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1.
Calmodulin regulates fodrin susceptibility to cleavage by calcium-dependent protease I 总被引:4,自引:0,他引:4
The intracellular calcium-dependent proteolysis of fodrin has been postulated to be central to the regulation of plasticity of the cortical cytoskeleton of many eukaryotic cells. The close proximity of the sites of calmodulin (CaM) binding and calcium-dependent protease I (CDP-I) cleavage in mammalian alpha-fodrin suggested that their action may be linked. In hypotonic and isotonic buffers, CDP-I proteolysis of the beta subunit of fodrin was absolutely dependent upon the presence of active CaM. The stimulation by CaM was inhibited by CaM antagonists. The rate of CDP-I proteolysis of both subunits was enhanced by CaM, while the rate of fodrin proteolysis with other proteases was not influenced by CaM. The increase in the susceptibility of fodrin to CDP-I proteolysis was half-maximal at 80 nM CaM, and maximal at 200 nM CaM. The unusual and differential susceptibility of alpha- and beta-fodrin to proteolysis by CDP-I in the absence of CaM was exploited to investigate the quaternary structure of fodrin in which only the alpha subunit was cleaved. Cleavage of the alpha subunit alone did not destroy the tetrameric form of the molecule, whereas CDP-I cleavage of both subunits rendered the molecule incapable of reforming tetramers. These results provide structural and functional evidence that CaM and CDP-I act synergistically in the regulated proteolysis of fodrin. 相似文献
2.
Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/50 +/- 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x 10(-7) M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 x 10(-6) M with 1 mol fodrin bound/80 +/- 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 x 10(-7) M and the maximum binding was 1 mol fodrin/55 +/- 10 mol GFP. 相似文献
3.
C F Chang L M Gutierrez C Mundina-Weilenmann M M Hosey 《The Journal of biological chemistry》1991,266(25):16395-16400
Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are multisubunit proteins and are regulated by protein phosphorylation. The purpose of this study was to determine: 1) which subunits are the preferential targets of various protein kinases when the channels are phosphorylated in vitro in their native membrane-bound state and 2) the consequences of these phosphorylations in functional assays. Using as substrates channels present in purified transverse (T) tubule membranes, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a multifunctional Ca2+/calmodulin-dependent protein kinase (CaM protein kinase) preferentially phosphorylated the 165-kDa alpha 1 subunit to an extent that was 2-5-fold greater than the 52-kDa beta subunit. A protein kinase endogenous to the skeletal muscle membranes preferentially phosphorylated the beta peptide and showed little activity toward the alpha 1 subunit; however, the extent of phosphorylation was low. Reconstitution of partially purified channels into liposomes was used to determine the functional consequences of phosphorylation by these kinases. Phosphorylation of channels by PKA or PKC resulted in an activation of the channels that was observed as increases in both the rate and extent of Ca2+ influx. However, phosphorylation of channels by either the CaM protein kinase or the endogenous kinase in T-tubule membranes was without effect. Phosphorylation did not affect the sensitivities of the channels toward the dihydropyridines. Taken together, the results demonstrate that the alpha 1 subunit is the preferred substrate of PKA, PKC, and CaM protein kinase when the channels are phosphorylated in the membrane-bound state and that phosphorylation of the channels by PKA and PKC, but not by CaM protein kinase or an endogenous T-tubule membrane protein kinase, results in activation of the dihydropyridine-sensitive Ca2+ channels from skeletal muscle. 相似文献
4.
Fodrin, an actin and calmodulin binding and spectrin-like protein present in many nonerythrocyte tissues, could be phosphorylated up to more than 1.5 mol of phosphate/mol of protein by a highly purified non-receptor-associated protein tyrosine kinase from bovine spleen. The protein phosphorylation was not affected by Ca2+/calmodulin or by F-actin. Km and Vmax values of the reaction were 91 nM and 0.35 nmol of P2 min-1 (mg of kinase)-1, respectively. Both subunits A and B of fodrin were phosphorylated, with the rate of subunit A phosphorylation much greater than that of subunit B phosphorylation. Tryptic phosphopeptide mapping of the phosphorylated subunits suggested that there were three major phosphorylation sites in subunit A and one in subunit B. Phosphotyrosylfodrin could be dephosphorylated by the calmodulin-stimulated phosphatase (calcineurin) in the presence of activating metal ions; Ni2+ was a much more effective activator than Mn2+ for this reaction. Fodrin phosphorylation by the spleen protein tyrosine kinase did not appear to alter the actin and calmodulin binding properties of the protein. On the other hand, the calmodulin-dependent stimulation of smooth muscle actomyosin Mg2+-ATPase by fodrin was enhanced by 101% +/- 3% (n = 3) upon fodrin phosphorylation. Ni2+-calcineurin, which was shown to effectively dephosphorylate the phosphotyrosyl residues on fodrin, could reverse the phosphorylation-enhanced Mg2+-ATPase stimulatory activity of fodrin. 相似文献
5.
Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells 总被引:6,自引:0,他引:6
cDNAs containing the entire coding regions of the alpha and beta subunits of calmodulin-dependent protein kinase II (CaM kinase II) were isolated from a rat cerebrum cDNA library, ligated into an expression vector under the control of SV40 early promoter and introduced into Chinese hamster ovary (CHO) cells. To investigate the role of the alpha and beta subunits and their functional domains in CaM kinase II activity, the properties of the kinases expressed in the transfected cells were studied. CaM kinase II activity was detected in the transfected cells when the alpha and beta cDNAs were introduced into CHO cells simultaneously. RNA transfer blot and protein immunoblot analyses demonstrated the expression of the mRNAs and proteins of both alpha and beta subunits in the cloned cells. When alpha or beta cDNA was introduced into CHO cells separately, a significant level of the enzyme activity was also expressed, indicating that the alpha and beta subunits exhibited enzyme activity individually. The apparent Km values for ATP and MAP 2 were almost the same for the alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II. However, there was a slight difference in the affinity for calmodulin between the expressed proteins. The alpha and beta subunits expressed in the same cells polymerized to form alpha beta complex of a size similar to that of brain CaM kinase II. The alpha subunit also polymerized to form an oligomer, which showed almost the same S value as that of alpha beta complex and brain CaM kinase II. In contrast, the beta subunit did not polymerize. The alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II were autophosphorylated with [gamma-32P]ATP in the presence of Ca2+ and calmodulin, which resulted in the appearance of Ca2+-independent activity. The Ca2+-independent activity was 60-75% of the total activity as measured in the presence of Ca2+ plus calmodulin. To examine the functional relationship of peptide domains of the subunits of CaM kinase II, deleted cDNAs were introduced into CHO cells and the properties of the expressed proteins were studied. In cells transfected with alpha or beta cDNA from which the association domain was deleted, a significant level of kinase activity was expressed. However, the expressed proteins showed hardly any autophosphorylation and the appearance of Ca2+-independent enzyme activity was very low, indicating that the association domain was essential for the autophosphorylation and for the appearance of the Ca2+-independent activity. 相似文献
6.
Regulation of the Ca2+-dependent neutral proteinases from rabbit liver by an endogenous inhibitor 总被引:1,自引:0,他引:1
E Melloni F Salamino B Sparatore M Michetti S Pontremoli B L Horecker 《Archives of biochemistry and biophysics》1984,232(2):513-519
An endogenous inhibitor of neutral Ca2+-dependent proteinases has been isolated from rabbit liver cytosol. The inhibitor is a heat-stable, 240-kDa, tetrameric protein. It is dissociated into its 60-kDa subunits by high concentrations of Ca2+ (0.1-1 mM), but not by lower concentrations in the physiological range. Inhibition of the 150-kDa proteinase of rabbit liver [Melloni, E., Pontremoli, S., Salamino, F., Sparatore, B., Michetti, M. and Horecker, B.L. (1984) Arch. Biochem. Biophys. 232, 505-512] requires the monomeric form of the inhibitor, and occurs only at the high concentrations of Ca2+ which also cause dissociation of the dimeric 150-kDa proteinase into its 80-kDa subunits. The molecular weight of the inactive proteinase-inhibitor complex was estimated by the equilibrium gel penetration method to be 140 kDa, suggesting that it contains one subunit of proteinase and one of inhibitor. The mechanism of interaction of the inhibitor with the 200-kDa proteinase at high concentrations of Ca2+ is identical to that observed for the 150-kDa proteinase, namely dissociation of both proteinase and inhibitor into subunits and formation of an inactive 160-kDa proteinase-inhibitor complex. However, unlike the 150-kDa proteinase, which does not interact with the inhibitor at low Ca2+ concentrations, the 200-kDa proteinase is also inhibited at low concentrations of Ca2+. Under these conditions, the high-molecular-weight complex (greater than 400 kDa) formed between the tetrameric inhibitor and the dimeric proteinase prevents conversion of the 200-kDa proenzyme to the active, low-Ca2+-requiring form. 相似文献
7.
Regulation of cAMP concentration by calmodulin-dependent cyclic nucleotide phosphodiesterase 总被引:2,自引:0,他引:2
Bovine brain contains two major calmodulin (CaM) dependent phosphodiesterase isozymes which are homodimeric proteins with subunit molecular masses of 60 and 63 kilodaltons (kDa), respectively. The 60-kDa subunit isozyme can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme affinity towards CaM. The phosphorylation is blocked by Ca2+ and CaM and reversed by the CaM-stimulated phosphatase (calcineurin). The 63-kDa subunit isozymes can also be phosphorylated, but in this case by a CaM-dependent protein kinase(s). This phosphorylation is also accompanied by a decrease in the isozyme affinity towards CaM and can be reversed by the CaM-dependent phosphatase. Analysis of the complex regulatory properties of the phosphodiesterase isozymes has led to the suggestion that fluxes of cAMP and Ca2+ during cell activations are closely coupled and that the CaM-dependent phosphodiesterase isozymes play key roles in this signal coupling phenomenon. 相似文献
8.
Autophosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II in intact nerve terminals 总被引:5,自引:0,他引:5
F S Gorelick J K Wang Y Lai A C Nairn P Greengard 《The Journal of biological chemistry》1988,263(33):17209-17212
The autophosphorylation of purified Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) on a threonine-containing phosphopeptide common to both the alpha and beta subunits was previously shown to convert this enzyme into a catalytically active Ca2+-independent species. We now have examined the phosphorylation and activation of Ca2+/CaM kinase II in synaptosomes, a Ca2+-dependent neurosecretory system consisting of isolated nerve terminals. Synaptosomes were prelabeled with 32Pi and the alpha subunit of Ca2+/CaM kinase II was immunoprecipitated. Under basal incubation conditions the alpha subunit was phosphorylated. Depolarization of synaptosomes produced a rapid (2-5 s) Ca2+-dependent increase of about 50% in the state of phosphorylation of the alpha subunit. This was followed by a slower increase in the 32P content of the alpha subunit over the next 5 min of depolarization. The enhanced phosphorylation was characterized by an initial rise (2 s) and subsequent decrease (30 s) in the phosphothreonine content of the alpha subunit. In contrast, the phosphoserine content of the alpha subunit slowly increased during the course of depolarization. Thermolytic two-dimensional phosphopeptide maps of the alpha subunit demonstrated that depolarization stimulated the labeling of a phosphopeptide associated with autoactivation. In parallel experiments, unlabeled synaptosomes were depolarized, and lysates of these synaptosomes were assayed for Ca2+/CaM kinase II activity. Depolarization produced a rapid (less than or equal to 2 s) increase in Ca2+-independent Ca2+/CaM kinase II activity. This activity returned to basal levels by 60 s. Thus, depolarization of intact synaptosomes is associated with the transient phosphorylation of Ca2+/CaM kinase II on threonine residues, presumably involving an autophosphorylation mechanism and concomitantly the transient generation of the Ca2+-independent form of Ca2+/CaM kinase II. 相似文献
9.
KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II 总被引:17,自引:0,他引:17
H Tokumitsu T Chijiwa M Hagiwara A Mizutani M Terasawa H Hidaka 《The Journal of biological chemistry》1990,265(8):4315-4320
1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo. 相似文献
10.
Serum and growth factors rapidly elicit phosphorylation of the Ca2+/calmodulin-dependent protein kinase II in intact quiescent rat 3Y1 cells 总被引:2,自引:0,他引:2
A 50-kDa protein was recognized in rat embryo fibroblast 3Y1 cells with an affinity-purified antibody against rat brain Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). When the cytosolic extract from quiescent 3Y1 cells was immunoprecipitated with the antibody, the 50-kDa protein in the immunoprecipitate became phosphorylated in a Ca2+- and calmodulin-dependent manner following exposure to [gamma-32P]ATP. Moreover, the reaction proceeded through an intramolecular mechanism. These results suggest that the 50-kDa protein is a subunit of CaM kinase II in rat 3Y1 cells. The addition of 10% fetal calf serum to quiescent 3Y1 cells caused a rapid increase in the phosphorylation of the 50-kDa protein, which was immunoprecipitated with the affinity-purified anti-CaM kinase II antibody. The phosphorylation of CaM kinase II was detected as early as 20 s after the addition of serum, reached the maximal level at 2 min, and decreased to the basal level within 60 min. Platelet-derived growth factor and epidermal growth factor also elicited the phosphorylation of the 50-kDa protein in quiescent 3Y1 cells, while neither insulin nor 12-O-tetradecanoylphorbol-13-acetate did. Calcium ionophores, A23187 and ionomycin, also caused the phosphorylation of the protein in 3Y1 cells. Moreover, phosphopeptide mappings of the phosphorylated 50-kDa subunit generated in response to serum, EGF, and A23187 yielded patterns similar to that generated from the immunoprecipitated 50-kDa subunit phosphorylated in vitro. Phosphoamino acid analysis of the phosphorylated subunit demonstrated that serine residue was the major amino acid labeled under any condition. These results suggest that CaM kinase II undergoes phosphorylation in response to various stimuli that can increase the free Ca2+ concentration in the cytoplasm of quiescent fibroblast cells and therefore probably mediates at least some of the biological actions of growth factors. 相似文献
11.
In vitro phosphorylation of purified spectrin dimer was studied in the presence of Ca2+-calmodulin (CaM). CaM inhibited autophosphorylation of the beta subunit of spectrin. The inhibitory effect (65% at a 32-fold molar excess) appeared to be due to a weak interaction of CaM with spectrin. CaM was similarly effective in a phosphatase-stimulated autothiophosphorylation of the beta subunit with [gamma-35S]ATP. Hence, its inhibitory effect was not due to stimulation of a spectrin-associated phosphatase activity. Phosphorylation of spectrin by the catalytic subunit of a cAMP-dependent protein kinase occurred in both subunits (1984, FEBS Lett. 169, 323). CaM selectively inhibited a cAMP-dependent phosphorylation of the alpha subunit of spectrin to 30% at two CaM per spectrin. It was ineffective on the cAMP-dependent phosphorylation of the beta subunit up to a 32-fold molar excess. These results yield functional evidence for a CaM-spectrin interaction. They further suggest that CaM can regulate the extent of a cAMP-dependent phosphorylation of the alpha subunit of spectrin. 相似文献
12.
Lee WS Ngo-Anh TJ Bruening-Wright A Maylie J Adelman JP 《The Journal of biological chemistry》2003,278(28):25940-25946
Small conductance Ca2+-activated K+ channels (SK channels) are heteromeric complexes of pore-forming alpha subunits and constitutively bound calmodulin (CaM). The binding of CaM is mediated in part by the electrostatic interaction between residues Arg-464 and Lys-467 of SK2 and Glu-84 and Glu-87 of CaM. Heterologous expression of the double charge reversal in SK2, SK2 R464E/K467E (SK2:64/67), did not yield detectable surface expression or channel activity in whole cell or inside-out patch recordings. Coexpression of SK2:64/67 with wild type CaM or CaM1,2,3,4, a mutant lacking the ability to bind Ca2+, rescued surface expression. In patches from cells coexpressing SK2:64/67 and wild type CaM, currents were recorded immediately following excision into Ca2+-containing solution but disappeared within minutes after excision or immediately upon exposure to Ca2+-free solution and were not reactivated upon reapplication of Ca2+-containing solution. Channel activity was restored by application of purified recombinant Ca2+-CaM or exposure to Ca2+-free CaM followed by application of Ca2+-containing solution. Coexpression of the double charge reversal E84R/E87K in CaM (CaM:84/87) with SK2:64/67 reconstituted stable Ca2+-dependent channel activity that was not lost with exposure to Ca2+-free solution. Therefore, Ca2+-independent interactions with CaM are required for surface expression of SK channels, whereas the constitutive association between the two channel subunits is not an essential requirement for gating. 相似文献
13.
S Pontremoli B Sparatore F Salamino M Michetti E Melloni 《Archives of biochemistry and biophysics》1985,239(2):517-522
Mn2+ (50 microM) satisfies the requirement for activity of the purified Ca2+-dependent neutral proteinase from human erythrocytes. Unlike the activation by Ca2+ [E. Melloni et al. (1984) Biochem. Int. 8, 477-489], the effect of Mn2+ is fully reversible and does not involve autodigestion of the native 80-kDa catalytic subunit. However, the native dimeric proenzyme (procalpain), which contains both the 80-kDa subunit and a smaller 30-kDa subunit, is not activated by Mn2+ alone but also requires the presence of micromolar concentrations of Ca2+. Under these conditions, 40% of the maximum activity is expressed without dissociation of the 80- and 30-kDa subunits. Mn2+, but not micromolar Ca2+, can also partially satisfy the metal requirement of the native 80-kDa subunit isolated after dissociation of the heterodimer. This activity is further enhanced by the addition of 5 microM Ca2+, which is ineffective in the absence of Mn2+. After procalpain is converted to active calpain by incubation with Ca2+ and substrate [S. Pontremoli et al. (1984) Biochem. Biophys. Res. Commun. 123, 331-337] full activity is observed with 5 microM Mn2+, which now substitutes completely for Ca2+. Activation of procalpain by Mn2+ represents a new mechanism for modulation of the Ca2+-dependent proteinase activity. 相似文献
14.
Calcium-dependent conformational changes in the 36-kDa subunit of intestinal protein I related to the cellular 36-kDa target of Rous sarcoma virus tyrosine kinase 总被引:24,自引:0,他引:24
Protein I from intestinal epithelium is biochemically and immunologically related to the fibroblast 36-kDa substrate of the Rous sarcoma virus-encoded tyrosine protein kinase (Gerke and Weber (1984) EMBO J. 3, 227-233). Protein I is a Ca2+-binding protein containing two copies each of a 36- and 10-kDa subunit. Denaturation/renaturation experiments show that the 36-kDa subunit is a monomer, whereas the 10-kDa subunit forms a dimer. Mixing of the subunits leads to reconstituted protein I. Physicochemical properties of protein I and its isolated subunits reveal a Ca2+-dependent conformational change in the 36-kDa subunit which involves the exposure of 1 or more tyrosine residues to a more aqueous environment. This change points to a Ca2+ binding constant of about 10(4) M-1 in the presence of 2 mM Mg2+ and induces the ability of protein I and the 36-kDa subunit to bind in vitro to F-actin and nonerythroid spectrin. The same high Ca2+ requirement has been reported for the in vitro tyrosine phosphorylation of a 35-kDa protein from A-431 carcinoma cells by the epidermal growth factor receptor kinase (Fava and Cohen (1984) J. Biol. Chem. 259, 2636-2645). Here we show that this 35-kDa substrate is biochemically and immunologically related to the 36-kDa subunit of protein I, which in turn corresponds to the substrate of the Rous sarcoma virus kinase. The protein of A-431 cells exists not only as a monomer but also as a dimer. The latter fraction contains a 10-kDa polypeptide immunologically related to the corresponding subunit of protein I. Given past results on the A-431 system, we speculate that the monomer rather than the dimer is the preferred in vitro substrate for the epidermal growth factor receptor kinase. Thus, the 10-kDa subunit, which induces dimerization of the phosphorylatable large subunit, may act as an inhibitor. 相似文献
15.
The cDNAs encoding the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) were ligated into the bacterial expression vector pET and expressed in Escherichia coli. The bacterially expressed alpha and beta subunits exhibited Ca2+/calmodulin-dependent activity and were easily purified to apparent homogeneity from cell extracts. To determine the minimum size required for catalytic activity and the properties of the calmodulin-binding domain, mutated CaM kinase II cDNAs were expressed in E. coli and the enzymatic property of expressed proteins was examined. The replacement of Thr-286 of the alpha subunit with the negatively charged amino acid Asp or that of Arg-283 with the neutral amino acid Gly induced the partially Ca2+ independent activity. The mutant enzymes alpha-I(delta 283-478) and alpha-II(delta 359-478), which truncated the C-terminal region of the alpha subunit, exhibited CaM kinase II activity and the activities of alpha-I(delta 283-478) and alpha-II(delta 359-478) were completely independent of and partially dependent on Ca2+ and calmodulin, respectively. However, the truncated protein alpha(delta 250-478), which was only 33 amino acids shorter than the alpha-I(delta 283-478) protein had no enzymatic activity, indicating that alpha-I(delta 283-478) was close to the minimum size of the active form. The mutant enzyme alpha(delta 291-315), which lacked the calmodulin-binding domain exhibited Ca2+ independent activity. The molecular mass was, however, smaller than that expected from the amino acid sequence. The mutant enzyme alpha(delta 304-315), which lacked the C-terminal half of the calmodulin-binding domain of the alpha subunit, however, exhibited Ca(2+)-independent activity without a reduction in molecular size, indicating that residues 304-315 of the alpha subunit constituted the core calmodulin-binding domain. 相似文献
16.
Functional analysis of Ca2+/calmodulin-dependent protein kinase II expressed in bacteria 总被引:3,自引:0,他引:3
The cDNA encoding the 50-kDa subunit of Ca2+/calmodulin (CaM)-dependent protein kinase II from adult rat brain was cloned into the bacterial expression vector pK223-2 and produced in bacteria. Extensive modification of the cDNA was required to express detectable levels of enzyme. The activity of the bacterially expressed kinase was stringently dependent on Ca2+/CaM but did not exhibit cooperative activation kinetics characteristic of the forebrain enzyme and required 10-fold greater amounts of CaM for half-maximal activation. The bacterially expressed enzyme displayed an apparent Km for a synthetic peptide substrate similar to that of the forebrain enzyme (12 and 10 microM, respectively). Limited proteolysis maps of autophosphorylated peptides, and Western blot analysis demonstrated that the bacterially expressed enzyme was structurally and immunologically indistinguishable from the 50-kDa subunit of the rat forebrain holoenzyme. The bacterially expressed enzyme became Ca2+/CaM-independent after Ca2+/CaM-dependent autophosphorylation in a fashion identical to the forebrain enzyme. 相似文献
17.
Calmodulin-dependent protein phosphatase (CaM-PPase) was isolated from bovine parotid gland by sequential application of DEAE-52, Affi-gel blue and calmodulin-affinity chromatography followed by gel filtration and high performance liquid chromatography. The enzyme was activated in the simultaneous presence of Ni2+ or Mn2+ and Ca2+ plus calmodulin. Ca2+/calmodulin-dependent activation of CaM-PPase was antagonized by inhibitors of calmodulin action, such as W-7 and trifluoperazine. Tryptophan fluorescence was quenched in the presence of Ni2+. CaM-PPase was a heterodimer. The molecular weights of large subunits which bound calmodulin (CaM) were 68 kD and 58 kD - the 68 kD subunit was predominant. Polyclonal antibodies against bovine calcineurin cross-reacted with both types of larger subunits. Using polyclonal antibodies against bovine calcineurin or the monoclonal antibody against subunit B of bovine calcineurin, the smaller molecular weight subunit (19 kD) was found to be immunologically identical to subunit B of bovine calcineurin. In bovine parotid gland, CaM-PPase was found both in acinar and duct cells. 相似文献
18.
Phosphorylase kinase (PhK) is a large hexadecameric enzyme consisting of four copies of four subunits: (alphabetagammadelta)4. An intrinsic calmodulin (CaM, the delta subunit) binds directly to the gamma protein kinase chain. The interaction site of CaM on gamma has been localized to a C-terminal extension of the kinase domain. Two 25-mer peptides derived from this region, PhK5 and PhK13, were identified previously as potential CaM-binding sites. Complex formation between Ca2+/CaM with these two peptides was characterized using analytical gel filtration and NMR methods. NMR chemical shift perturbation studies showed that while PhK5 forms a robust complex with Ca2+/CaM, no interactions with PhK13 were observed. 15N relaxation characteristics of Ca2+/CaM and Ca2+/CaM/PhK5 complexes were compared with the experimentally determined structures of several Ca2+/CaM/peptide complexes. Good fits were observed between Ca2+/CaM/PhK5 and three structures: Ca2+/CaM complexes with peptides from endothelial nitric oxide synthase, with smooth muscle myosin light chain kinase and CaM kinase I. We conclude that the PhK5 site is likely to have a direct role in Ca2+-regulated control of PhK activity through the formation of a classical 'compact' CaM complex. 相似文献
19.
Calcium/calmodulin modulation of olfactory and rod cyclic nucleotide-gated ion channels 总被引:6,自引:0,他引:6
Cyclic nucleotide-gated (CNG) ion channels mediate sensory transduction in olfactory sensory neurons and retinal photoreceptor cells. In these systems, internal calcium/calmodulin (Ca2+/CaM) inhibits CNG channels, thereby having a putative role in sensory adaptation. Functional differences in Ca2+/CaM-dependent inhibition depend on the different subunit composition of olfactory and rod CNG channels. Recent evidence shows that three subunit types (CNGA2, CNGA4, and CNGB1b) make up native olfactory CNG channels and account for the fast inhibition of native channels by Ca2+/CaM. In contrast, two subunit types (CNGA1 and CNGB1) appear sufficient to mirror the native properties of rod CNG channels, including the inhibition by Ca2+/CaM. Within CNG channel tetramers, specific subunit interactions also mediate Ca2+/CaM-dependent inhibition. In olfactory CNGA2 channels, Ca2+/CaM binds to an N-terminal region and disrupts an interaction between the N- and C-terminal regions, causing inhibition. Ca2+/CaM also binds the N-terminal region of CNGB1 subunits and disrupts an intersubunit, N- and C-terminal interaction between CNGB1 and CNGA1 subunits in rod channels. However, the precise N- and C-terminal regions that form these interactions in olfactory channels are different from those in rod channels. Here, we will review recent advances in understanding the subunit composition and the mechanisms and roles for Ca2+/CaM-dependent inhibition in olfactory and rod CNG channels. 相似文献
20.
Fukunaga K Muller D Ohmitsu M Bakó E DePaoli-Roach AA Miyamoto E 《Journal of neurochemistry》2000,74(2):807-817
Using autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) as substrate, we now find that long-term potentian (LTP) induction and maintenance are also associated with a significant decrease in calyculin A-sensitive protein phosphatase (protein phosphatase 2A) activity, without changes in Mg2+-dependent protein phosphatase (protein phosphatase 2C) activity. This decrease in protein phosphatase 2A activity was prevented when LTP induction was inhibited by treatment with calmidazolium or D-2-amino-5-phosphonopentanoic acid. In addition, the application of high-frequency stimulation to 32P-labeled hippocampal slices resulted in increases in the phosphorylation of a 55-kDa protein immunoprecipitated with anti-phosphatase 2A antibodies. Use of a specific antibody revealed that the 55-kDa protein is the B'alpha subunit of protein phosphatase 2A. Following purification of brain protein phosphatase 2A, the B'alpha subunit was phosphorylated by CaM kinase II, an event that led to the reduction of protein phosphatase 2A activity. These results suggest that the decreased activity in protein phosphatase 2A following LTP induction contributes to the maintenance of constitutively active CaM kinase II and to the long-lasting increase in phosphorylation of synaptic components implicated in LTP. 相似文献