67 (10) ◽  
pp. 5386-5394 ◽  
Author(s):  
S. Timothy Motley ◽  
Stephen Lory
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Catalytic Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Amino Acid ◽  
Migration Behavior

ABSTRACT Protein kinases play a key role in signal transduction pathways in both eukaryotic and prokaryotic cells. Using in vivo expression technology, we have identified several promoters in Pseudomonas aeruginosa which are preferentially activated during infection of neutropenic mice. One of these promoters directs the transcription of a gene encoding a putative protein kinase similar to the enzymes found in eukaryotic cells. The full characterization of this protein, termed PpkA, is presented in this communication. The ppkA gene encodes a 1,032-amino-acid polypeptide with an N-terminal catalytic domain showing all of the conserved residues of protein kinases with the substrate phosphorylation specificities for serine and threonine residues. The catalytic domain is linked to the rest of the protein by a short proline-rich segment. The enzymes showed anomalous migration behavior when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be attributed to autophosphorylation activity. The full-length enzyme was expressed as an oligohistidine fusion protein and was shown to phosphorylate several artificial protein substrates. Both autophosphorylation and phosphorylation of added substrates were strongly reduced by a single-amino-acid substitution in the catalytic domain of PpkA. Although PpkA appears to be differentially phosphorylated by autocatalysis, the levels of phosphorylation have minimal effect on its overall enzymatic activity. Our results, therefore, indicate the operation of a novel protein phosphorylation mechanism during transduction of signals in P. aeruginosa, and this pathway may be important in regulating the expression of virulence factors by this pathogen during certain phases of infection.

Characterization of a dual plant protein kinase (FsPK1) up-regulated by abscisic acid and calcium and specifically expressed in dormant seeds ofFagus sylvaticaL.

2003 ◽  
Vol 13 (4) ◽  
pp. 261-271 ◽  
Author(s):  
O. Lorenzo ◽  
C. Nicolás ◽  
G. Nicolás ◽  
D. Rodríguez
Keyword(s):  
Abscisic Acid ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Catalytic Domain ◽  
Consensus Sequence ◽  
Calcium Dependent ◽  
Localization Signal ◽  
Rapid Amplification

An abscisic acid (ABA)-induced cDNA fragment encoding a putative protein kinase (PK) was obtained by differential screening of a cDNA library fromFagus sylvaticaseeds. The full-length clone, named FsPK1, was produced by 5′ rapid amplification of cDNA ends (RACE) extension. This clone contained the 11 catalytic domains present in all protein kinases, but displayed unusual characteristics found only in a few plant PKs. FsPK1 exhibits features of both serine/threonine and tyrosine protein kinases within the catalytic domain, a putative nuclear localization signal within the regulatory domain and the consensus sequence involved in binding of 14-3-3 proteins. The catalytic domain, expressed inEscherichia colias a fusion protein, showed Ca2+-dependentin vitrokinase activity and dual serine/threonine and tyrosine specificity. Transcription of theFsPK1gene was reduced by seed stratification at 4°C, and clearly increased when seeds were treated with 0.1 mM ABA, correlating with the inhibition of germination. Interestingly,FsPK1transcripts were enhanced when ABA (0.1 mM) and calcium (1 mM) were added together, while the addition of EGTA (calcium chelator) and 3,4,5,-trimethoxibenzoic acid 8-(diethylamino) octyl ester (TMB-8, a calcium antagonist) decreased its expression. Furthermore,FsPK1transcript expression was tissue specific and accumulated only in ABA-treated seeds, but not in any ABA-treated vegetative tissues examined. These results suggest that the expression of the corresponding protein could be related to the inhibition of germination mediated by ABA in a calcium-dependent pathway.

scholarly journals EXPLICIT-Kinase: a gene expression predictor for dissecting the functions of the Arabidopsis kinome

2021 ◽  
Author(s):  
Yuming Peng ◽  
Wanzhu Zuo ◽  
Yue Qin ◽  
Shisong Ma
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Stress Responses ◽  
Expression Patterns ◽  
Gene Expression Patterns ◽  
The Novel ◽  
Cellular Processes ◽  
Bona Fide ◽  
Kinase A

Protein kinases regulate virtually all cellular processes, but it remains challenging to determine the functions of all protein kinases, collectively called the kinome, in any species. We developed an approach called EXPLICIT-Kinase to predict the functions of the Arabidopsis kinome. Because the activities of many kinases can be regulated transcriptionally, their gene expression patterns provide clues to their functions. A universal gene expression predictor for Arabidopsis was constructed to predict the expression of 30,172 non-kinase genes based on the expression of 994 protein kinase genes. The model reconstituted highly accurate transcriptomes for diverse Arabidopsis samples. It identified the significant kinases as predictor kinases for predicting the expression of Arabidopsis genes and pathways. Strikingly, these predictor kinases were often known regulators of the related pathways, as exemplified by those involved in cytokinesis, tissue development, and stress responses. Comparative analyses have revealed that portions of these predictor kinases, including the novel ones, are shared and conserved between Arabidopsis and maize. The conservation between species provide additional evidence to support the novel predictor kinases as bona fide regulators of the pathways involved. Thus our approach enables the systematic dissection of the functions of the Arabidopsis kinome.

scholarly journals NPK1, a tobacco gene that encodes a protein with a domain homologous to yeast BCK1, STE11, and Byr2 protein kinases

1993 ◽  
Vol 13 (8) ◽  
pp. 4745-4752
Author(s):  
H Banno ◽  
K Hirano ◽  
T Nakamura ◽  
K Irie ◽  
S Nomoto ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Stationary Phase ◽  
Protein Kinases ◽  
Catalytic Domain ◽  
Signal Transduction Pathway ◽  
Yeast Cells ◽  
Transduction Pathway ◽  
Pheromone Response ◽  
Tobacco Cells

We have isolated a cDNA (cNPK1) that encodes a predicted protein kinase of 690 amino acids from suspension cultures of tobacco cells. The deduced sequence is closely related to those of the protein kinases encoded by the STE11 and BCK1 genes of Saccharomyces cerevisiae and the byr2 gene of Schizosaccharomyces pombe. STE11 and Byr2 function in the yeast mating pheromone response pathways, and BCK1 acts downstream of the yeast protein kinase C homolog encoded by the PKC1 gene, which is essential for normal growth and division of yeast cells. Overexpression in yeast cells of a truncated form of cNPK1, which encodes only the putative catalytic domain, replaced the growth control functions of BCK1 and PKC1 but not the mating pheromone response function of STE11. Thus, the catalytic domain of NPK1 specifically activates the signal transduction pathway mediated by BCK1 in yeast. In tobacco cells in suspension culture, the NPK1 gene is transcribed during logarithmic phase and early stationary phase but not during late stationary phase. In a tobacco plant, it is also transcribed in stems and roots but not in mature leaves, which rarely contain growing cells. The present results suggest that a signal transduction pathway mediated by this BCK1- and STE11-related protein kinase is also conserved in plants and that a function of NPK1 is controlled at least in part at a transcriptional level.

scholarly journals Isolation and Preliminary Characterization of a Receptor-like Protein Kinase from Cold Acclimated Peach Bark

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 585d-585
Author(s):  
Carole Bassett ◽  
Mike Nickerson ◽  
Tim Artlip ◽  
Michael Wisniewski
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Catalytic Domain ◽  
Genomic Sequence ◽  
Signal Transduction Pathway ◽  
Pcr Analysis ◽  
S Locus ◽  
Transmembrane Region ◽  
Amino Terminal

To identify components of a possible signal transduction pathway associated with woody plant cold acclimation, a cDNA library prepared from peach bark collected in December was screened using a small DNA fragment from a maize receptor-like protein kinase (ZMPK1) as probe. Six isolates were obtained and partially sequenced to confirm protein kinase identity. One isolate (PPPKB5) contained a relatively large insert (about 1.9 kbp) and was selected for further analysis. Both strands of PPPKB5 were sequenced and the derived amino acid sequence was compared with a variety of known plant protein kinases, leading to the tentative identification of this clone as encoding a partial cDNA for a serine/threonine class protein kinase. PPPKB5 has a classic protein kinase catalytic domain with all the features required for phosphorylation activity and with greater similarity to the known plant receptor-like protein kinases (RLKs) than other classes of these enzymes. In addition, it contains a potential transmembrane region separating the catalytic portion of the molecule from the receptor region. The receptor region has greatest identity with the class of RLKs known as the S-locus type based on conservation of a stretch of 10 cysteines on the amino-terminal side of the transmembrane region. PPPKB5 shares greater amino acid identity with ZMPK1 (50%) than with the Brassica, rice or Arabidopsis S-locus RLKs (34%–40%). Furthermore, PCR analysis of `Loring' suggests that, like maize ZMPK1, the genomic sequence encoding PPPKB5 contains no introns, in contrast to the genomic sequences of Brassica and Arabidopsis which contain six.

scholarly journals Phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-dependent activation and biological signaling.

1997 ◽  
Vol 17 (8) ◽  
pp. 4509-4516 ◽  
Author(s):  
B Diaz ◽  
D Barnard ◽  
A Filson ◽  
S MacDonald ◽  
A King ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Wild Type ◽  
Kinase Activation ◽  
Oncogenic Ras ◽  
Protein Kinase Activation ◽  
In The Wild ◽  
Transforming Activity ◽  
Phosphopeptide Mapping ◽  
Antipeptide Antibody

Activation of the Raf serine/threonine protein kinases is tightly regulated by multiple phosphorylation events. Phosphorylation of either tyrosine 340 or 341 in the catalytic domain of Raf-1 has been previously shown to induce the ability of the protein kinase to phosphorylate MEK. By using a combination of mitogenic and enzymatic assays, we found that phosphorylation of the adjacent residue, serine 338, and, to a lesser extent, serine 339 is essential for the biological and enzymatic activities of Raf-1. Replacement of S338 with alanine blocked the ability of prenylated Raf-CX to transform Rat-1 fibroblasts. Similarly, the loss of S338-S339 in Raf-1 prevented protein kinase activation in COS-7 cells by either oncogenic Ras[V12] or v-Src. Consistent with phosphorylation of S338-S339, acidic amino acid substitutions of these residues partially restored transforming activity to Raf-CX, as well as kinase activation of Raf-1 by Ras[V12] or v-Src. Two-dimensional phosphopeptide mapping of wild-type Raf-CX and Raf-CX[A338A339] confirmed the presence of a phosphoserine-containing peptide with the predicted mobility in the wild-type protein which was absent from the mutant. This peptide could be quantitatively precipitated by an antipeptide antibody specific for the 18-residue tryptic peptide containing S338-S339 and was demonstrated to contain only phosphoserine. Phosphorylation of this peptide in Raf-1 was significantly increased by coexpression with Ras[V12]. These data demonstrate that Raf-1 residues 338 to 341 constitute a unique phosphoregulatory site in which the phosphorylation of serine and tyrosine residues contributes to the regulation of Raf by Ras, Src, and Ras-independent membrane localization.

Structure and function of MK5/PRAK: the loner among the mitogen-activated protein kinase-activated protein kinases

2013 ◽  
Vol 394 (9) ◽  
pp. 1115-1132 ◽  
Author(s):  
Ugo Moens ◽  
Sergiy Kostenko
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Cellular Processes ◽  
Mapk Pathways ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Apoptosis Gene ◽  
And Function

Abstract Mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways that control pivotal cellular processes including proliferation, differentiation, survival, apoptosis, gene regulation, and motility. MAPK pathways consist of a relay of consecutive phosphorylation events exerted by MAPK kinase kinases, MAPK kinases, and MAPKs. Conventional MAPKs are characterized by a conserved Thr-X-Tyr motif in the activation loop of the kinase domain, while atypical MAPKs lack this motif and do not seem to be organized into the classical three-tiered kinase cascade. One functional group of conventional and atypical MAPK substrates consists of protein kinases known as MAPK-activated protein kinases. Eleven mammalian MAPK-activated protein kinases have been identified, and they are divided into five subgroups: the ribosomal-S6-kinases RSK1-4, the MAPK-interacting kinases MNK1 and 2, the mitogen- and stress-activated kinases MSK1 and 2, the MAPK-activated protein kinases MK2 and 3, and the MAPK-activated protein kinase MK5 (also referred to as PRAK). MK5/PRAK is the only MAPK-activated protein kinase that is a substrate for both conventional and atypical MAPK, while all other MAPKAPKs are exclusively phosphorylated by conventional MAPKs. This review focuses on the structure, activation, substrates, functions, and possible implications of MK5/PRAK in malignant and nonmalignant diseases.

scholarly journals Phosphoproteomics Meets Chemical Genetics: Approaches for Global Mapping and Deciphering the Phosphoproteome

2020 ◽  
Vol 21 (20) ◽  
pp. 7637
Author(s):  
Jan Jurcik ◽  
Barbara Sivakova ◽  
Ingrid Cipakova ◽  
Tomas Selicky ◽  
Erika Stupenova ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Chemical Genetics ◽  
Genetically Engineered ◽  
Specific Substrate ◽  
Dynamic Nature ◽  
Cellular Processes ◽  
Global Mapping ◽  
Bona Fide ◽  
Kinase Substrates

Protein kinases are important enzymes involved in the regulation of various cellular processes. To function properly, each protein kinase phosphorylates only a limited number of proteins among the thousands present in the cell. This provides a rapid and dynamic regulatory mechanism that controls biological functions of the proteins. Despite the importance of protein kinases, most of their substrates remain unknown. Recently, the advances in the fields of protein engineering, chemical genetics, and mass spectrometry have boosted studies on identification of bona fide substrates of protein kinases. Among the various methods in protein kinase specific substrate identification, genetically engineered protein kinases and quantitative phosphoproteomics have become promising tools. Herein, we review the current advances in the field of chemical genetics in analog-sensitive protein kinase mutants and highlight selected strategies for identifying protein kinase substrates and studying the dynamic nature of protein phosphorylation.

scholarly journals Specific loss of protein kinase activities in senescent erythrocytes

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1479-1487 ◽  
Author(s):  
HK Jindal ◽  
Z Ai ◽  
P Gascard ◽  
C Horton ◽  
CM Cohen
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Phosphoglycerate Kinase ◽  
Type I ◽  
Isolated Cells ◽  
Cellular Processes ◽  
Cytosolic Protein ◽  
Time Period ◽  
Age Dependent ◽  
Senescent Erythrocytes

Rabbit erythrocytes of progressively increasing age were isolated using an avidin-biotin affinity technique and the activity of protein kinases and other enzymes was analyzed in cytosols and membranes from the isolated cells. The activities of cytosolic protein kinase C (PKC), cAMP-dependent kinase (PKA), and casein kinase type I and II (CKI and II) were all found to undergo an age-dependent decrease of twofold to fourfold over the 8-week lifespan of the cells. Membrane-associated tyrosine kinase showed little or no decrease, but membrane-associated CKI showed a dramatic eightfold decrease over the 8-week period. By contrast, various cytosolic enzymes, including lactate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and acid phosphatase, showed no change in activity over the same time period. Density-separated human erythrocytes showed qualitatively similar decreases in cytosolic protein kinase activities in the densest fractions, which contain the oldest cells. Our results show that aging erythrocytes undergo progressive loss of protein kinases that may adversely affect various cellular processes. The age-dependent loss of kinase activity reported here is one of the most striking manifestations of erythrocyte senescence yet to be reported.

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