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1.
Charles M. Schworer Thomas R. Soderling 《Biochemical and biophysical research communications》1983,116(2):412-416
A number of proteins were tested as potential substrates for purified rabbit liver calmodulin-dependent glycogen synthase kinase. It was found that liver phenylalanine hydroxylase and several brain proteins including tyrosine hydroxylase, microtubule-associated protein 2, and synapsin I were readily phosphorylated. Brain tubulin was very poorly phosphorylated. These results suggest that calmodulin-dependent glycogen synthase kinase may be a more general protein kinase involved in the regulation of several cellular Ca2+-dependent functions. 相似文献
2.
Calcium/calmodulin-dependent protein kinase II (CaMKII), a major component of the postsynaptic density (PSD) of excitatory synapses, plays a key role in the regulation of synaptic function in the mammalian brain. Although many postsynaptic substrates for CaMKII have been characterized in vitro, relatively little is known about their phosphorylation in vivo. By tagging synaptic proteins with a peptide substrate specific for CaMKII and expressing them in cultured neurons, we have visualized substrate phosphorylation by CaMKII at intact synapses. All substrates tested were strongly phosphorylated by CaMKII in HEK293 cells. However, activity-dependent phosphorylation of substrates at synapses was highly selective in that the glutamate receptor subunits NR2B and GluR1 were poorly phosphorylated whereas PSD-95 and Stargazin, proteins implicated in the scaffolding and trafficking of AMPA receptors, were robustly phosphorylated. Phosphatase activity limited phosphorylation of Stargazin but not NR2B and GluR1. These results suggest that the unique molecular architecture of the PSD results in highly selective substrate discrimination by CaMKII. 相似文献
3.
Protein kinase C (PKC) exhibits both negative and positive cross-talk with multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in PC12 cells. PKC effects negative cross-talk by inhibiting the mobilization of intracellular Ca2+ stores and by inhibiting Ca2+ influx through voltage-sensitive Ca2+ channels. In the absence of cross-talk, Ca2+ influx induced by depolarization with 56 mM K+ stimulates CaM kinase and its autophosphorylation and converts up to 50% of the enzyme to a Ca(2+)-independent or autonomous species. Acute treatment with phorbol myristate acetate (PMA) elicits a parallel reduction in depolarization-induced Ca2+ influx and in generation of autonomous CaM kinase. Negative cross-talk also occurs during stimulation of the phosphatidylinositol signaling system with bradykinin, which activates both PKC and CaM kinase. The extent of CaM kinase activation is attenuated by the simultaneous activation of PKC; it is enhanced by prior down-regulation of PKC. PKC also exhibits positive cross-talk with CaM kinase. Submaximal activation of CaM kinase by ionomycin is potentiated by concurrent activation of PKC with PMA. Such PMA treatment is found to increase the level of cytosolic calmodulin. Enhanced activation of CaM kinase by PKC may result from PKC-mediated phosphorylation of calmodulin-binding proteins, such as neuromodulin and MARCKS, and the subsequent increase in the availability of previously bound calmodulin for activation of CaM kinase. 相似文献
4.
5.
Purification and properties of a multifunctional calcium/calmodulin-dependent protein kinase from rat pancreas 总被引:3,自引:0,他引:3
J A Cohn B Kinder J D Jamieson N G Delahunt F S Gorelick 《Biochimica et biophysica acta》1987,928(3):320-331
A calcium/calmodulin-dependent protein kinase (Ca/calmodulin protein kinase) was purified from rat pancreas using hydrophobic chromatography followed by gel filtration and affinity chromatography. Ca/calmodulin protein kinase from pancreas resembled previously described multifunctional Ca/calmodulin protein kinases from other tissues with respect to substrate specificity, autophosphorylation on serine and threonine residues, and catalytic and hydrodynamic properties. While Ca/calmodulin protein kinase from other tissues contains subunits of 53-60 kDa with variable proportions of a smaller 50-52 kDa subunit, pancreatic Ca/calmodulin protein kinase was found to contain a single component of 51 kDa. Experiments mixing brain Ca/calmodulin protein kinase with pancreatic homogenate suggest that the absence of a larger subunit in the pancreatic Ca/calmodulin protein kinase is not due to proteolytic degradation during enzyme preparation. Ca/calmodulin protein kinase binding to 125I-labeled calmodulin in solution was demonstrated using the photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azidobenzoate. 125I-labeled calmodulin binding to Ca/calmodulin protein kinase was also demonstrated using filters containing Ca/calmodulin protein kinase transferred from polyacrylamide gels after two-dimensional gel electrophoresis. Finally, the ribosomal substrate for Ca/calmodulin protein kinase was identified as the ribosomal protein, S6. The purification procedure presented in this study promises to be useful in characterizing Ca/calmodulin protein kinase in other tissues and in clarifying the role of these enzymes in cellular function. 相似文献
6.
Mechanism of autophosphorylation of the multifunctional Ca2+/calmodulin-dependent protein kinase 总被引:10,自引:0,他引:10
The multifunctional Ca2+/calmodulin-dependent protein kinase purified from rat brain cytosol undergoes a self-phosphorylation or autophosphorylation reaction. Our conclusion that this reaction is autocatalytic is based on the following lines of evidence: The autophosphorylation reaction and the protein kinase activity toward other substrates are absolutely dependent on the presence of both Ca2+ and calmodulin; autophosphorylation and phosvitin kinase activity show a similar time course and indistinguishable heat lability; the reaction is a consistent property of every preparation of rat brain kinase; the reaction is present in both crude and highly purified preparations of similar kinases or isozymes from rat lung, spleen, heart, bovine brain, and a neuronal tissue from Aplysia californica, a marine mollusk; phosphorylation of the kinase subunits is not mimicked by addition of cAMP, cGMP, Ca2+ plus diglyceride, or addition of the cAMP-dependent protein kinase, and is not blocked by the heat-stable inhibitor protein of the cAMP-dependent protein kinase; and the reaction is intramolecular. Autophosphorylation results in the stoichiometric incorporation of phosphate into both the 51,000- and 60,000-dalton subunits. 相似文献
7.
Purification and characterization of a brain-specific multifunctional calmodulin-dependent protein kinase from rat cerebellum. 总被引:10,自引:0,他引:10
A brain-specific multifunctional calmodulin-dependent protein kinase, calmodulin-dependent protein kinase IV, which exhibited characteristic properties quite different from those of calmodulin-dependent protein kinase II, was purified approximately 230-fold from rat cerebellum. The purified preparation gave two protein bands with molecular weights of 63,000 (alpha) and 66,000 (beta) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both of which showed protein kinase activity as examined by the activity gel method. The molecular weight of the enzyme was estimated as about 67,000 from sedimentation coefficient (3.2 S) and Stokes radius (50 A), indicating a monomeric structure of the enzyme. The enzyme phosphorylated smooth muscle myosin light chain, synapsin I, microtubule-associated protein 2, tau protein, myelin basic protein, histone H1, and tyrosine hydroxylase in a Ca2+/calmodulin dependent manner, suggesting that the enzyme is a multifunctional calmodulin-dependent protein kinase capable of phosphorylating a large number of substrates. A synthetic peptide, Lys-Ser-Asp-Gly-Gly-Val-Lys-Lys-Arg-Lys-Ser-Ser-Ser-Ser, was found to be a specific substrate for this kinase and, using this peptide as substrate, the distribution of the enzyme activity in various rat tissues was examined. The activity was found in cerebral cortex, brain stem, and cerebellum, most abundantly in cerebellum, but other tissues tested, including liver, spleen, kidney, lung, heart, skeletal muscle, and adrenal gland showed very little activity. 相似文献
8.
A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene. 相似文献
9.
Beullens M Vancauwenbergh S Morrice N Derua R Ceulemans H Waelkens E Bollen M 《The Journal of biological chemistry》2005,280(48):40003-40011
Maternal embryonic leucine zipper kinase (MELK) is a protein Ser/Thr kinase that has been implicated in stem cell renewal, cell cycle progression, and pre-mRNA splicing, but its substrates and regulation are not yet known. We show here that MELK has a rather broad substrate specificity and does not appear to require a specific sequence surrounding its (auto)phosphorylation sites. We have mapped no less than 16 autophosphorylation sites including serines, threonines, and a tyrosine residue and show that the phosphorylation of Thr167 and Ser171 is required for the activation of MELK. The expression of MELK activity also requires reducing agents such as dithiothreitol or reduced glutathione. Furthermore, we show that MELK is a Ca2+-binding protein and is inhibited by physiological Ca2+ concentrations. The smallest MELK fragment that was still catalytically active comprises the N-terminal catalytic domain and the flanking ubiquitin-associated domain. A C-terminal fragment of MELK functions as an autoinhibitory domain. Our data show that the activity of MELK is regulated in a complex manner and offer new perspectives for the further elucidation of its biological function. 相似文献
10.
Purification and characterization of calmodulin-dependent multifunctional protein kinase from smooth muscle: isolation of caldesmon kinase 总被引:1,自引:0,他引:1
Previously, it was reported that smooth muscle caldesmon is a protein kinase and is autophosphorylated [Scott-Woo, G.C., & Walsh, M.P. (1988) Biochem. J. 252, 463-472]. We separated a Ca2+/calmodulin-dependent protein kinase from caldesmon in the presence of 15 mM MgCl2. The Ca2+/calmodulin-dependent caldesmon kinase was purified by using a series of liquid chromatography steps and was characterized. The subunit molecular weight (MW) of the kinase was 56K by SDS gel electrophoresis and was autophosphorylated. After the autophosphorylation, the kinase became active even in the absence of Ca2+/calmodulin. The substrate specificity of caldesmon kinase was similar to the rat brain calmodulin-dependent multifunctional protein kinase II (CaM PK-II) and phosphorylated brain synapsin and smooth muscle 20-kDa myosin light chain. The purified kinase bound to caldesmon, and the binding was abolished in the presence of high MgCl2. Enzymological parameters were measured for smooth muscle caldesmon kinase, and these were KCaM = 32 nM, KATP = 12 microM, Kcaldesmon = 4.9 microM, and KMg2+ = 1.1 mM. Optimum pH was 7.5-9.5. The observed properties were similar to brain CaM PK-II, and, therefore, it was concluded that smooth muscle caldesmon kinase is the isozyme of CaM PK-II in smooth muscle. 相似文献
11.
Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation 总被引:15,自引:0,他引:15
Autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase converts it from a Ca2(+)-dependent to a Ca2(+)-independent or autonomous kinase, a process that may underlie some long-term enhancement of transient Ca2+ signals. We demonstrate that the neuronal alpha subunit clone expressed in COS-7 cells (alpha-CaM kinase) is sufficient to encode the regulatory phenomena characteristic of the multisubunit kinase isolated from brain. Activity of alpha-CaM kinase is highly dependent on Ca2+/calmodulin. It is converted by autophosphorylation to an enzyme capable of Ca2(+)-independent (autonomous) substrate phosphorylation and autophosphorylation. Using site-directed mutagenesis, we separately eliminate five putative autophosphorylation sites within the regulatory domain and directly examine their individual roles. Ca2+/calmodulin-dependent kinase activity is fully retained by each mutant, but Thr286 is unique among the sites in being indispensable for generation of an autonomous kinase. 相似文献
12.
Ishida A Shigeri Y Tatsu Y Endo Y Kameshita I Okuno S Kitani T Takeuchi M Yumoto N Fujisawa H 《Journal of biochemistry》2001,129(5):745-753
Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases. In order to elucidate the mechanism of substrate recognition by CaMKPase, we chemically synthesized a variety of phosphopeptide analogs and carried out kinetic analysis using them as CaMKPase substrates. This is the first report using systematically synthesized phosphopeptides as substrates for kinetic studies on substrate specificities of protein Ser/Thr phosphatases. CaMKPase was shown to be a protein Ser/Thr phosphatase having a strong preference for a phospho-Thr residue. A Pro residue adjacent to the dephosphorylation site on the C-terminal side and acidic clusters around the dephosphorylation site had detrimental effects on dephosphorylation by CaMKPase. Deletion analysis of a model substrate peptide revealed that the minimal length of the substrate peptide was only 2 to 3 amino acid residues including the dephosphorylation site. The residues on the C-terminal side of the dephosphorylation site were not essential for dephosphorylation, whereas the residue adjacent to the dephosphorylation site on the N-terminal side was essential. Ala-scanning analysis suggested that CaMKPase did not recognize a specific motif around the dephosphorylation site. Myosin light chain phosphorylated by protein kinase C and Erk2 phosphorylated by MEK1 were poor substrates for CaMKPase, while a synthetic phosphopeptide corresponding to the sequence around the phosphorylation site of the former was not dephosphorylated by CaMKPase but that of the latter was fairly good substrate. These data suggest that substrate specificity of CaMKPase is determined by higher-order structure of the substrate protein rather than by the primary structure around its dephosphorylation site. Use of phosphopeptide substrates also revealed that poly-L-lysine, an activator for CaMKPase, activated the enzyme mainly through increase in the V(max) values. 相似文献
13.
Regulation of human CLC-3 channels by multifunctional Ca2+/calmodulin-dependent protein kinase 总被引:12,自引:0,他引:12
Huang P Liu J Di A Robinson NC Musch MW Kaetzel MA Nelson DJ 《The Journal of biological chemistry》2001,276(23):20093-20100
The multifunctional calcium/calmodulin-dependent protein kinase II, CaMKII, has been shown to regulate chloride movement and cellular function in both excitable and non-excitable cells. We show that the plasma membrane expression of a member of the ClC family of Cl(-) channels, human CLC-3 (hCLC-3), a 90-kDa protein, is regulated by CaMKII. We cloned the full-length hCLC-3 gene from the human colonic tumor cell line T84, previously shown to express a CaMKII-activated Cl(-) conductance (I(Cl,CaMKII)), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of I(Cl,CaMKII). Biotinylation experiments demonstrated plasma membrane expression of hCLC-3 in the stably transfected cells. In whole cell patch clamp experiments, autonomously active CaMKII was introduced into tsA cells stably transfected with hCLC-3 via the patch pipette. Cells transfected with the hCLC-3 gene showed a 22-fold increase in current density over cells expressing the vector alone. Kinase-dependent current expression was abolished in the presence of the autocamtide-2-related inhibitory peptide, a specific inhibitor of CaMKII. A mutation of glycine 280 to glutamic acid in the conserved motif in the putative pore region of the channel changed anion selectivity from I(-) > Cl(-) to Cl(-) > I(-). These results indicate that hCLC-3 encodes a Cl(-) channel that is regulated by CaMKII-dependent phosphorylation. 相似文献
14.
Substrate specificity of rat brain calcium-activated and phospholipid-dependent protein kinase 总被引:3,自引:0,他引:3
K F Chan G L Stoner G A Hashim K P Huang 《Biochemical and biophysical research communications》1986,134(3):1358-1364
A synthetic peptide ArgThrProProProSerGly with sequence similar to the threonine sites of phosphorylation in both myelin basic protein and simian virus 40 T antigen could be phosphorylated in vitro by a purified rat brain Ca2+-activated and phospholipid-dependent protein kinase, protein kinase C. The apparent Km and Vm values of this heptapeptide for the enzyme were determined to be 240 microM and 60 nmol/min/mg, respectively. Up to 0.8 mol 32P could be incorporated into the peptide, mainly at the threonine residue. Substitution of the L-threonine residue in the heptapeptide by its D-enantiomer abolished the phosphorylatability of the peptide by protein kinase C. However, this (D)Thr-containing peptide could act as a competitive inhibitor for the kinase with an apparent Ki value of approximately 320 microM. These findings suggest that a triprolyl sequence may act as a recognition site for protein kinase C. 相似文献
15.
The Chk1 protein kinase plays a critical role in a DNA damage checkpoint pathway conserved between fission yeast and animals. We have developed a quantitative assay for Chk1 activity, using a peptide derived from a region of Xenopus Cdc25C containing Ser-287, a known target of Chk1. Variants of this peptide were used to determine the residues involved in substrate recognition by Chk1, revealing the phosphorylation motif Phi-X-beta-X-X-(S/T)*, where * indicates the phosphorylated residue, Phi is a hydrophobic residue (M>I>L>V), beta is a basic residue (R>K) and X is any amino acid. This motif suggests that Chk1 is a member of a group of stress-response protein kinases which phosphorylate target proteins with related specificities. 相似文献
16.
Phosphorylation of smooth myosin light chain kinase by smooth muscle Ca2+/calmodulin-dependent multifunctional protein kinase 总被引:2,自引:0,他引:2
Smooth muscle myosin light chain kinase (MLC kinase) was phosphorylated by smooth muscle calmodulin-dependent protein kinase II (CaM protein kinase II). When MLC kinase was free from calmodulin, two sites were phosphorylated. The phosphorylation at the one site was much faster than the other site; however, the phosphorylation at the first site was completely blocked by calmodulin binding to MLC kinase. Phosphorylation of MLC kinase by CaM protein kinase II increased the dissociation constant of MLC kinase for calmodulin about 10 times without changing the Vmax. The location of the phosphorylation sites was identified by isolating and sequencing the tryptic phosphopeptides of MLC kinase. The preferred site was identified as serine 512 and the second site as serine 525. These sites are the same as the sites phosphorylated by cAMP-dependent protein kinase. 相似文献
17.
Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells. 总被引:3,自引:0,他引:3
We report that the rat pituitary cell line GH3 contains a Ca2(+)- and calmodulin-dependent protein kinase with properties characteristic of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) from rat brain. The GH3 kinase exhibits the hallmark of authentic CaM kinase: conversion from Ca2(+)-dependent to Ca2(+)-independent activity following a brief initial phosphorylation in vitro. This phosphorylation occurs at a site which is similar or identical to that of the "autonomy" site of the rat brain enzyme and thus may be an autophosphorylation event. GH3 CaM kinase is phosphorylated and becomes Ca2(+)-independent in situ. Depolarization of intact cells with K+ opens calcium channels and leads to the phosphorylation of CaM kinase at the autonomy site, and the kinase becomes significantly and persistently Ca2(+)-independent. Treatment of cells with thyrotropin-releasing hormone (TRH), which activates the phosphatidylinositol signaling pathway, also generates a Ca2(+)-independent CaM kinase in situ. The primary effect of TRH on CaM kinase activity is transient and correlates with the spike of Ca2+ released from intracellular stores and the rapid phase of prolactin release from GH3 cells. This study demonstrates that CaM kinase is able to detect and respond to both calcium that enters the cell through voltage-sensitive Ca2+ channels and calcium released from internal stores via the phosphatidylinositol pathway. We find that TRH, a hormone that causes release of prolactin and was previously believed to activate primarily protein kinase C, also significantly activates CaM kinase in intact cells. 相似文献
18.
19.
Initial autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) occurs at Thr286 (the "autonomy" site) and converts the kinase from a Ca(2+)-dependent to a partially Ca(2+)-independent or autonomous enzyme. After removal of Ca2+/calmodulin, the autonomous kinase undergoes a "burst" of inhibitory autophosphorylation at sites distinct from the autonomy site which may be masked in the presence of bound calmodulin. This burst of Ca(2+)-independent autophosphorylation blocks the ability of calmodulin to activate the kinase. We have used site-directed mutagenesis to replace putative inhibitory autophosphorylation sites within the calmodulin binding domain of recombinant alpha-CaM kinase with nonphosphorylatable alanines and examined the effects on autophosphorylation, kinase activity, and calmodulin binding. Although prominent Ca(2+)-independent autophosphorylation occurs within the calmodulin binding domain at Thr305, Thr306, and Ser314 in wild-type alpha-CaM kinase, the inhibitory effect on kinase activity and calmodulin binding is retained in mutants lacking any one of these three sites. However, when both Thr305 and Thr306 are converted to alanines the kinase does not display inhibition of either activity or calmodulin binding. Autophosphorylation at either Thr305 or Thr306 is therefore sufficient to block both binding and activation of the kinase by Ca2+/calmodulin. Thr306 is also slowly autophosphorylated in a basal reaction in the continuous absence of Ca2+/calmodulin. Autophosphorylation of Thr306 by the kinase in either its basal or autonomous state suggests that in the absence of bound calmodulin, the region of the autoregulatory domain surrounding Thr306, rather than the region near the autonomy site, lies nearest the peptide substrate binding site of the kinase. 相似文献
20.
Phosphorylation of tubulin by a calmodulin-dependent protein kinase 总被引:16,自引:0,他引:16
F Wandosell L Serrano M A Hernández J Avila 《The Journal of biological chemistry》1986,261(22):10332-10339
Calmodulin-dependent protein kinase was purified from porcine brain cytosol through sequential steps involving acid precipitation, DEAE-chromatography, and calmodulin-Sepharose chromatography. The purified enzyme contained a major Mr 50,000 and a minor Mr 60,000 peptide. Porcine brain tubulin was a major substrate for this kinase. Under optimal conditions 2.6 mol of phosphate were incorporated per mol of tubulin. The kinase phosphorylated both tubulin subunits at their carboxyl-terminal region. Limited proteolysis, using trypsin and chymotrypsin, of phosphorylated and unphosphorylated tubulins resulted in different cleavage patterns as determined by peptide mapping. Phosphorylated tubulin was unable to bind to microtubule-associated protein or to polymerize, but regained its assembly capacity after phosphatase treatment. 相似文献