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Manganese-Dependent Serine/Threonine/Tyrosine Kinase From Arabidopsis Thaliana : Role Of Serine And Threonine Residues In The Regulation Of Kinase ActivityReddy, Mamatha M 08 1900 (has links)
Protein phosphorylation is an important post-translational modification of proteins, which can either activate or inhibit the function of a given protein. The enzymes, protein kinases and protein phosphatases catalyze the phosphorylation and dephosphorylation of target proteins, respectively. Protein kinases catalyze the transfer of γ-phosphate from ATP to serine, threonine or tyrosine residues in target proteins. They are traditionally classified as protein serine/threonine kinases and
protein tyrosine kinases based on the amino acid to which they transfer the phosphate group. Protein tyrosine kinases play vital roles in numerous pathways that regulate growth, development and oncogenesis in animals. However, no protein tyrosine kinase has been cloned so far from plants. The sequence motif, CW(X)6RPXF of sub-domain XI is well conserved among biochemically characterized protein tyrosine kinases from human, rat, mice, worm, fruitfly and
Dictyostelium. To seek plant genes encoding tyrosine kinase, we have performed extensive
genome-wide analysis of Arabidopsis thaliana using the delineated tyrosine kinase from animal systems. Repetitive database mining with CW(X)6RPXF sequence motif revealed the presence of 57 different protein kinases that have tyrosine kinase motifs. Myosin light chain protein kinase was identified as false positive with this motif. Multiple sequence alignment of all the 57 kinases
indicated the presence of twelve conserved sub-domains in their kinase catalytic domain. Out of the 12 sub-domains present in protein kinases, sub-domain VIb confers serine/threonine kinase Specificity and sub-domains VIII and XI confer tyrosine kinase specificity. All the 57 kinases were Verified to contain CW(X) 6RPXF in sub-domain XI. However, the catalytic domain of all the catalogued kinases contain KXXN motif in sub-domain VIb, which is indicative of serine/threonine Kinase specificity. None of the kinases had the tyrosine kinase consensus motif RAA or ARR in sub-domain VIb. Thus, the catalytic domains of all the identified Arabidopsis protein kinases have
motifs for serine/threonine specificity in sub-domain VIb and tyrosine kinase motif in sub-domain XI. These results indicate that perhaps all the kinases belong to the dual-specificity kinase family.
Hence, we have tentatively named these protein sequences as STY (serine/threonine/tyrosine) protein kinases. To examine the relationships of Arabidopsis STY protein kinases, a topographic cladogram was constructed. Casein kinase 1 was used as an outgroup to perceive the true class of STY protein kinase family. Neighbor joining tree was constructed with the full-length protein sequences following the alignments. Dendrogram of STY protein kinases suggested that the kinases are mainly clustered into four groups. Group I includes kinases related to ATN1-like kinases, peanut STY related kinases, soybean GmPK6-like kinases and ATMRK1-like kinases. Group II consists of MAP3K-like kinases, CTR1 and EDR1 related kinases. Group III includes protein kinases that harbor ankyrin domain repeat motifs. These kinases are related to Medicago sativa ankyrin kinase, MsAPK1. Group IV consists of light sensory kinases that are related to
Ceratodon purpureus phytochrome kinase. C. purpureus light sensory kinase has both
phytochrome and protein kinase domains. However, the protein kinases of group IV do not have a phytochrome domain.
BLAST analysis was performed using CW(X)6RPXF motif against all the available plant
sequences in the database. We retrieved 11 rice protein kinases and 14 Dictyostelium kinases. In addition, we obtained STY protein kinases from wheat, barley, soybean, tomato, beech and alfalfa. Dendrogram analysis indicated that the plant STY protein kinases are clustered in similar manner as observed for Arabidopsis. Large number of sequences were retrieved when the tyrosine kinase motif CW(X)6RPXF was used to perform BLAST analysis against all the known sequences from animals. As large numbers of protein tyrosine kinases are available in animals, we have used representative kinases of each family towards the construction of phylogenetic tree. The main difference between the animal and plant tyrosine kinases is in the consensus motif conferring the tyrosine and serine/threonine specificity in the sub-domain VIb. Animal tyrosine kinases have consensus ARR/RAA in sub-domain VIb and plant kinases have KXXN which is indicative of serine/threonine specificity.
Expression analysis of Arabidopsis STY protein kinases was performed using
Genevestigator online search tool Meta-Analyzer. Genes were grouped based on their relative expression levels during a specific growth stage, in a particular organ or following different environmental stresses. Various kinases are highly expressed in stamens and seeds while some kinases are expressed ubiquitously. A number of biotic and abiotic factors upregulated plant STY
protein kinases. Gene expression data suggests the importance of STY protein kinases in plant growth and development.
Genome-wide analysis is supported by phosphoproteomics in Arabidopsis seedlings.
Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, phosphoamino acid analysis and peptide mass fingerprinting. Alkaline treatment detected two proteins corresponding to 46 and 37.5 kD. Phosphoamino acids analysis confirmed their dual-specificity nature. MALDI mass spectrometry and peptide mass fingerprinting analysis identified these two proteins as ATN1 and peanut serine/threonine/tyrosine protein kinase like protein from Arabidopsis.
To further support the in silico approach, we have overexpressed one of the identified
Arabidopsis thaliana serine/threonine/tyrosine protein kinases (AtSTYPK) in E. coli. The recombinant kinase was induced with IPTG and purified by using nickel-nitrilotriacetic acid affinity chromatography. AtSTYPK exhibited a strong preference for manganese over magnesium for kinase activity. The autophosphorylation activity of AtSTYPK was inhibited by the addition of calcium to reaction buffer containing manganese. The rate of autophosphorylation reaction was linear with increasing time and protein concentration. The AtSTYPK phosphorylated histone H1
(type III-S), and myelin basic protein (MBP) in substrate phosphorylation reaction and it did not phosphorylate casein or enolase. To see whether calcium or magnesium inhibits phosphorylation of MBP, we have performed the reaction in the presence of combination of different metal ions. The MBP phosphorylation reaction is more efficient in the presence of Mg2++ Mn2+ than Ca2++ Mn2+
under the same conditions. The recombinant kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK harbors a manganese-dependent serine/threonine kinase domain, COG3642. H248 and S265 on COG3642 are conserved in AtSTYPK and the site-directed mutation of H248 to alanine resulted in loss of serine/threonine kinase activity, but the mutation of S265 to alanine showed a slight increase in its kinase activity.
The protein kinase activity is regulated by various mechanisms that include
autophosphorylation, protein phosphorylation by other kinases and by the action of regulatory domains or subunits. The role of tyrosine residues in the regulation of peanut dual-specificity kinase activity is well documented, but the importance of serine and threonine residues in the
regulation of dual-specificity protein kinase is not studied so far. The kinase activity is generally regulated by phosphorylation of one or more residues within the kinase activation loop. The role of threonine residues in the kinase activation loop and the TEY motif of AtSTYPK were investigated in the present study. Four threonine residues in the activation loop and a T208 in the TEY sequence motif were converted to alanine to study their role in the regulation of kinase activity. The protein kinase activity was abolished when T208 and T293 of the activation loop were converted to alanine. Interestingly, the conversion of T284 in the activation loop to alanine resulted in an increase in both auto- and substrate phosphorylations. The mutation of T288 and T291 to alanine had no effect on kinase activity.
There are eight serine residues in the kinase catalytic domain of AtSTYPK and surprisingly there is no serine residue in the kinase activation loop. So it is worthwhile to see how phosphorylation of serine residues regulates the dual-specificity protein kinase activity. The role of each serine residue was studied individually by substituting them with alanine. Serines at positions 215, 259, 269 and 315 regulate the kinase activity both in terms of autophosphorylation and substrate phosphorylation of myelin basic protein. The mutation of serine 265 to alanine resulted in
slight increase in auto- and substrate phosphorylations, suggesting that it could be autoinhibitory in function. The other serine residues at positions 165, 181 and 360 did not show any change in the
phosphorylation status when compared to wild-type AtSTYPK. In conclusion, this data suggests the importance of serine and threonine residues in the regulation of dual-specificity protein kinase activity and emphasizes the complexity of regulation of dual-specificity protein kinases in plants.
To summarise, this study suggests that tyrosine phosphorylation in plants could be brought about only by dual-specificity protein kinases that phosphorylate on serine, threonine and tyrosine residues. This raises an interesting possibility that plants lack classical tyrosine kinases. The results provide a first report of manganese-dependent dual-specificity kinase from plant systems. This data also suggests that plant dual-specificity kinases undergo phosphorylation at multiple amino acid
residues and their activity is regulated by various mechanisms, suggesting that they could be regulated by different mechanisms at different stages of plant growth and development.
However, the role of dual-specificity kinases in planta has to be understood by mutant analysis in order to assign the physiological roles to these kinases. Further studies are needed to identify the upstream kinase(s) and downstream targets. Determination of physiological functions
for dual-specificity protein kinases raises an important challenge in future in the area of plant signal transduction.
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Signalizační působení adenylát-cyklázového toxinu na fagocyty / Signaling effects of adenylate cyclase toxin action on phagocytesČerný, Ondřej January 2015 (has links)
The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...
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Funkční analýza populačně specifických sekvenčních variant genu pro kinázu kontrolního bodu buněčného cyklu CHEK2 / Functional analysis of the population-specific checkpoint kinase gene CHEK2 sequence variantsStolařová, Lenka January 2015 (has links)
CHEK2 gene codes for serin/threonine kinase Chk2 (Checkpoint kinase 2). In response to genomic DNA damage, Chk2 phosphorylates its substrates (proteins Cdc25C, BRCA1 or p53), whose activation leads either to cell cycle arrest, DNA damage repair or induction of apoptosis. Germline mutations in CHEK2 gene increase risk of cancer development. Analysis of high risk breast cancer patients in Czech Republic reveals rare CHEK2 mutations (mainly missense) with yet unknown clinical significance. This work focuses on functional impact of these variants and analysis of kinase activity of variant isoforms of Chk2 kinase. For this purpose, recombinant constructs were expressed in bacterial cells of E. coli. Enzymatic activity of Chk2 kinase isoforms in crude cell lysates was measured by the phosphorylation of Chk2 arteficial substrate spectrophotometrically. Results of in vitro kinase assay were correlated to the results of in silico prediction software. The results show that from 15 analyzed mutations (together with one in frame deletion), kinase activity was abrogated in all variants affecting the kinase domain of Chk2, in concordance with in silico predictions. The same result has been found for a FHA domain variant p.R145Q. No significant changes in kinase activity were observed in case of two FHA domain variants...
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Venous malformation causative mutations affect TIE2 receptor trafficking, downstream signaling and vascular endothelial cell functionsNätynki, M. (Marjut) 29 March 2016 (has links)
Abstract
Venous malformations (VMs) are localized defects in vascular morphogenesis which can seriously impede or even threaten the patient’s life. VMs are characterized by enlarged, torturous vein-like channels lined by unevenly distributed smooth muscle cells. A large number of mutations in the endothelial TIE2 receptor tyrosine kinase have been found from more than half of the lesions screened, thus providing a common genetic cause. TIE2 has a crucial role in vascular development, remodeling and quiescence. However, the molecular and cellular abnormalities caused by TIE2-mutations in endothelial cells and how they relate to VM formation have been unknown.
The aim of this study was to examine how VM-specific mutations affect the molecular characteristics of TIE2-receptor downstream signaling and cellular functions. Because no effective treatment has been available for VMs, a better understanding of the molecular basis of their pathology should enable the development of more potent and non-invasive treatments as well as provide a better understanding of vascular morphogenesis in general.
The results demonstrate that the TIE2-VM forms have both common and specific effects on TIE2 and the endothelial cells (ECs) expressing them. Mutation-induced TIE2 autoactivation leading to loss of normal EC monolayer organization due to extracellular matrix (ECM) fibronectin deficiency was found to be a common change. This was shown to occur through chronic activation of the mitogen-activated protein kinase (MAPK) pathway, which also caused activation of the proteolytic plasminogen system. Also, most mutations altered TIE2 trafficking and angiopoietin ligand regulated TIE2 functions, albeit through different mechanisms. Using RNA-screening we showed that the most common sporadic TIE2-VM mutation dysregulates genes affecting vascular development, cell migration and ECM remodeling. PDGFB, a major attractant of vascular mural cells, was found to be strongly attenuated due to chronic activation of Akt, which also increases EC survival, by the TIE2 mutant receptors.
To conclude, the results in this thesis reveal genetic, molecular and cellular alterations which may potentiate VM formation. This data provides new information on the pathological mechanisms behind abnormal vascular morphogenesis and should assist the development of new molecular treatment strategies for VM patients. / Tiivistelmä
Laskimoepämuodostumat ovat paikallisia verisuoniston kehityksen häiriöitä. Riippuen niiden koosta ja anatomisesta sijainnista ne voivat aiheuttaa merkittävää haittaa. Epämuodostumat koostuvat laajentuneista, laskimonkaltaisista verisuonista, joissa sileiden lihassolujen kerros on puutteellisesti järjestäytynyt. Yli puolessa tutkituista laskimoepämuodostumista havaitaan mutaatioita verisuonten sisäpinnan endoteelisoluissa ilmenevässä TIE2 reseptorityrosiinikinaasissa, joka säätelee verisuonten kehitystä, muokkausta ja fysiologista toimintaa. TIE2-mutaatioiden aiheuttamia molekyyli- ja solutason muutoksia tai niiden yhteyttä epämuodostumien syntyyn ei ole aikaisemmin tunnettu.
Tämän tutkimuksen tarkoituksena oli selvittää, miten laskimoepämuodostumista löydetyt mutaatiot vaikuttavat TIE2-reseptorin toimintaan molekyyli- ja solutasolla sekä TIE2-reseptorista alkavaan solunsisäiseen viestintään. Koska pysyvää hoitomuotoa laskimoepämuodostumille ei tunneta, voisi tieto niiden taustalla olevista patologisista mekanismeista edesauttaa parempien, ei-kajoavien hoitomuotojen kehittämisessä ja antaa myös yleisesti uutta tietoa verisuoniston kehityksestä.
Väitöskirjan tulokset osoittavat, että mutaatiot vaikuttavat TIE2-reseptoriin ja sitä ilmentäviin endoteelisoluihin mutaatioille yhteisillä sekä mutaatiokohtaisilla tavoilla. Mutaatioille tyypillinen TIE2-reseptorin ligandista riippumaton aktivaatio aiheutti aktivaation nousun myös TIE2:sta alavirtaan olevissa viestinvälittäjissä. Tämä puolestaan johti fibronektiini-proteiinin häviämiseen soluväliaineesta, sileitä lihassoluja säätelevän PDGFB-kasvutekijän ilmenemisen laskuun ja solujen ohjelmoidun solukuoleman vähenemiseen. Useimmat tutkitut mutaatiot muuttivat myös TIE2-reseptorin sijaintia soluissa häiriten TIE2:n angiopoietiini-ligandien säätelemiä toimintoja usean eri mekanismin kautta. Transkriptomin laajuiset RNA-tutkimukset osoittivat monien verisuonten kehitykseen, solujen liikkumiseen ja soluväliaineen muokkaukseen liittyvien geenien ilmentymisen muuttuneen.
Lopputuloksena tutkimus paljasti geeni-, molekyyli-, ja solutason muutoksia, jotka saattavat vaikuttaa laskimoepämuodostumien syntyyn. Tulokset antavat lisätietoa sairautta aiheuttavista mekanismeista verisuoniston kehityksen häiriöiden taustalla ja ovat hyödyksi kehitettäessä uusia lääkkeitä laskimoepämuodostumien molekulaarisia hoitoja varten.
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Casein kinase 1 isoforms in degenerative disordersKannanayakal, Theresa Joseph 01 December 2004 (has links)
No description available.
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L'activador del CDK2 relacionat amb l'apoptosi: clonatge i estudi bioquímic del seu paper regulador de la mort cel·lular programadaBrunet Roig, Maurici 14 July 2006 (has links)
L´apoptosi, o mort cel.lular programada, és un procés actiu que mobilitza els recursos cel.lulars amb l´objectiu de mantenir l´homeostasi de l´organisme a expenses del suïcidi de cèl.lules individuals. Diferents estudis han mostrat un increment de l´activiat d´algunes cdk, especialment Cdk1 i Cdk2, en correlació amb la progressió dels primers estadis apoptòtics. En el nostre laboratori l´estudi de l´apoptosi en timòcits, els quals no tenen una activitat cdk significativa degut a l´aturada del cicle cel.lular en G1, demostren que la inducció de l´activitat de Cdk2 després del tractament amb radiació gamma o amb glucocorticoides és necessària per l´inici de l´apoptosi. Mentre cap de les ciclines conegudes sembla ser la proteïna activadora de Cdk2 en apoptosi, en el nostre laboratori hem identificat un nou membre de la família de les ciclines, denominada Ciclina O, capaç d´activar aquesta kinasa in vivo en línies cel.lulars. L´expressió d´aquesta nova ciclina en el timus, i altres teixits, s´indueix ràpidament després del tractament amb radiació gamma i coincideix amb l´aparició de l´apoptosi. Aquests resultats posicionen la Ciclina O com a millor candidat a ser l´activador de Cdk2 necessari per induïr la mort cel.lular programada en el timus, i probablement també en altres òrgans. / The apoptosis, also called programmed cell death, is an active process able to use the cellular mechanisms to kill individual cells in order to keep the functional homeostasis of the whole organism. Different studies had shown a correlation between the first apoptotic events and the induction of some cdk proteins, particularly Cdk1 and Cdk2. The studies of thymocytes in our laboratory, wich lacks the most amount of cdk activity related to the cell cycle because of its arrest in G1, had shown that the induction of Cdk2 activity after the treatment with gamma radiation or glucocorticoids is a necessary step for the apoptosis induction. While any of the cyclins described at the moment seems to be the Cdk2 activator for apoptosis a new member of the cyclin family able to activate the kinase Cdk2 in vivo in cell lines has been identified in our laboratory. The expresion of this cyclin, known as Cyclin O, is quickly induced in the thymus after the treatment with gamma radiation and correlates with the induction of apoptosis. These results position Cyclin O as the best candidate to activate Cdk2 and inuce the programmed cell death in the thymus, and probably other tissues.
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DISTINCT ROLES OF THE aD HELIX IN aCAMKII ACTIVATION CHARACTERIZED USING A DE NOVO MUTATION FROM CHILDREN WITH LEARNING DISABILITIESWalter Saide (16650807) 07 August 2023 (has links)
<p>This dissertation describes the effects of a <i>de novo</i> mutation of CaMKII found in children with learning disabilities and describes its effect on catalytic activity. We develop a malachite green assay for the measurement of CaMKII activation and use it for high-throughput chemical screening to identify CaMKII inhibitors and enhancers. We also propose a new mechanism of regulation of CaMKII activity by ADP.</p><p><br></p>
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