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1.
Hsp70蛋白自身磷酸化对其分子伴侣功能的影响 总被引:1,自引:0,他引:1
近年对分子伴侣蛋白Hsp70作用机制的研究发现,其ATP功能区域X光晶体结构有一个新的钙离子结合区域,这个新的功能区域与Hsp70分子的ADP结合、ATP水解及合成有关.有报道认为Hsp70蛋白的NDP激酶样作用,通过形成酸不稳定性自身磷酸化中间体催化γ 磷酸基团在ATP和ADP间传递,组氨酸H89与这个新的区域有密切关系,有可能与Hsp70蛋白形成自身磷酸化中间体有关.本研究运用基因定位诱导突变技术,将89位组氨酸以丝氨酸替代(H89S),通过比较Hsp70野生型及突变型蛋白的自身磷酸化过程的改变,及其对Hsp70蛋白体外荧光素酶活性影响的不同,初步探讨Hsp70作用机制.结果发现,突变的H89S蛋白自身磷酸化过程及体外变性荧光素酶重折叠受到抑制.野生型蛋白未受到影响,野生型Hsp70可以形成酸不稳定的自身磷酸化中间体,产生CDP依赖性解磷酸反应,而H89S突变型蛋白不能形成这种反应.89位组氨酸点突变能显著降低ATP酶交换反应及体外变性荧光素酶重折叠水平,但它的自身磷酸化可能并非唯一必需的介导位点或只是一个选择性的功能侧链. 相似文献
2.
The chaperone activity of Hsp70 in protein folding and its conformational switching are regulated through the hydrolysis of ATP and the ATP-ADP exchange cycle. It was reported that, in the presence of physiological concentrations of ATP (approximately 5 mM) and ADP (approximately 0.5 mM), Hsp70 catalyzes ATP-ADP exchange through transfer of gamma-phosphate between ATP and ADP, via an autophosphorylated intermediate, whereas it only catalyzes the hydrolysis of ATP in the absence of ADP. To clarify the functional domain of the ATP-ADP exchange activity of Hsp70, we isolated the 44-kD ATPase domain of Hsp70 after limited proteolysis with alpha-chymotrypsin (EC 3.4.21.1). The possibility of ATP-ADP exchange activity of a contaminating nucleoside diphosphate kinase (EC 2.7.4.6) was monitored throughout the experiments. The purified 44-kD ATPase domain exhibited intrinsic ATP-ADP exchange by catalyzing the transfer of gamma-phosphate between ATP and ADP with acid-stable autophosphorylation at Thr204. 相似文献
3.
General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core, and the equivalent Lys72 in cAMP-dependent kinase has often been used to generate a "dead" kinase. Here, we have analyzed the consequences of this mutation on kinase structure and function. Mutation of Lys72 to histidine (K72H) generated an inactive enzyme, which was unphosphorylated. Treatment with an exogenous kinase (PDK-1) resulted in a mutant that was phosphorylated only at Thr197 and remained inactive but nevertheless capable of binding ATP. Ser338 in K72H cannot be autophosphorylated, nor can it be phosphorylated in an intermolecular process by active wild type C-subunit. The Lys72 mutant, once phosphorylated on Thr197, can bind with high affinity to the RIalpha subunits. Thus a dead kinase can still act as a scaffold for binding substrates and inhibitors; it is only phosphoryl transfer that is defective. Using a potent inhibitor of C-subunit activity, H-89, Escherichia coli-expressed C-subunit was also obtained in its unphosphorylated state. This protein is able to mature into its active form in the presence of PDK-1 and is able to undergo secondary autophosphorylation on Ser338. Unlike the H-89-treated wild type protein, the mutant protein (K72H) cannot undergo the subsequent cis autophosphorylation following phosphorylation at Thr197. Using these two substrates and mammalian-expressed PDK-1, we can elucidate a possible two-step process for the activation of the C-subunit: initial phosphorylation on the activation loop at Thr197 by PDK-1, or a PDK-1-like enzyme, followed by second cis autophosphorylation step at Ser338. 相似文献
4.
分子伴侣蛋白Hsp70氮端核苷酸结合域(NBD, nucleotide-binding domain)的ATP酶活性变化对其行使分子伴侣功能具有重要作用。本文采用分子动力学模拟方法研究酵母分子伴侣Hsp70氮端NBD内残基A17,R23,G32和R167点突变对其ATP酶活性区域构象影响及功能关系。结果表明,突变体A17V,T23H,G32S的ATP结合口袋袋口的loopl(第一个转角,连接p1与p2)结构柔性增强,活性残基T11侧链明显向内移动,从而更加接近ATP的γ-磷酸基团,更容易使ATP水解。这可能蕞终导致ATP酶活性增强,从而引起分子伴侣功能的变化。 相似文献
5.
The cyanobacterial circadian oscillator can be reconstituted in vitro; mixing three clock proteins (KaiA, KaiB, and KaiC) with ATP results in an oscillation of KaiC phosphorylation with a periodicity of ~24 h. The hexameric ATPase KaiC hydrolyzes ATP bound at subunit interfaces. KaiC also exhibits autokinase and autophosphatase activities, the latter of which is particularly noteworthy because KaiC is phylogenetically distinct from typical protein phosphatases. To examine this activity, we performed autodephosphorylation assays using (32)P-labeled KaiC. The residual radioactive ATP bound to subunit interfaces was removed using a newly established method, which included the dissociation of KaiC hexamers into monomers and the reconstitution of KaiC hexamers with nonradioactive ATP. This approach ensured that only the signals derived from (32)P-labeled KaiC were examined. We detected the transient formation of [(32)P]ATP preceding the accumulation of (32)P(i). Together with kinetic analyses, our data demonstrate that KaiC undergoes dephosphorylation via a mechanism that differs from those of conventional protein phosphatases. A phosphate group at a phosphorylation site is first transferred to KaiC-bound ADP to form ATP as an intermediate, which can be regarded as a reversal of the autophosphorylation reaction. Subsequently, the ATP molecule is hydrolyzed to form P(i). We propose that the ATPase active site mediates not only ATP hydrolysis but also the bidirectional transfer of the phosphate between phosphorylation sites and the KaiC-bound nucleotide. On the basis of these findings, we can now dissect the dynamics of the KaiC phosphorylation cycle relative to ATPase activity. 相似文献
6.
Incubation of a highly purified bovine spleen protein tyrosine kinase with [gamma-32P]ATP and Mg2+ resulted in a gradual radioactive labeling of the protein kinase (50 kDa) with no change in the protein kinase activity toward angiotensin II. On the other hand, treatment of the protein tyrosine kinase with an immobilized alkaline phosphatase caused essentially complete loss in the kinase activity, which could be restored by incubation of the enzyme with ATP and Mg2+. By using the alkaline phosphatase-treated kinase, time courses of the protein phosphorylation and the enzyme activation were demonstrated to correlate closely. These results indicate that this protein tyrosine kinase relies on autophosphorylation for activity and that the purified enzyme usually exists in a fully phosphorylated state. The radioactive labeling of the purified kinase during incubation with [gamma-32P]ATP resulted from a phosphate exchange reaction: the exchange of [gamma-32P]phosphate of ATP with the protein bound phosphate as previously suggested (Kong, S.K., and Wang, J.H. (1987) J. Biol. Chem. 262, 2597-2603). It could be shown that the autophosphorylation of phosphatase-treated tyrosine kinase was strongly inhibited by the substrate angiotensin II, whereas the exchange reaction carried out with untreated tyrosine kinase was not. Autophosphorylation is suggested to be an intermolecular reaction since its initial rate is proportional to the square of the protein concentration. 相似文献
7.
Autophosphorylation of Arabidopsis nucleoside diphosphate kinase 2 occurs only on its active histidine residue 总被引:1,自引:0,他引:1
Arabidopsis nucleoside diphosphate kinase 2 (NDPK2) is a component in the phytochrome-mediated light signaling. In the present study, its autophosphorylation was investigated. Acid-stable and alkali-stable phosphorylated residues were analyzed under two different conditions. Results revealed that NDPK2 is phosphorylated only on its active histidine residue His197 and the presence of serine/threonine phosphorylation is an experimental artifact due to the harsh condition applied in the treatment of the phosphorylated protein sample. To resolve the controversy of whether serine/threonine phosphorylation of NDPK occurs as has been suggested by other NDPK studies, NDPK2 putative phosphorylation site mutants were generated and examined. No serine/threonine phosphorylation was identified in NDPK2 or implicated in its enzymatic activity. Further studies indicated that the low enzymatic activity and autophosphorylation level of NDPK2 mutant S199A are shown to be due to a damaged H-bonding with the active histidine residue His197 in the nucleotide-binding pocket. In addition, NDPK2 Kpn loop mutant T182A was found to possess an extremely low enzymatic activity and almost no autophosphorylation, suggesting the importance of the oligomeric states of NDPK2 in NDPK2 functioning. 相似文献
8.
The Ser122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/Nm23-H2 was recently found in melanoma cells. In comparison to the wild-type enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to analyze phosphoryl transfer between the mutant enzyme and various pairs of nucleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine residue (His118) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the rate-limiting step of the NDK-BS122P-catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser120 --> Gly mutation in human NDK-A/Nm23-H1, the Ser122 --> Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes. 相似文献
9.
Nucleoside diphosphate (NDP) kinases have been found to be involved in a wide range of fundamental biological processes ranging from developmental control to signal transduction and metastasis. We have recently cloned and sequenced a cDNA encoding an NDP-kinase of the rat mucosal mast cell line RBL-2H3 [Hemmerich, S., Yarden, Y., & Pecht, I. (1992) Biochemistry (preceding paper in this issue)]. The enzyme itself has been isolated by means of its affinity to the bischromone cromoglycate. Here we report several of its biochemical characteristics: A structural model for the native protein is proposed in which two disulfide-linked pairs of similar 18-kDa subunits (p18) associate to form a 72-kDa tetramer (p72). This is based on the migration properties of the purified enzyme on gel filtration columns, sodium dodecylsulfate gel electrophoresis, and two-dimensional electrophoresis, together with peptide mapping data. In the absence of NDP, both intact p72 and the dissociated 18-kDa subunits (p18) were shown to undergo Mg(2+)-dependent stoichiometric autophosphorylation utilizing adenosine and guanosine triphosphate or gamma-thiotriphosphate as phosphate donor. This autophosphorylation activity was found to be retained by the 18-kDa subunits even following fractionation by SDS-PAGE and electrophoretic transfer to nitrocellulose. The Michaelis constant of this autophosphorylation reaction with either ATP, ATP gamma S, GTP, or GTP gamma S was determined to be 6.5 +/- 1 microM, and maximally 2 mol of phosphate were found to be incorporated per p72 molecule, thus indicating that phosphorylation occurs at a single site on only two of the four 18-kDa subunits of the holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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11.
Ohji G Hidayat S Nakashima A Tokunaga C Oshiro N Yoshino K Yokono K Kikkawa U Yonezawa K 《Journal of biochemistry》2006,139(1):129-135
Heat shock protein 90 (Hsp90) was co-immunoprecipitated with raptor, the binding partner of the mammalian target of rapamycin (mTOR) from HEK293 cells. Hsp90 was detected in the anti-raptor antibody immunoprecipitates prepared from the cell extract by immunoblot analysis using the anti-Hsp90 antibody, and the association of these two proteins was confirmed by immunoprecipitation from the cells co-expressing Hsp90 and raptor as epitope-tagged molecules. Geldanamycin, a potent inhibitor of Hsp90, disrupted the in vivo binding of Hsp90 to raptor without affecting the association of raptor and mTOR, and suppressed the phosphorylation by mTOR of the downstream translational regulators p70 S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The protein kinase activity of S6K as well as the phosphorylation of the substrate, 40S ribosomal protein S6, were lowered in the geldanamycin-treated cells. These results indicate that Hsp90 is involved in the regulation of protein translation by facilitating the phosphorylation reaction of 4E-BP1 and S6K catalyzed by the mTOR/raptor complex through the association with raptor, and that the mTOR signaling pathway is a novel target of geldanamycin. 相似文献
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13.
Formation of a hetero-oligomeric complex between Hsp70 and Hsp80 of Neurospora crassa was observed previously by means of chemical crosslinking and enzyme-linked immunosorbent assays (ELISA). The present study documents the effect of nucleotides on the subunit structure of Hsp70 and Hsp80 by crosslinking with bifunctional reagents: glutaraldehyde, dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). The inter-protomer crosslinking of Hsp80 with DMA and DMS was suppressed by ATP and to a lesser extent by ADP, CTP, and NAD. Crosslinking of purified Hsp70 by glutaraldehyde yielded dimers and higher order oligomers. Binding of ATP, ADP, CTP, and NAD, but not NADH, led to a marked reduction in the yield of oligomers. Similarly, crosslinking by DMA and DMS was suppressed by ADP, ATP, and CTP. Both Hsp70 and Hsp80 exhibited intrinsic ATPase activity. Interestingly, ATP levels exceeding 25 microM resulted in pronounced inhibition of the ATPase activity of Hsp80 and 0.5 mM and 0.25 mM ATP led to a prolonged lag in the reaction. Addition of NAD resulted in the abolition of the lag period. The binding of 2-p-toluidinylnapthalene-6-sulfonate (TNS) to Hsp70 and its displacement by ATP and other nucleotides demonstrated the hydrophobic nature of the nucleotide-binding region. 相似文献
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15.
Milda Pleckaityte Lina Mistinaite Edita Mistiniene Gervydas Dienys Gintas Zvirblis 《Biocatalysis and Biotransformation》2005,23(3):191-200
The guanidine-hydrochloride (Gdn-HCl) and thermally induced unfolding of Hsp70 from Meiothermus ruber (Mru.Hsp70) were analysed using tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. The ANS binding to Mru.Hsp70 showed both the increase in fluorescence intensity and a shift in emission maximum. Analysis of the unfolding profile of Mru.Hsp70 indicated that Gdn-HCl induced unfolding of Mru.Hsp70 occurred through intermediate species. The tryptophan and ANS fluorescence emission spectra revealed that ATP induced conformational change increased the thermal stability of Mru.Hsp70. The data obtained are similar to those of Escherichia coli DnaK. The ATP-ase activity of chaperones is fundamental for their biological activity. It this paper we demonstrate that, in contrast to Thermus thermophilus, both Mru.Hsp40 and Mru.Hsp22 co-chaperones affect the ATP-ase activity of Mru.Hsp70. The use of truncated Mru.Hsp40 proteins showed that full-length Mru.Hsp40 is required for stimulation of ATP-ase activity of Mru.Hsp70. E. coli GrpE could act as nucleotide exchange factor the in thermophilic Hsp70 ATP hydrolysis reaction. However, the role of E. coli DnaJ in the M. ruber ATP cycle needs further analysis. We selected the new substrate laccA suitable for determination of refolding activity of thermophilic chaperones. 相似文献
16.
Milda Pleckaityte Lina Mistinaite Edita Mistiniene Gervydas Dienys Gintas Zvirblis 《Biocatalysis and Biotransformation》2013,31(3-4):191-200
The guanidine-hydrochloride (Gdn-HCl) and thermally induced unfolding of Hsp70 from Meiothermus ruber (Mru.Hsp70) were analysed using tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. The ANS binding to Mru.Hsp70 showed both the increase in fluorescence intensity and a shift in emission maximum. Analysis of the unfolding profile of Mru.Hsp70 indicated that Gdn-HCl induced unfolding of Mru.Hsp70 occurred through intermediate species. The tryptophan and ANS fluorescence emission spectra revealed that ATP induced conformational change increased the thermal stability of Mru.Hsp70. The data obtained are similar to those of Escherichia coli DnaK. The ATP-ase activity of chaperones is fundamental for their biological activity. It this paper we demonstrate that, in contrast to Thermus thermophilus, both Mru.Hsp40 and Mru.Hsp22 co-chaperones affect the ATP-ase activity of Mru.Hsp70. The use of truncated Mru.Hsp40 proteins showed that full-length Mru.Hsp40 is required for stimulation of ATP-ase activity of Mru.Hsp70. E. coli GrpE could act as nucleotide exchange factor the in thermophilic Hsp70 ATP hydrolysis reaction. However, the role of E. coli DnaJ in the M. ruber ATP cycle needs further analysis. We selected the new substrate laccA suitable for determination of refolding activity of thermophilic chaperones. 相似文献
17.
T Leustek D Amir-Shapira H Toledo N Brot H Weissbach 《Cellular and molecular biology, including cyto-enzymology》1992,38(1):1-10
Several members of the 70 kDa heat shock protein group are known to be phosphorylated in vivo and have recently been found to undergo a Ca(2+)-stimulated autophosphorylation. The characteristics of the autophosphorylation reaction with Escherichia coli DnaK the mitochondrial and chloroplast homologs, and the endoplasmic reticulum Bip/Grp78 are discussed. Some common features are a requirement for Ca2+, inhibition by Mg2+ and phosphorylation solely on a threonine residue. Although the role of autophosphorylation of these proteins is not clear, it is known that the level of phosphorylation of some Hsp70 proteins in vivo is responsive to stress and other cellular conditions. 相似文献
18.
The plant genome encodes at least two distinct and evolutionary conserved plastidial starch-related dikinases that phosphorylate a low percentage of glucosyl residues at the starch granule surface. Esterification of starch favours the transition of highly ordered α-glucans to a less ordered state and thereby facilitates the cleavage of interglucose bonds by hydrolases. Metabolically most important is the phosphorylation at position C6, which is catalysed by the glucan, water dikinase (GWD). The reactions mediated by recombinant wild-type GWD from Arabidopsis thaliana (AtGWD) and from Solanum tuberosum (StGWD) were studied. Two mutated proteins lacking the conserved histidine residue that is indispensible for glucan phosphorylation were also included. The wild-type GWDs consume approximately 20% more ATP than is required for glucan phosphorylation. Similarly, although incapable of phosphorylating α-glucans, the two mutated dikinase proteins are capable of degrading ATP. Thus, consumption of ATP and phosphorylation of α-glucans are not strictly coupled processes but, to some extent, occur as independent phosphotransfer reactions. As revealed by incubation of the GWDs with [γ-(33) P]ATP, the consumption of ATP includes the transfer of the γ-phosphate group to the GWD protein but this autophosphorylation does not require the conserved histidine residue. Thus, the GWD proteins possess two vicinal phosphorylation sites, both of which are transiently phosphorylated. Following autophosphorylation at both sites, native dikinases flexibly use various terminal phosphate acceptors, such as water, α-glucans, AMP and ADP. A model is presented describing the complex phosphotransfer reactions of GWDs as affected by the availability of the various acceptors. 相似文献
19.
TcrX/Y is one of the twelve two component system (TCS) present in Mycobacterium tuberculosis. We have investigated the TcrX/Y interaction by in silico studies, pull down assay, radioactive phosphotransfer, surface plasmon resonance as well as crosstalk analysis of TcrY with TcrA – a non-cognate response regulator. Sequence alignment of TcrY with other histidine kinases revealed His256 as the residue responsible for autophosphorylation. The modeled structure of TcrX/Y was docked with each other by GRAMM-X revealing the interaction of TcrY/His256 with TcrX/Asp54. TcrY dimerization via the formation of four helix bundle was also observed by protein–protein docking. Autophosphorylation of TcrY has been observed followed by the phosphate transfer from TcrY to TcrX. The phosphorylation process required divalent metal ions like Mg2+ or Ca2+ ions as evident from the radioactive phosphorylation studies. Interaction was not observed between TcrY and TcrA suggesting the signal transduction process is specific in TcrX/Y system. TcrY hydrolyzes ATP and the Km value has been found to be 10 mM which is comparable to that of Hsp104. TcrX/Y interaction has been determined by surface plasmon resonance and dissociation constant (KD) was evaluated to be 3.6 μM. We conclude from our results that TcrX and TcrY are part of the same signal transduction pathway without their involvement in crosstalk with non-cognate counterpart. 相似文献
20.
C Tomomori T Tanaka R Dutta H Park S K Saha Y Zhu R Ishima D Liu K I Tong H Kurokawa H Qian M Inouye M Ikura 《Nature structural biology》1999,6(8):729-734
Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase. 相似文献