Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.17 (CaMKII)
4,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Widespread localization, responsiveness to numerous signal transduction systems, and broad substrate specificity enable the multifunctional CaM kinase to mediate regulation of many cellular functions. The abundance and diversity of CaM kinase substrates attest to its role as a multifunctional kinase. However, expanded identification of its in situ substrates as well as the consequences of their regulation by phosphorylation needs to be accomplished. Recently identified substrates have contributed to the list of potential functions for the CaM kinase. CREB is a hormonally stimulated transcriptional activator, and CaM kinase may lie on the pathway to its activation. This pathway could provide an interface between the potentiation of Ca2+ signals by CaM kinase and longer-term modifications of neuronal gene expression. The ryanodine receptor, as well as phospholamban, are involved in cardiac Ca2+ homeostasis, and their regulation by CaM kinase phosphorylation suggests the possibility of some feedback control of intracellular Ca2+ levels by CaM kinase. Regulation of neuronal plasticity by phosphorylation of synapsin I and of postsynaptic substrates necessary for long-term potentiation is another dynamic area of investigation. The study of substrates and their functions promises to continue providing exciting insights into the control of cellular signalling by Ca2+. Molecular cloning has enabled structural comparison of neuronal isoforms of the kinase, and has revealed the existence of closely related subunits. Subunits identified to data differ substantially only in two small variable domains, yet their expression in various tissues and during the course of development is precisely controlled. What unique properties do these small variable domains impart to the different isoforms? What directs high concentrations of kinase to a particular subcellular localization, and especially to the PSD? Further molecular cloning will undoubtedly determine whether other multifunctional CaM kinases with unique structures and properties exist. Finally, studies on the autoregulatory properties of CaM kinase have provided a fascinating picture of how this molecule can alone encode responses to Ca2+ signals, potentiating both the duration and magnitude of its activity. Autophosphorylation of the Thr286 autonomy site both traps calmodulin and permits Ca(2+)-independent activity after calmodulin dissociates. Further analysis of the role of the holoenzyme structure in these modulations will help clarify remaining mechanistic questions. Studies performed during the past few years have clearly established that this Ca(2+)-independent activity is generated in situ in response to a variety of cell stimuli.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Neuronal Ca2+/calmodulin-dependent protein kinases. 132 38

The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)-associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70-80% of the SR-associated proteins present in the tissue. At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser-16 (the residue labelled by cAMP-dependent protein kinase, PK-A) or Thr-17 (the residue phosphorylated by an SR-associated Ca2+/calmodulin-dependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity. A major form of cardiac PP1, present in comparable amounts in both the extract and Triton fraction, was similar, if not identical, to skeletal muscle protein phosphatase 1G (PP1G), which is composed of the PP1 catalytic (C) subunit complexed to a G subunit of approximately 160 kDa, responsible for targeting PP1 to both the SR and glycogen particles of skeletal muscle. This conclusion was based on immunoblotting experiments using antibody to the G subunit, ability to bind to glycogen and the release of PP1 activity from glycogen upon incubation with PK-A and MgATP. PP1 accounted for approximately 90% of the phospholamban (Ser-16 or Thr-17) phosphatase activity in the material sedimented by centrifugation at 45,000 x g, a fraction prepared from cardiac extracts which is enriched in SR membranes. The G subunit in this fraction could be solubilised by Triton X-100, but not with 0.5 M NaCl or digestion with alpha-amylase, indicating that it is bound to membranes and not to glycogen. By analogy with the situation in skeletal muscle, the PK-A catalysed phosphorylation of the G subunit, with ensuing release of the C subunit from the SR, may prevent PP1 from dephosphorylating SR-bound substrates and represent one of the mechanisms by which adrenalin increases the phosphorylation of cardiac phospholamban (Ser-16 and Thr-17) in vivo. Hearts left in situ post mortem lose 85-95% of their PP1 activity within 20-30 min. This remarkable disappearance of PP1 may partly explain why the importance of this enzyme in cardiac muscle metabolism has not been recognized previously.
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PMID:Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban. 184 81

Phospholamban is the major membrane protein of the heart phosphorylated in response to beta-adrenergic stimulation. In cell-free systems, cAMP-dependent protein kinase catalyzes exclusive phosphorylation of serine 16 of phospholamban, whereas Ca2+/calmodulin-dependent protein kinase gives exclusive phosphorylation of threonine 17 (Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341). In this work we have localized the sites of phospholamban phosphorylation in intact ventricles treated with the beta-adrenergic agonist isoproterenol. Isolation of phosphorylated phospholamban from 32P-perfused guinea pig ventricles, followed by partial acid hydrolysis and phosphoamino acid analysis, revealed phosphorylation of both serine and threonine residues. At steady state after isoproterenol exposure, phospholamban contained approximately equimolar amounts of these two phosphoamino acids. Two major tryptic phosphopeptides containing greater than 90% of the incorporated radioactivity were obtained from phospholamban labeled in intact ventricles. The amino acid sequences of these two tryptic peptides corresponded exactly to residues 14-25 and 15-25 of canine cardiac phospholamban, thus localizing the sites of in situ phosphorylation to serine 16 and threonine 17. Phosphorylation of phospholamban at two sites in heart perfused with isoproterenol was supported by detection of 11 distinct mobility forms of the pentameric protein by use of the Western blotting method, consistent with each phospholamban monomer containing two phosphorylation sites, and with each pentamer containing from 0 to 10 incorporated phosphates. Our results localize the sites of in situ phospholamban phosphorylation to serine 16 and threonine 17 and, furthermore, are consistent with the phosphorylations of these 2 residues being catalyzed by cAMP- and Ca2+/calmodulin-dependent protein kinases, respectively.
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PMID:Phospholamban phosphorylation in intact ventricles. Phosphorylation of serine 16 and threonine 17 in response to beta-adrenergic stimulation. 254 95

A monoclonal antibody, A1, was produced against sodium dodecyl sulfate-polyacrylamide gel electrophoresis purified canine phospholamban and isolated from mouse ascites by chromatography on a hydroxylapatite column. Western immunoblotting experiments showed that the antibody was specific for phospholamban and cross-reacted with the protein from a bovine source. Incubation of bovine cardiac sarcoplasmic reticulum (SR) vesicles with the antibody resulted in a marked increase in the ATP-dependent Ca2+ pump activity which was slightly higher than that brought about by cyclic AMP-dependent protein kinase. This observation provides direct proof for the involvement of phospholamban as a SR Ca2+ pump regulatory protein. In addition to stimulating the Ca2+ pump activity, antibody A1 was capable of blocking the phosphorylation of phospholamban by cyclic AMP-dependent protein kinase and by an endogenous SR Ca2+/calmodulin-dependent protein kinase. It was also capable of blocking the dephosphorylation of phosphorylated phospholamban by an endogenous SR protein phosphatase. From these observations, it may be suggested that the antigenic site of A1 antibody is proximal to the phosphorylation sites of phospholamban.
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PMID:Stimulation of bovine cardiac sarcoplasmic reticulum Ca2+ pump and blocking of phospholamban phosphorylation and dephosphorylation by a phospholamban monoclonal antibody. 293 76

Purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles was subjected to proteolysis and peptide mapping to localize the different sites of phosphorylation on the protein and to gain further information on its subunit structure. Five different proteases (trypsin, papain, chymotrypsin, elastase, and Pronase) degraded the oligomeric 27-kDa phosphoprotein into a major 21-22-kDa protease-resistant fragment. No 32P was retained by this protease-resistant fragment, regardless of whether phospholamban had been phosphorylated by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, or protein kinase C. Phosphoamino acid analysis and thin-layer electrophoresis of liberated phosphopeptides revealed that 1 threonine and 2 serine residues were phosphorylated in phospholamban and that 1 of these serine residues and the threonine residue were in close proximity. Only serine was phosphorylated by cAMP-dependent protein kinase, whereas Ca2+-calmodulin-dependent protein kinase phosphorylated exclusively threonine. The results demonstrate that phospholamban has a large protease-resistant domain and a smaller protease-sensitive domain, the latter of which contains all of the sites of phosphorylation. The 21-22-kDa protease-resistant domain, although devoid of incorporated 32P, was completely dissociated into identical lower molecular weight subunits by boiling in sodium dodecyl sulfate, suggesting that this region of the molecule promotes the relatively strong interactions that hold the subunits together. The data presented lend further support for a model of phospholamban structure in which several identical low molecular weight subunits are noncovalently bound to one another, each containing one site of phosphorylation for cAMP-dependent protein kinase and another site of phosphorylation for Ca2+/calmodulin-dependent protein kinase.
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PMID:Proteolytic cleavage of phospholamban purified from canine cardiac sarcoplasmic reticulum vesicles. Generation of a low resolution model of phospholamban structure. 300 93

Phospholamban, the cardiac sarcoplasmic reticulum proteolipid, is phosphorylated by cAMP-dependent protein kinase, by Ca2+/phospholipid-dependent protein kinase, and by an endogenous Ca2+/calmodulin-dependent protein kinase, the identity of which remains to be defined. The aim of this study was therefore to characterize the latter kinase, called phospholamban kinase. Phospholamban kinase was purified approximately 42-fold with a yield of 11%. The purified fraction exhibits a specific activity of 6.5 nmol of phosphate incorporated into exogenous phospholamban per minute per milligram of protein. Phospholamban kinase appears to be a high molecular weight enzyme and presents a broad substrate specificity, synapsin-1, glycogen synthase, and smooth muscle myosin regulatory light chain being the best substrates. Phospholamban kinase phosphorylates synapsin-1 on a Mr 30 000 peptide. The enzyme exhibits an optimum pH of 8.6, a Km for ATP of 9 microM, and a requirement for Mg2+ ions. These data suggest that phospholamban kinase might be an isoenzyme of the multifunctional Ca2+/calmodulin-dependent protein kinase. Consequently we have searched for Mr 50 000-60 000 phosphorylatable subunits among cardiac sarcoplasmic reticulum proteins. A Mr 56 000 protein was found to be phosphorylated in the presence of Ca2+/calmodulin. Such phosphorylation alters the electrophoretic migration velocity of the protein. In addition, this protein that binds calmodulin was always found to be present in fractions containing phospholamban kinase activity. This Mr 56 000 protein is therefore a good candidate for being a subunit of phospholamban kinase. However, the Mr 56 000 calmodulin-binding protein and the Mr 53 000 intrinsic glycoprotein which binds ATP are two distinct entities.
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PMID:Characterization and partial purification of cardiac sarcoplasmic reticulum phospholamban kinase. 373 Mar 67

Phospholamban is a regulatory protein in cardiac sarcoplasmic reticulum that is phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. In this report, we present the partial amino acid sequence of canine cardiac phospholamban and the identification of the sites phosphorylated by these two protein kinases. Gas-phase protein sequencing was used to identify 20 NH2-terminal residues. Overlap peptides produced by trypsin or papain digestion extended the sequence 16 residues to give the following primary structure: Ser-Ala-Ile-Arg-Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-Ala- Arg-Gln-Asn-Leu-Gln-Asn-Leu-Phe-Ile-Asn-Phe-(Cys)-Leu-Ile-Leu-Ile-(Cys)- Leu-Leu-Leu-Ile-. Phospholamban phosphorylated by either cAMP-dependent or Ca2+/calmodulin-dependent protein kinase was cleaved with trypsin, and the major phosphorylated peptide (comprising greater than 70% of the incorporated 32P label) was purified by reverse-phase high performance liquid chromatography. The identical sequence was revealed for the radioactive peptide obtained from phospholamban phosphorylated by either kinase: Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-. The adjacent residues Ser7 and Thr8 of phospholamban were identified as the unique sites phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinases, respectively. These results establish that phospholamban is an oligomer of small, identical polypeptide chains. A hydrophilic, cytoplasmically oriented NH2-terminal domain on each monomer contains the unique, adjacent residues phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. Analysis by hydropathic profiling and secondary structure prediction suggests that phospholamban monomers also contain a hydrophobic domain, which could form amphipathic helices sufficiently long to traverse the sarcoplasmic reticulum membrane. A model of phospholamban as a pentamer is presented in which the amphipathic alpha-helix of each monomer is a subunit of the pentameric membrane-anchored domain, which is comprised of an exterior hydrophobic surface and an interior hydrophilic region containing polar side chains.
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PMID:Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains. 375 68

In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca(2+)-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca(2+)-ATPase. Studies comparing the effects of PKA and CaM kinase on cardiac Ca(2+)-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca(2+)-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 microM) directly to the Ca2+ transport assay medium results in minimal (approximately 112-130% of control) stimulation of Ca2+ uptake activity when the Ca2+ uptake reaction is initiated by the addition or either ATP or Ca2+/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca2+ transport assay medium results in stimulation of Ca2+ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca2+ uptake markedly (215% of control) when the Ca2+ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca2+/EGTA but minimally (130% of control) when the Ca2+ uptake reaction is initiated by the addition of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of the effects of the membrane-associated Ca2+/calmodulin-dependent protein kinase on Ca(2+)-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum. 777 65

Effects of KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine), a specific Ca++/calmodulin (CaM)-dependent protein kinase inhibitor, were examined on the rate of spontaneous beating and the intracellular Ca++ transient of cultured myocytes from fetal mouse ventricle. KN-62 depressed the rate of beating in a dose-dependent fashion. Spontaneous beating ceased 10 min after the administration of 1 microM KN-62 and recovered gradually after washing with cultured medium. Addition of KN-04 [N-(1-1[P-(5-isoquinolinsulfonyl)benzyl]-2-(4- phenylpiperazinyl)ethyl)-5-isoquinolinsulfonamide; 1 microM], an analog of KN-62, did not change the rate of beating. In the experiment using an intracellular Ca++ fluorescence indicator, fluo-3, KN-62 depressed the fluo-3 intensity at a systolic phase. The kinase activity to syntide-2 of Ca++/CaM kinase II purified from the rabbit heart was inhibited by KN-62, but not by KN-04. Addition of KN-62 inhibited the phosphorylation of phospholamban by Ca++/CaM kinase II in a dose-dependent manner. KN-62 depressed the Ca(++)-pumping ATPase activity in the presence of Ca++ and CaM by 32%. These findings indicate that Ca++/CaM kinase II changes an intracellular Ca++ transient and modulates the rate of beating at least in part.
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PMID:KN-62, a specific Ca++/calmodulin-dependent protein kinase inhibitor, reversibly depresses the rate of beating of cultured fetal mouse cardiac myocytes. 793 85

We have demonstrated recently that in cardiac sarcoplasmic reticulum (SR), a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase) phosphorylates and activates the Ca(2+)-pumping ATPase (Ca(2+)-ATPase) in addition to phosphorylating the previously characterized substrates, phospholamban, and Ca2+ release channel (ryanodine receptor) (Xu, A., Hawkins, C., and Narayanan, N. (1993) J. Biol. Chem. 268, 8394-8397). The present study shows that a CaM kinase regulatory system capable of modulating SR Ca2+ pump activity through direct phosphorylation of the Ca(2+)-ATPase is functional in slow twitch but not fast twitch skeletal muscle. Incubation of SR vesicles isolated from rabbit slow twitch (soleus) and fast twitch (adductor magnus) skeletal muscles in the presence of Ca2+ and calmodulin resulted in phosphorylation of the Ca(2+)-ATPase in slow twitch muscle SR but not in fast twitch muscle SR. Exogenous CaM kinase II, which stimulated phosphorylation of the cardiac and slow twitch muscle SR Ca(2+)-ATPase, failed to phosphorylate fast twitch muscle SR Ca(2+)-ATPase. These observations demonstrate that CaM kinase-catalyzed phosphorylation of the Ca2+ pump is isoform-specific since heart and slow twitch muscle express the same Ca(2+)-ATPase isoform (SERCA2a), which is distinct from that of fast twitch muscle (SERCA1). As in the case of cardiac SR Ca(2+)-ATPase, phosphorylation of the slow twitch muscle SR Ca(2+)-ATPase (occurring at a serine residue) resulted in a 2-fold increase in catalytic activity of the enzyme without alteration in its Ca2+ sensitivity. In addition, Ca2+/calmodulin-dependent prephosphorylation of slow twitch muscle SR resulted in a greater than 2-fold increase in its Ca2+ transport activity. In both cardiac and slow twitch muscle SR, phosphorylation of the Ca(2+)-ATPase by the endogenous CaM kinase occurred rapidly (maximum within 2 min at 37 degrees C), had similar pH optimum (8.5-9.0), temperature optimum (30 degrees C), and calmodulin concentration-dependence (k0.5 50-60 nM). cAMP-dependent protein kinase did not phosphorylate the Ca(2+)-ATPase appreciably in either cardiac or slow twitch muscle SR. These findings suggest a muscle-specific role for the membrane-associated CaM kinase in the modulation of Ca2+ uptake and release functions of the SR. In cardiac and slow twitch muscle, phosphorylation of the SR Ca(2+)-ATPase by CaM kinase might provide a novel mechanism for the modulation of the enzymatic and Ca2+ transport functions of this enzyme.
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PMID:Sarcoplasmic reticulum calcium pump in cardiac and slow twitch skeletal muscle but not fast twitch skeletal muscle undergoes phosphorylation by endogenous and exogenous Ca2+/calmodulin-dependent protein kinase. Characterization of optimal conditions for calcium pump phosphorylation. 798 62


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