共查询到20条相似文献,搜索用时 31 毫秒
1.
Smal C Vertommen D Bertrand L Rider MH van den Neste E Bontemps F 《Nucleosides, nucleotides & nucleic acids》2006,25(9-11):1141-1146
Compelling evidence suggests that deoxycytidine kinase (dCK), a key enzyme in the salvage of deoxyribonucleosides and in the activation of clinically relevant nucleoside analogues, can be regulated by reversible phosphorylation. In this study, we show that dCK overexpressed in HEK-293T cells was labelled after incubation of the cells with [32P]orthophosphate. Tandem mass spectrometry allowed the identification of 4 in vivo phosphorylation sites, Thr3, Ser11, Ser15, and Ser74. These results provide the first evidence that dCK is constitutively multiphosphorylated in intact cells. In addition, site-directed mutagenesis demonstrated that phosphorylation of Ser74, the major in vivo phosphorylation site, is crucial for dCK activity. 相似文献
2.
Rachid Amsailale Maxime Beyaert Caroline Smal Veerle Janssens Eric Van Den Neste Françoise Bontemps 《FEBS letters》2014
Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn2+-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity. 相似文献
3.
Smal C Vertommen D Bertrand L Ntamashimikiro S Rider MH Van Den Neste E Bontemps F 《The Journal of biological chemistry》2006,281(8):4887-4893
Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [32P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of 32P-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity. 相似文献
4.
Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant. 相似文献
5.
Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the kcat values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme. 相似文献
6.
Caroline Smal Didier Vertommen Rachid Amsailale Angélique Arts Pierre Morsomme Eric Van Den Neste Françoise Bontemps 《Archives of biochemistry and biophysics》2010,502(1):44-52
Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI δ. We showed that recombinant CKI δ phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI δ correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI δ, strengthening the key role of this residue in the control of dCK activity. However, neither CKI δ inhibitors nor CKI δ siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI δ in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI δ could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme. 相似文献
7.
Yuri L. Bunimovich Evan Nair-Gill Mireille Riedinger Melissa N. McCracken Donghui Cheng Jami McLaughlin Caius G. Radu Owen N. Witte 《PloS one》2014,9(8)
Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [18F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy. 相似文献
8.
Chunying Yang Michael Lee Jianwei Hao Xiaoli Cui Xiaojing Guo Caroline Smal Fran?oise Bontemps Shumei Ma Xiaodong Liu David Engler William B. Parker Bo Xu 《Nucleic acids research》2012,40(19):9621-9632
Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. dCK is activated in response to DNA damage; however, how it functions in the DNA damage response is largely unknown. Here, we report that dCK is required for the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR). We demonstrate that the ataxia–telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate it in response to DNA damage. We further demonstrate that Serine 74 phosphorylation is required for initiation of the G2/M checkpoint. Using mass spectrometry, we identified a protein complex associated with dCK in response to DNA damage. We demonstrate that dCK interacts with cyclin-dependent kinase 1 (Cdk1) after IR and that the interaction inhibits Cdk1 activity both in vitro and in vivo. Together, our results highlight the novel function of dCK and provide molecular insights into the G2/M checkpoint regulation in response to DNA damage. 相似文献
9.
10.
11.
Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. 总被引:38,自引:9,他引:29
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
D R Alessi Y Saito D G Campbell P Cohen G Sithanandam U Rapp A Ashworth C J Marshall S Cowley 《The EMBO journal》1994,13(7):1610-1619
Many growth factors whose receptors are protein tyrosine kinases stimulate the MAP kinase pathway by activating first the GTP-binding protein Ras and then the protein kinase p74raf-1. p74raf-1 phosphorylates and activates MAP kinase kinase (MAPKK). To understand the mechanism of activation of MAPKK, we have identified Ser217 and Ser221 of MAPKK1 as the sites phosphorylated by p74raf-1. This represents the first characterization of sites phosphorylated by this proto-oncogene product. Ser217 and Ser221 lie in a region of the catalytic domain where the activating phosphorylation sites of several other protein kinases are located. Among MAPKK family members, this region is the most conserved, suggesting that all members of the family are activated by the phosphorylation of these sites. A 'kinase-dead' MAPKK1 mutant was phosphorylated at the same residues as the wild-type enzyme, establishing that both sites are phosphorylated directly by p74raf-1, and not by autophosphorylation. Only the diphosphorylated form of MAPKK1 (phosphorylated at both Ser217 and Ser221) was detected, even when the stoichiometry of phosphorylation by p74raf-1 was low, indicating that phosphorylation of one of these sites is rate limiting, phosphorylation of the second then occurring extremely rapidly. Ser217 and Ser221 were both phosphorylated in vivo within minutes when PC12 cells were stimulated with nerve growth factor. Analysis of MAPKK1 mutants in which either Ser217 or Ser221 were changed to glutamic acid, and the finding that inactivation of maximally activated MAPKK1 required the dephosphorylation of both serines, shows that phosphorylation of either residue is sufficient for maximal activation. 相似文献
12.
Keszler G Spasokoukotskaja T Sasvári-Székely M Eriksson S Staub M 《Nucleosides, nucleotides & nucleic acids》2006,25(9-11):1147-1151
The activity of deoxycytidine kinase (dCK) has been shown to be enhanced upon genotoxic stress in human lymphocytes, and reversible phosphorylation of the enzyme has been implicated in the activation process. Here, we provide compelling evidence that dCK is a cytosolic phosphoprotein. Two-dimensional gel electrophoresis revealed that dCK has several differentially charged isoforms in cells. One-third of total cellular dCK was bound to a phosphoprotein-binding column irrespective of its activity levels, indicating that other mechanisms rather than phosphorylation alone might also be involved in the stimulation of enzyme activity. We excluded the possibility that activated dCK is translocated to the nucleus, but identified a dCK isoform of low abundance with a higher molecular weight in the nuclear fractions. 相似文献
13.
G. Keszler T. Spasokoukotskaja M. Sasvári-Székely S. Eriksson M. Staub 《Nucleosides, nucleotides & nucleic acids》2013,32(9-11):1147-1151
The activity of deoxycytidine kinase (dCK) has been shown to be enhanced upon genotoxic stress in human lymphocytes, and reversible phosphorylation of the enzyme has been implicated in the activation process. Here, we provide compelling evidence that dCK is a cytosolic phosphoprotein. Two-dimensional gel electrophoresis revealed that dCK has several differentially charged isoforms in cells. One-third of total cellular dCK was bound to a phosphoprotein-binding column irrespective of its activity levels, indicating that other mechanisms rather than phosphorylation alone might also be involved in the stimulation of enzyme activity. We excluded the possibility that activated dCK is translocated to the nucleus, but identified a dCK isoform of low abundance with a higher molecular weight in the nuclear fractions. 相似文献
14.
Hilde Herrema Jaemin LeeYingjiang Zhou Kyle D. CoppsMorris F. White Umut Ozcan 《Biochemical and biophysical research communications》2014
Increased mammalian target of rapamycin complex 1 (mTORC1) activity has been suggested to play important roles in development of insulin resistance in obesity. mTORC1 hyperactivity also increases endoplasmic reticulum (ER) stress, which in turn contributes to development of insulin resistance and glucose intolerance. Increased IRS1 phosphorylation at Ser307 in vitro is correlated with mTORC1- and ER stress-induced insulin resistance. This phosphorylation site correlates strongly with impaired insulin receptor signaling in diabetic mice and humans. In contrast, evidence from knock-in mice suggests that phosphorylation of IRS1 at Ser307 is actually required to maintain insulin sensitivity. To study the involvement of IRS1Ser307 phosphorylation in mTORC1-mediated glucose intolerance and insulin sensitivity in vivo, we investigated the effects of liver specific TSC1 depletion in IRS1Ser307Ala mice and controls. Our results demonstrate that blockade of IRS1Ser307 phosphorylation in vivo does not prevent mTORC1-mediated glucose intolerance and insulin resistance. 相似文献
15.
R T Waldron O Rey T Iglesias T Tugal D Cantrell E Rozengurt 《The Journal of biological chemistry》2001,276(35):32606-32615
The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) mu) activity has become controversial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser(748) in PKD (pS748). Western blot analysis with the pS748 antibody, carried out with a variety of PKD forms and in a variety of cell types including full-length PKD transfected in COS-7 and HEK 293 cells, a green fluorescent protein-PKD fusion protein transfected in either Swiss 3T3 fibroblasts or Madin-Darby canine kidney epithelial cells, and endogenous PKD expressed in A20 lymphocytes and Rat-1 fibroblasts, indicated that Ser(748) phosphorylation was absent from unstimulated cells. In contrast, dramatic increases in Ser(748) phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKCepsilon or PKCeta. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser(744), revealed that Ser(744) phosphorylation accompanies Ser(748) phosphorylation during PKD activation in vivo. Ser(744)/Ser(748) phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser(744) and Ser(748) occurs during PKD activation in vivo and support the notion of a PKC-PKD phosphorylation cascade. 相似文献
16.
17.
Coccetti P Zinzalla V Tedeschi G Russo GL Fantinato S Marin O Pinna LA Vanoni M Alberghina L 《Biochemical and biophysical research communications》2006,346(3):786-793
We have previously identified Ser201 of Sic1, a yeast cyclin-dependent kinase inhibitor, as an in vitro target of protein kinase CK2. Here we present new evidence, by using specific anti-P-Ser201 antibodies and 2-D gel electrophoresis coupled to MALDI mass spectrometry analysis, that Sic1 is phosphorylated in vivo on Ser201 shortly after its de novo synthesis, during late anaphase in glucose-grown cells. This phosphorylation is also detected in Sic1 immunopurified from G1 cells. In agreement with these data we also show that the catalytic alpha' subunit of CK2, whose function is required for cell cycle progression, is detected in Sic1 immunopurified complexes, and that phosphorylation on Ser201 is reduced after CK2 inactivation at the non-permissive temperature in a cka1delta cka2(ts) yeast strain. These data strongly support the notion that CK2 phosphorylates Sic1 in vivo. 相似文献
18.
Krawiec K Kierdaszuk B Shugar D 《Biochemical and biophysical research communications》2003,301(1):192-197
Inorganic tripolyphosphate (PPP(i)) and pyrophosphate (PP(i)) were examined as potential phosphate donors for human deoxynucleoside kinase (dCK), deoxyguanosine kinase (dGK), cytosolic thymidine kinase (TK1), mitochondrial TK2, and the deoxynucleoside kinase (dNK) from Drosophila melanogaster. PPP(i) proved to be a good phosphate donor for dGK, as well as for dCK with dCyd, but not dAdo, as acceptor substrate, illustrating also the dependence of donor properties on acceptor. Products of phosphorylation were shown to be 5(')-phosphates. In striking contrast to ATP, the phosphorylation reaction follows strict Michaelis-Menten kinetics, with K(m) values of 74 and 92 microM for dCK and dGK, respectively, and V(max) values 40-50% that for ATP. With the other three enzymes, as well as for dCK with dAdo as acceptor, no, or only low levels (=1% of that for ATP) of activity were observed. PP(i) was inactive (<0.1%) as a phosphate donor with all enzymes, but was a competitive inhibitor vs ATP, as was PPP(i) in systems with no or low donor activity. This is the first report on inorganic tripolyphosphate as a phosphate donor for nucleoside kinases, in particular human deoxyribonucleoside kinases. 相似文献
19.
20.
Phosphorylation of CD44 in vivo requires both Ser323 and Ser325, but does not regulate membrane localization or cytoskeletal interaction in epithelial cells.
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
CD44 has been implicated to play an important role in a diverse range of physiological processes, which involve cell-matrix recognition, cell-cell adhesion and cell motility. There is increasing evidence that the highly conserved intracellular domain of CD44 may be involved in influencing these activities. CD44 is phosphorylated in vivo on serine residue(s). In view of the importance that phosphorylation has been accorded in a multitude of cellular regulatory processes, we have investigated the role of phosphorylation in the control of CD44. In this report we identify the sites of human CD44 phosphorylation by mutating the three conserved cytoplasmic serine residues. We show that both Ser323 and Ser325, but not Ser316, are required for phosphorylation in vivo and demonstrate that this event is not stimulated by phorbol esters. Clonal MDCK cell lines expressing both the single and double CD44 phosphorylation mutants have been generated. These cell lines have been used to directly assess the role of phosphorylation on CD44 localization in polarized epithelial cells and its association with the cytoskeleton. 相似文献