scholarly journals Alternative splicing variants of dual specificity tyrosine phosphorylated and regulated kinase 1B exhibit distinct patterns of expression and functional properties

2003 ◽  
Vol 372 (3) ◽  
pp. 881-888 ◽  
Author(s):  
Susanne LEDER ◽  
Hanna CZAJKOWSKA ◽  
Barbara MAENZ ◽  
Katrin de GRAAF ◽  
Andreas BARTHEL ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Expression Patterns ◽  
Neuronal Cell ◽  
Dual Specificity ◽  
Mouse Tissues ◽  
Splicing Variants

The dual specificity tyrosine phosphorylated and regulated kinase (DYRK) family of protein kinases is a group of evolutionarily conserved protein kinases that have been characterized as regulators of growth and development in mammals, Drosophila and lower eukaryotes. In the present study, we have characterized three splicing variants of DYRK1B (DYRK1B-p65, DYRK1B-p69 and DYRK1B-p75) with different expression patterns and enzymic activities. DYRK1B-p65 and DYRK1B-p69 exhibited similar, but not identical, patterns of expression in mouse tissues, with the highest protein levels found in the spleen, lung, brain, bladder, stomach and testis. In contrast, DYRK1B-p75 was detected specifically in skeletal muscles, in the neuronal cell line GT1-7 and also in differentiated, adipocyte-like 3T3-L1 cells, but not in undifferentiated 3T3-L1 preadipocytes. A comparison of the mouse and human Dyrk1b genomic and cDNA sequences defined the alternative splicing events that produce the variants of DYRK1B. In DYRK1B-p75, transcription starts with exon 1B instead of exon 1A, generating a new translation start, which extends the open reading frame by 60 codons. This gene structure suggests that alternative promoters direct the expression of DYRK1B-p69 and DYRK1B-p75. Both splicing variants exhibited kinase activity in vitro and contained phosphotyrosine when expressed in COS-7 cells. Owing to differential recognition of the 3′-splice site in exon 9, DYRK1B-p65 differs from DYRK1B-p69 by the absence of 40 amino acids within the catalytic domain. DYRK1B-p65 lacked kinase activity in vitro and did not contain phosphotyrosine. DYRK1B-p69 and DYRK1B-p75 stimulated reporter gene activity driven by the forkhead in rhabdosarcoma (FKHR)-dependent glucose-6-phosphatase promoter more strongly when compared with DYRK1B-p65, indicating that the DYRK1B splicing variants exhibit functional differences.

scholarly journals Characterization and comparison of four serine- and arginine-rich (SR) protein kinases

1997 ◽  
Vol 326 (3) ◽  
pp. 693-700 ◽  
Author(s):  
Oliver NAYLER ◽  
Stefan STAMM ◽  
Axel ULLRICH
Keyword(s):  
Alternative Splicing ◽  
Catalytic Domain ◽  
Expression Patterns ◽  
Sr Proteins ◽  
Amino Acid Sequences ◽  
Sr Protein ◽  
High Sequence Identity ◽  
Mouse Tissues ◽  
Transformed Cell Lines

Phosphorylated serine- and arginine-rich (SR) proteins are components of the spliceosomal complex, and have been implicated in the control of alternative splicing. Kinases that regulate the phosphorylation and possibly the intranuclear distribution of SR proteins may therefore contribute to changes in choice of splice site. We have cloned three mouse cDNAs with high sequence identity to the family of LAMMER kinases (i.e. kinases carrying the conserved signature EHLAMMERILG in the catalytic domain). A comparison of their amino acid sequences revealed two related subfamilies with high evolutionary conservation. We have compared the expression patterns of these proteins in mouse tissues and transformed cell lines with that of a previously cloned family member (mCLK1/STY), and detected various transcripts for each gene. This underlines previous findings of alternative splicing of mclk1/STY. Our results suggest that the proportions of products for each gene are regulated independently. We further demonstrate that all variants encode autophosphorylating proteins that can phosphorylate several biochemically purified SR proteins in vitro, leading to hyperphosphorylation of at least one SR protein in vivo. The observed tissue distributions and substrate specificities suggest that these kinases may all be constituents of a network of regulatory mechanisms that enable SR proteins to control RNA splicing.

scholarly journals Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14.

1994 ◽  
Vol 5 (3) ◽  
pp. 273-282 ◽  
Author(s):  
S Kornbluth ◽  
B Sebastian ◽  
T Hunter ◽  
J Newport
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Schizosaccharomyces Pombe ◽  
Protein Kinases ◽  
Membrane Fraction ◽  
Kinase Activity ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Dual Specificity ◽  
Detergent Extraction

The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2. This kinase is regulated both by association with cyclins and by phosphorylation at several sites. Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2. In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15. It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14. In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated. Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15. In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases. The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated. This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14.

Sequence, expression and localization of calmodulin-domain protein kinases inEimeria tenellaandEimeria maxima

Parasitology ◽  
1996 ◽  
Vol 113 (5) ◽  
pp. 439-448 ◽  
Author(s):  
P. P. J. Dunn ◽  
J. M. Bumstead ◽  
F. M. Tomley
Keyword(s):  
Host Cell ◽  
Protein Kinases ◽  
Catalytic Domain ◽  
Sequence Data ◽  
Host Cells ◽  
Cdna Clones ◽  
Host Cell Membrane ◽  
Amino Terminal ◽  
Domain Protein

SUMMARYWe have isolated and sequenced cDNA clones fromEimeria tenellaandEimeria maximawhich encode proteins that share homology with a recently described family of calmodulin-domain protein kinases. The primary sequence data show that each of the protein kinases can be divided into 2 main functional domains – an amino-terminal catalytic domain typical of serine/threonine protein kinases and a carboxy-terminal domain homologous to calmodulin, which is capable of binding calcium ions at 4 ‘EF-hand’ motifs. Expression of theE. tenellacalmodulin-domain protein kinase (EtCDPK) increased towards the end of oocyst sporulation, as judged by Northern and Western blotting, and indirect immunofluorescent antibody labelling showed that within a few minutes of adding sporozoites to target host cells inin vitroculture EtCDPK was found to be specifically associated with a filament-like structure that converges at the apical end of the parasite. Once the parasite entered the host cell EtCDPK appeared to be left on the host cell membrane at the point of entry, indicating a brief yet specific role for this molecule in the invasion of host cells byE. tenella.

Alternative transcription initiation and splicing variants of the DHRS4 gene cluster

2009 ◽  
Vol 29 (1) ◽  
pp. 47-56 ◽  
Author(s):  
Qiaoxia Zhang ◽  
Yifan Li ◽  
Gefei Liu ◽  
Xiaoyuan Xu ◽  
Xuhong Song ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Gene Cluster ◽  
Transcription Initiation ◽  
Expression Patterns ◽  
Cpg Islands ◽  
Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
Alternative Transcription ◽  
Splicing Variants ◽  
Copy Gene

The DHRS4 (short-chain dehydrogenase/reductase superfamily member 4) gene cluster, consisting of DHRS4 and its copy gene DHRS4L2, is localized on 14q11.2. The DHRS4 gene product NADP(H)-dependent retinol oxidoreductase participates in the metabolism of retinoids. The expression patterns of the DHRS4 gene cluster were investigated in human neuroblastoma cells. Transcript analysis of the DHRS4 gene cluster using 3′- and 5′-RACE (rapid amplification of cDNA ends), reverse transcription-PCR and bioinformatics approaches showed an alternative transcription start site in the copy gene DHRS4L2 which generates two transcripts, DHRS4A1 (GenBank® nucleotide sequence database accession number AY616183) and DHRS4A2 (AY943857), together with at least six alternative splicing variants (DHRS4A_v1–6) (AY920361, AY920362, DN237886, DN237887, DN237890 and DN237892 respectively), resulted from alternative splicing. DHRS4A1 and DHRS4A2 were specifically transcribed in neuroblastoma cells. RNA structural analysis of DHRS4A1 and DHRS4A2 suggested that they are non-coding RNAs. Expression analysis of DHRS4 by quantitative real-time PCR and Western blotting showed a lack of correlation between the levels of transcription and translation in the tissues examined. Bisulfite genomic sequencing PCR experiments indicated that the expression of DHRS4L2 was regulated by methylation of its CpG islands.

scholarly journals Activation of a Nuclear Cdc2-Related Kinase within a Mitogen-Activated Protein Kinase-Like TDY Motif by Autophosphorylation and Cyclin-Dependent Protein Kinase-Activating Kinase

2005 ◽  
Vol 25 (14) ◽  
pp. 6047-6064 ◽  
Author(s):  
Zheng Fu ◽  
Melanie J. Schroeder ◽  
Jeffrey Shabanowitz ◽  
Philipp Kaldis ◽  
Kasumi Togawa ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Mitogen Activated Protein Kinase ◽  
Intestinal Cell ◽  
Dependent Protein Kinase ◽  
Mitogen Activated Protein ◽  
Dependent Protein

ABSTRACT Male germ cell-associated kinase (MAK) and intestinal cell kinase (ICK) are nuclear Cdc2-related kinases with nearly identical N-terminal catalytic domains and more divergent C-terminal noncatalytic domains. The catalytic domain is also related to mitogen-activated protein kinases (MAPKs) and contains a corresponding TDY motif. Nuclear localization of ICK requires subdomain XI and interactions of the conserved Arg-272, but not kinase activity or, surprisingly, any of the noncatalytic domain. Further, nuclear localization of ICK is required for its activation. ICK is activated by dual phosphorylation of the TDY motif. Phosphorylation of Tyr-159 in the TDY motif requires ICK autokinase activity but confers only basal kinase activity. Full activation requires additional phosphorylation of Thr-157 in the TDY motif. Coexpression of ICK with constitutively active MEK1 or MEK5 fails to increase ICK phosphorylation or activity, suggesting that MEKs are not involved. ICK and MAK are related to Ime2p in budding yeast, and cyclin-dependent protein kinase-activating kinase Cak1p has been placed genetically upstream of Ime2p. Recombinant Cak1p phosphorylates Thr-157 in the TDY motif of recombinant ICK and activates its activity in vitro. Coexpression of ICK with wild-type CAK1 but not kinase-inactive CAK1 in cells also increases ICK phosphorylation and activity. Our studies establish ICK as the prototype for a new group of MAPK-like kinases requiring dual phosphorylation at TDY motifs.

scholarly journals Complex Effects of Naturally Occurring Mutations in the JAK3 Pseudokinase Domain: Evidence for Interactions between the Kinase and Pseudokinase Domains

2000 ◽  
Vol 20 (3) ◽  
pp. 947-956 ◽  
Author(s):  
Min Chen ◽  
Alan Cheng ◽  
Fabio Candotti ◽  
Yong-Jie Zhou ◽  
Anka Hymel ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Intramolecular Interaction ◽  
Severe Combined Immunodeficiency ◽  
Kinase Domain ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The structure of Janus kinases (JAKs) is unique among protein tyrosine kinases in having tandem, nonidentical kinase and pseudokinase domains. Despite its conservation in evolution, however, the function of the pseudokinase domain remains poorly understood. Lack of JAK3 expression results in severe combined immunodeficiency (SCID). In this study, we analyze two SCID patients with mutations in the JAK3 pseudokinase domain, which allows for protein expression but disrupts the regulation of the kinase activity. Specifically, these mutant forms of JAK3 had undetectable kinase activity in vitro but were hyperphosphorylated both in patients' Epstein-Barr virus-transformed B cells and when overexpressed in COS7 cells. Moreover, reconstitution of cells with these mutants demonstrated that, although they were constitutively phosphorylated basally, they were unable to transmit cytokine-dependent signals. Further analysis showed that the isolated catalytic domain of JAK3 was functional whereas either the addition of the pseudokinase domain or its deletion from the full-length molecule reduced catalytic activity. Through coimmunoprecipitation of the isolated pseudokinase domain with the isolated catalytic domain, we provide the first evidence that these two domains interact. Furthermore, whereas the wild-type pseudokinase domain modestly inhibited kinase domain-mediated STAT5 phosphorylation, the patient-derived mutants markedly inhibited this phosphorylation. We thus conclude that the JAK3 pseudokinase domain is essential for JAK3 function by regulating its catalytic activity and autophosphorylation. We propose a model in which this occurs via intramolecular interaction with the kinase domain and that increased inhibition of kinase activity by the pseudokinase domain likely contributes to the disease pathogenesis in these two patients.

scholarly journals Differential induction of Pax genes by NGF and BDNF in cerebellar primary cultures.

1994 ◽  
Vol 125 (2) ◽  
pp. 417-425 ◽  
Author(s):  
C Kioussi ◽  
P Gruss
Keyword(s):  
Gene Family ◽  
Expression Patterns ◽  
Primary Cultures ◽  
Neuronal Cell ◽  
Cell Types ◽  
Early Gene ◽  
Specific Expression ◽  
Pax Genes ◽  
Cell Type Specific Expression

The Pax genes encode sequence-specific DNA binding transcription factors that are expressed in embryonic development of the nervous system. Primary neuronal cell cultures derived from the cerebellar cortex of embryonic day 14, newborn and 7-d old mice, were used to investigate the cell-type specific expression patterns of three members of the murine paired box containing gene family (Pax gene family), in vitro. Cell types which express Pax-2, Pax-3, and Pax-6 RNA in primary cultures correspond to those found in regions of the cerebellum which show RNA signals in sections of the developing mouse brain. To find mechanisms regulating Pax gene expression during cerebellar development, the differential regulation of Pax-2, Pax-3, and Pax-6 by NGF and BDNF, two structurally related neurotrophins, was studied in such primary cultures. Pax-2 and Pax-6 RNA increased slightly by 1 h and remained elevated throughout a 24-h treatment with BDNF and NGF. Pax-3 RNA was not detected in newborn cultures, but underwent a rapid (1 h) and transient (2 h) induction upon treatment with either BDNF or NGF. No response was seen with EGF or FGF. Cycloheximide treatment amplified Pax-3 induction and prolonged the signal. Thus, Pax-3 induction resembles that of the immediate-early gene c-fos, which transduces growth factor signals during the development of particular neuronal/glial cell types. The changes in Pax expression were inductive rather than trophic.

scholarly journals Deletion of 11 Amino Acids in p90rsk-mo-1Abolishes Kinase Activity

1999 ◽  
Vol 19 (1) ◽  
pp. 317-320 ◽  
Author(s):  
Denise J. Spring ◽  
Edwin G. Krebs
Keyword(s):  
Ribosomal Protein ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Direct Sequencing ◽  
Conclusive Evidence ◽  
Full Length ◽  
Mitogen Activated Protein Kinase ◽  
Reverse Transcription Pcr ◽  
Ribosomal Protein S6 ◽  
Mouse Tissues

ABSTRACT p90 rsk is a distal member of the mitogen-activated protein kinase signaling pathway. It has been cloned from a variety of species including Xenopus laevis, mouse, chicken, rat, and human. The clone p90 rsk-mo-1, isolated by others from a mouse library, contains a unique 33-nucleotide deletion not found in the p90 rsk clones from any other species that have been examined. When p90 rsk-mo-1 was expressed in Cos-7 cells that were subsequently stimulated with epidermal growth factor, the immunoprecipitated p90 rsk-mo-1 protein showed no measurable kinase activity toward the ribosomal protein S6 peptide. By comparison, expression of rat p90 rsk-1 resulted in significant kinase activity. Deletion of the 33-nucleotide region missing in the p90 rsk-mo-1clone from the p90 rsk-rat-1 cDNA abolished kinase activity in the resulting protein. When these 33 nucleotides were introduced into the p90 rsk-mo-1 cDNA, the expressed protein showed significant kinase activity. Reverse transcription-PCR and direct sequencing of mRNA isolated from several mouse tissues indicated the presence of the full-length form of p90 rsk-1 in the mouse and showed no conclusive evidence for a deletion-containing form. This study indicates the presence of a full-length p90 rsk-1 mRNA in mouse tissues that is homologous to that identified in other species and suggests that the deletion in p90 rsk-mo-1may be a cloning artifact. The findings provide additional support for the conclusion that the first catalytic domain of p90 rsk is responsible for its enzymatic activity toward ribosomal protein S6.

scholarly journals Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity.

1995 ◽  
Vol 6 (2) ◽  
pp. 215-226 ◽  
Author(s):  
T Izumi ◽  
J L Maller
Keyword(s):  
Kinase Activity ◽  
Cdc2 Kinase ◽  
Nuclear Envelope Breakdown ◽  
Dual Specificity ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The M-phase inducer, Cdc25C, is a dual-specificity phosphatase that directly phosphorylates and activates the cyclin B/Cdc2 kinase complex, leading to initiation of mitosis. Cdc25 itself is activated at the G2/M transition by phosphorylation on serine and threonine residues. Previously, it was demonstrated that Cdc2 kinase is capable of phosphorylating and activating Cdc25, suggesting the existence of a positive feedback loop. In the present study, kinases other than Cdc2 that can phosphorylate and activate Cdc25 were investigated. Cdc25 was found to be phosphorylated and activated by cyclin A/Cdk2 and cyclin E/Cdk2 in vitro. However, in interphase Xenopus egg extracts with no detectable Cdc2 and Cdk2, treatment with the phosphatase inhibitor microcystin activated a distinct kinase that could phosphorylate and activate Cdc25. Microcystin also induced other mitotic phenomena such as chromosome condensation and nuclear envelope breakdown in extracts containing less than 5% of the mitotic level of Cdc2 kinase activity. These findings implicate a kinase other than Cdc2 and Cdk2 that may initially activate Cdc25 in vivo and suggest that this kinase may also phosphorylate M-phase substrates even in the absence of Cdc2 kinase.

scholarly journals Budding and fission yeast casein kinase I isoforms have dual-specificity protein kinase activity.

1994 ◽  
Vol 5 (8) ◽  
pp. 877-886 ◽  
Author(s):  
M F Hoekstra ◽  
N Dhillon ◽  
G Carmel ◽  
A J DeMaggio ◽  
R A Lindberg ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Tyrosine Kinase Activity ◽  
Casein Kinase I ◽  
Tyrosine Residues ◽  
Dual Specificity ◽  
Specificity Protein ◽  
Protein Tyrosine Kinase Activity

We have examined the activity and substrate specificity of the Saccharomyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms. These four gene products are isotypes of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases. However, each of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analysis of 32P-labeled proteins showed phosphorylation on serine, threonine, and tyrosine residues. The E. coli produced forms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protein kinase synthetic peptide substrate polymer poly-E4Y1. Immune complex protein kinases assays from S. pombe cells showed that Hhp1-containing precipitates were associated with a protein-tyrosine kinase activity, and the Hhp1 present in these immunoprecipitates was phosphorylated on tyrosine residues. Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyrosine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold increase in the Km for poly-E4Y1 and casein. These data demonstrate that four different CKI isoforms from two different yeasts are capable of protein-tyrosine kinase activity and encode dual-specificity protein kinases.

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