共查询到20条相似文献,搜索用时 15 毫秒
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Y J Zhou M Chen N A Cusack L H Kimmel K S Magnuson J G Boyd W Lin J L Roberts A Lengi R H Buckley R L Geahlen F Candotti M Gadina P S Changelian J J O'Shea 《Molecular cell》2001,8(5):959-969
Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity. 相似文献
3.
Sanz A Ungureanu D Pekkala T Ruijtenbeek R Touw IP Hilhorst R Silvennoinen O 《PloS one》2011,6(4):e18522
Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the K(m) for ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or K(m) for ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches. 相似文献
4.
Janus (Jak) tyrosine kinases contain a tyrosine kinase (JH1) domain adjacent to a catalytically inactive pseudokinase domain (JH2). The JH2 domain has been implicated in regulation of Jak activity, but its function remains poorly understood. Here, we found that the JH2 domain negatively regulates the activity of Jak2 and Jak3. Deletion of JH2 resulted in increased tyrosine phosphorylation of the Jak2- and Jak3-JH2 deletion mutants as well as of coexpressed STAT5. In cytokine receptor signaling, the deletion of the Jak2- and Jak3-JH2 domains resulted in interferon-gamma and interleukin-2-independent STAT activation, respectively. However, cytokine stimulations did not further induce the JH2 deletion mutant-mediated STAT activation. The deletion of the Jak2 JH2 domain also abolished interferon-gamma-inducible kinase activation, although it did not affect the reciprocal Jak1-Jak2 interaction in 293T cells. Chimeric constructs, where the JH2 domains were swapped between Jak2 and Jak3, retained low basal activity and cytokine inducible signaling, indicating functional conservation between the two JH2 domains. However, the basal activity of Jak2 was significantly lower than that of Jak3, suggesting differences in the regulation of Jak2 and Jak3 activity. In conclusion, we found that the JH2 domain has a conserved function in Jak2 and Jak3. The JH2 domain is required for two distinct functions in cytokine signaling: (i) inhibition of the basal activity of Jak2 and Jak3, and (ii) cytokine-inducible activation of signaling. The Jak-JH2 deletion mutants are catalytically active, activate STAT5, and interact with another Jak kinase, but the JH2 domain is required to connect these signaling events to receptor activation. Thus, we propose that the JH2 domain contributes to both the uninduced and ligand-induced Jak-receptor complex, where it acts as a cytokine-inducible switch to regulate signal transduction. 相似文献
5.
F Kohlhuber N C Rogers D Watling J Feng D Guschin J Briscoe B A Witthuhn S V Kotenko S Pestka G R Stark J N Ihle I M Kerr 《Molecular and cellular biology》1997,17(2):695-706
Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains. 相似文献
6.
Suijkerbuijk SJ van Dam TJ Karagöz GE von Castelmur E Hubner NC Duarte AM Vleugel M Perrakis A Rüdiger SG Snel B Kops GJ 《Developmental cell》2012,22(6):1321-1329
Chromosomal stability is safeguarded by a mitotic checkpoint, of which BUB1 and Mad3/BUBR1 are core components. These paralogs have similar, but not identical, domain organization. We show that Mad3/BUBR1 and BUB1 paralogous pairs arose by nine independent gene duplications throughout evolution, followed by parallel subfunctionalization in which preservation of the ancestral, amino-terminal KEN box or kinase domain was mutually exclusive. In one exception, vertebrate BUBR1-defined by the KEN box-preserved the kinase domain but allowed nonconserved degeneration of catalytic motifs. Although BUBR1 evolved to a typical pseudokinase in some vertebrates, it retained the catalytic triad in humans. However, we show that putative catalysis by human BUBR1 is dispensable for error-free chromosome segregation. Instead, residues that interact with ATP in conventional kinases are essential for conformational stability in BUBR1. We propose that parallel evolution of BUBR1 orthologs rendered its kinase function dispensable in vertebrates, producing an unusual, triad-containing pseudokinase. 相似文献
7.
Arturo Sanz Sanz Yashavanthi Niranjan Henrik Hammarén Daniela Ungureanu Rob Ruijtenbeek Ivo P. Touw Olli Silvennoinen Riet Hilhorst 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(10):1835-1841
JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi–Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2–JH2 linker region participates in controlling activity by reducing the affinity for ATP. 相似文献
8.
Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase domain, and a complex interplay of the Jak domains is involved in regulation of catalytic activity and association to cytokine receptors. Most activating mutations are found in the pseudokinase domain. Here we present recently discovered mutations in the context of our structural models of the respective domains. We describe two structural hotspots in the pseudokinase domain of Jak2 that seem to be associated either to myeloproliferation or to lymphoblastic leukaemia, pointing at the involvement of distinct signalling complexes in these disease settings. The different domains of Jaks are discussed as potential drug targets. We present currently available inhibitors targeting Jaks and indicate structural differences in the kinase domains of the different Jaks that may be exploited in the development of specific inhibitors. Moreover, we discuss recent chemical genetic approaches which can be applied to Jaks to better understand the role of these kinases in their biological settings and as drug targets. 相似文献
9.
John S. Tokarski Adriana Zupa-Fernandez Jeffrey A. Tredup Kristen Pike ChiehYing Chang Dianlin Xie Lihong Cheng Donna Pedicord Jodi Muckelbauer Stephen R. Johnson Sophie Wu Suzanne C. Edavettal Yang Hong Mark R. Witmer Lisa L. Elkin Yuval Blat William J. Pitts David S. Weinstein James R. Burke 《The Journal of biological chemistry》2015,290(17):11061-11074
10.
Xiaoshan Min Daniela Ungureanu Sarah Maxwell Henrik Hammarén Steve Thibault Ellin-Kristina Hillert Merrill Ayres Brad Greenfield John Eksterowicz Chris Gabel Nigel Walker Olli Silvennoinen Zhulun Wang 《The Journal of biological chemistry》2015,290(45):27261-27270
JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) participates in signaling through cytokine receptors involved in immune responses and inflammation. JAKs are characterized by dual kinase domain: a tyrosine kinase domain (JH1) that is preceded by a pseudokinase domain (JH2). The majority of disease-associated mutations in JAKs map to JH2, demonstrating its central regulatory function. JH2s were considered catalytically inactive, but JAK2 JH2 was found to have low autoregulatory catalytic activity. Whether the other JAK JH2s share ATP binding and enzymatic activity has been unclear. Here we report the crystal structure of TYK2 JH2 in complex with adenosine 5′-O-(thiotriphosphate) (ATP-γS) and characterize its nucleotide binding by biochemical and biophysical methods. TYK2 JH2 did not show phosphotransfer activity, but it binds ATP and the nucleotide binding stabilizes the protein without inducing major conformational changes. Mutation of the JH2 ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling. The overall structural characteristics of TYK2 JH2 resemble JAK2 JH2, but distinct stabilizing molecular interactions around helix αAL in the activation loop provide a structural basis for differences in substrate access and catalytic activities among JAK family JH2s. The structural and biochemical data suggest that ATP binding is functionally important for both TYK2 and JAK2 JH2s, whereas the regulatory phosphorylation appears to be a unique property of JAK2. Finally, the co-crystal structure of TYK2 JH2 complexed with a small molecule inhibitor demonstrates that JH2 is accessible to ATP-competitive compounds, which offers novel approaches for targeting cytokine signaling as well as potential therapeutic applications. 相似文献
11.
Dusa A Staerk J Elliott J Pecquet C Poirel HA Johnston JA Constantinescu SN 《The Journal of biological chemistry》2008,283(19):12941-12948
Explaining the uniqueness of the acquired somatic JAK2 V617F mutation, which is present in more than 95% of polycythemia vera patients, has been a challenge. The V617F mutation in the pseudokinase domain of JAK2 renders the unmutated kinase domain constitutively active. We have performed random mutagenesis at position 617 of JAK2 and tested each of the 20 possible amino acids for ability to induce constitutive signaling in Ba/F3 cells expressing the erythropoietin receptor. Four JAK2 mutants, V617W, V617M, V617I, and V617L, were able to induce cytokine independence and constitutive downstream signaling. Only V617W induced a level of constitutive activation comparable with V617F. Also, only V617W stabilized tyrosine-phosphorylated suppressor of cytokine signaling 3 (SOCS3), a mechanism by which JAK2 V617F overcomes inhibition by SOCS3. The V617W mutant induced a myeloproliferative disease in mice, mainly characterized by erythrocytosis and megakaryocytic proliferation. Although JAK2 V617W would predictably be pathogenic in humans, the substitution of the Val codon, GTC, by TTG, the codon for Trp, would require three base pair changes, and thus it is unlikely to occur. We discuss how the predicted conformations of the activated JAK2 mutants can lead to better screening assays for novel small molecule inhibitors. 相似文献
12.
Atwell S Adams JM Badger J Buchanan MD Feil IK Froning KJ Gao X Hendle J Keegan K Leon BC Müller-Dieckmann HJ Nienaber VL Noland BW Post K Rajashankar KR Ramos A Russell M Burley SK Buchanan SG 《The Journal of biological chemistry》2004,279(53):55827-55832
Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine --> phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine. 相似文献
13.
Julijus Bogomolovas Alexander Gasch Felix Simkovic Daniel J. Rigden Siegfried Labeit Olga Mayans 《Open biology》2014,4(5)
Striated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK''s best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK''s active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds. 相似文献
14.
Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase. 总被引:8,自引:5,他引:8
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We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases. 相似文献
15.
It is known that the human Ras GTPase activating protein (GAP) p120-GAP can be phosphorylated by different members of the Src kinase family and recently phosphorylation of the GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 by proteins of the Src kinase family has been revealed in vivo [Kiyono, M., Kaziro, Y. & Satoh, T. (2000) J. Biol. Chem. 275, 5441-5446]. As it still remains unclear how these phosphorylations can influence the Ras pathway we have analyzed the ability of p60c-Src and Lck to phosphorylate these two Ras regulators and have compared the activity of the phosphorylated and unphosphorylated forms. Both kinases were found to phosphorylate full-length or truncated forms of GAP and GEF. The use of the catalytic domain of p60c-Src showed that its SH3/SH2 domains are not required for the interaction and the phosphorylation of both regulators. Remarkably, the phosphorylations by the two kinases were accompanied by different functional effects. The phosphorylation of p120-GAP by p60c-Src inhibited its ability to stimulate the Ha-Ras-GTPase activity, whereas phosphorylation by Lck did not display any effect. A different picture became evident with CDC25Mm; phosphorylation by Lck increased its capacity to stimulate the GDP/GTP exchange on Ha-Ras, whereas its phosphorylation by p60c-Src was ineffective. Our results suggest that phosphorylation by p60c-Src and Lck is a selective process that can modulate the activity of p120-GAP and CDC25Mm towards Ras proteins. 相似文献
16.
The Wiscott-Aldrich syndrome protein, WASP, is an effector through which cdc42, a Rho family GTPase, regulates the actin cytoskeleton in hematopoietic cells. We have found that WASP binds readily to a number of tyrosine protein kinases including the Src kinases and the Abl kinase when the proteins are coexpressed during transient transfection. Binding inhibited the activity of each of these kinases strikingly, both in vitro and in vivo. Surprisingly, the binding was not due to an interaction between the proline-rich domain of WASP and the SH3 domain of these kinases. Rather, residues 83-93 in WASP were found to bind to the catalytic domains of the kinases. Binding did not decrease the affinity of Src kinases for either ATP or a peptide substrate noticeably. Rather, the V(max) of substrate phosphorylation was reduced by the binding of the peptide. This inhibition represents a novel form of regulation of protein kinase activity and suggests that that the isolation of small molecules that exploit this inhibitory mechanism may be possible. 相似文献
17.
Autoinhibition of Jak2 tyrosine kinase is dependent on specific regions in its pseudokinase domain
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Jak tyrosine kinases have a unique domain structure containing a kinase domain (JH1) adjacent to a catalytically inactive pseudokinase domain (JH2). JH2 is crucial for inhibition of basal Jak activity, but the mechanism of this regulation has remained elusive. We show that JH2 negatively regulated Jak2 in bacterial cells, indicating that regulation is an intrinsic property of Jak2. JH2 suppressed basal Jak2 activity by lowering the V(max) of Jak2, whereas JH2 did not affect the K(m) of Jak2 for a peptide substrate. Three inhibitory regions (IR1-3) within JH2 were identified. IR3 (residues 758-807), at the C terminus of JH2, directly inhibited JH1, suggesting an inhibitory interaction between IR3 and JH1. Molecular modeling of JH2 showed that IR3 could form a stable alpha-helical fold, supporting that IR3 could independently inhibit JH1. IR2 (725-757) in the C-terminal lobe of JH2, and IR1 (619-670), extending from the N-terminal to the C-terminal lobe, enhanced IR3-mediated inhibition of JH1. Disruption of IR3 either by mutations or a small deletion increased basal Jak2 activity, but abolished interferon-gamma-inducible signaling. Together, the results provide evidence for autoinhibition of a Jak family kinase and identify JH2 regions important for autoregulation of Jak2. 相似文献
18.
Tsuyoshi Araki Linh Hai Vu Norimitsu Sasaki Takefumi Kawata Ludwig Eichinger Jeffrey G. Williams 《Molecular biology of the cell》2014,25(20):3222-3233
When Dictyostelium cells are hyperosmotically stressed, STATc is activated by tyrosine phosphorylation. Unusually, activation is regulated by serine phosphorylation and consequent inhibition of a tyrosine phosphatase: PTP3. The identity of the cognate tyrosine kinase is unknown, and we show that two tyrosine kinase–like (TKL) enzymes, Pyk2 and Pyk3, share this function; thus, for stress-induced STATc activation, single null mutants are only marginally impaired, but the double mutant is nonactivatable. When cells are stressed, Pyk2 and Pyk3 undergo increased autocatalytic tyrosine phosphorylation. The site(s) that are generated bind the SH2 domain of STATc, and then STATc becomes the target of further kinase action. The signaling pathways that activate Pyk2 and Pyk3 are only partially overlapping, and there may be a structural basis for this difference because Pyk3 contains both a TKL domain and a pseudokinase domain. The latter functions, like the JH2 domain of metazoan JAKs, as a negative regulator of the kinase domain. The fact that two differently regulated kinases catalyze the same phosphorylation event may facilitate specific targeting because under stress, Pyk3 and Pyk2 accumulate in different parts of the cell; Pyk3 moves from the cytosol to the cortex, whereas Pyk2 accumulates in cytosolic granules that colocalize with PTP3. 相似文献
19.
The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity 总被引:8,自引:1,他引:7
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Beamish HJ Jessberger R Riballo E Priestley A Blunt T Kysela B Jeggo PA 《Nucleic acids research》2000,28(7):1506-1513
DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines. 相似文献
20.
Thakur M Chaba R Mondal AK Chakraborti PK 《The Journal of biological chemistry》2008,283(12):8023-8033
Eukaryotic type Ser/Thr protein kinases have recently been shown to regulate a variety of cellular functions in bacteria. PknA, a transmembrane Ser/Thr protein kinase from Mycobacterium tuberculosis, when constitutively expressed in Escherichia coli resulted in cell elongation and therefore has been thought to be regulating morphological changes associated with cell division. Bioinformatic analysis revealed that PknA has N-terminal catalytic, juxtamembrane, transmembrane, and C-terminal extracellular domains, like known eukaryotic type Ser/Thr protein kinases from other bacteria. To identify the minimum region capable of exhibiting phosphorylation activity of PknA, we created several deletion mutants. Surprisingly, we found that the catalytic domain itself was not sufficient for exhibiting phosphorylation ability of PknA. However, the juxtamembrane region together with the kinase domain was necessary for the enzymatic activity and thus constitutes the catalytic core of PknA. Utilizing this core, we deduce that the autophosphorylation of PknA is an intermolecular event. Interestingly, the core itself was unable to restore the cell elongation phenotype as manifested by the full-length protein in E. coli; however, its co-expression along with the C-terminal region of PknA can associate them in trans to reconstitute a functional protein in vivo. Therefore, these findings argue that the transmembrane and extracellular domains of PknA, although dispensable for phosphorylation activities, are crucial in responding to signals. Thus, our results for the first time establish the significance of different domains in a bacterial eukaryotic type Ser/Thr kinase for reconstitution of its functionality. 相似文献