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)

Smooth muscle myosin light chain kinase (MLC-kinase) was rapidly phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) to a molar stoichiometry of 2.77 +/- 0.15 associated with a threefold increase in the concentration of calmodulin (CaM) required for half-maximal activation of MLC-kinase. Binding of CaM to MLC-kinase markedly reduced the phosphorylation stoichiometry to 0.21 +/- 0.05 and almost completely inhibited phosphorylation of sites in two peptides (32P-peptides P1 and P2) with reduced phosphorylation of peptide P3. By analogy, cAMP-dependent protein kinase phosphorylated MLC-kinase to a stoichiometry of 3.0 or greater in the absence of CaM with about a threefold decrease in the apparent affinity of MLC-kinase for CaM. Binding of CaM to MLC-kinase inhibited the phosphorylation to 0.84 +/- 0.13. Complete tryptic digests contained two major 32P-peptides as reported previously. One of the peptides, whose phosphorylation was inhibited in the presence of excess calmodulin, appeared to be the same as P2. Automated Edman sequence analysis suggested that both CaM-kinase II and cAMP-dependent protein kinase phosphorylated this peptide at the second of the two adjacent serine residues located at the C-terminal boundary of the CaM-binding domain. However, the other peptide phosphorylated by cAMP-dependent protein kinase, regardless of whether CaM was bound, was different from P1 and P3. Thus, MLC-kinase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of CaM-kinase II and is blocked by Ca2+/CaM-binding.
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PMID:Phosphorylation of smooth muscle myosin light chain kinase by Ca2+/calmodulin-dependent protein kinase II: comparative study of the phosphorylation sites. 215 62

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) that is transiently expressed in COS-7 cells is essentially inactive when assayed without Ca2+. Physiological activation of the kinase occurs by binding of Ca2+/calmodulin near a putative autoinhibitory subdomain that contains the sequence His282-Arg-Gln-Glu-Thr286. We have markedly increased the Ca2(+)-independent activity of CaM kinase by altering the charge of this sequence by site-directed mutagenesis. The mutant containing Asp282-Gly-Glu-Glu-Thr286 is 67% Ca2+ independent. We also mimicked the effect of autophosphorylation at Thr286 by the mutant containing His282-Arg-Gln-Glu-Asp286, which is 36% Ca2+ independent. In addition to delineating the autoinhibitory domain by use of mutations that disable it, these constructs are of immediate practical value for simulating CaM kinase action in vivo without elevating Ca2+. To this end, we show that nuclear microinjection of cDNA of a constitutive mutant, but not of the wild-type kinase, initiates maturation of Xenopus oocytes.
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PMID:Multifunctional Ca2+/calmodulin-dependent protein kinase made Ca2+ independent for functional studies. 215 11

Calcium, adenosine 3',5'-cyclic monophosphate (cAMP), and guanosine 3',5'-cyclic monophosphate (cGMP) can regulate the same or different ion transport processes within an epithelium, presumably via independent protein phosphorylation mechanisms. Because there have been few detailed studies characterizing these processes in epithelia, we examined the distribution of Ca-, cAMP-, and cGMP-specific protein kinases and substrates in vitro in a homogenous salt-absorbing epithelium, the winter flounder intestine. In this tissue cGMP and Ca inhibit Na-K-2Cl cotransport, cAMP increases anion permeability, and phorbol esters do not affect ion transport. The Ca-specific kinases are calmodulin (CaM) dependent. The tissue possesses type III Ca-CaM protein kinase and its specific substrate elongation factor 2 and type II but not type I Ca-CaM kinase. Addition of phosphatidylserine (PS) and Ca to crude or DEAE-cellulose-purified cytosol neither increased the phosphorylation of exogenous histone H1 substrate nor that of any endogenous substrates. Although these suggest the absence of Ca-phospholipid-dependent kinase (PKC), the cytosol has a 78-kDa protein recognizable by a highly specific polyclonal sheep antibody to rat brain PKC. Both the particulate and cytosolic fractions possess cAMP-specific binding proteins and cAMP-specific phosphoprotein substrates. The particulate fraction cAMP-binding proteins are of molecular mass 50 kDa (pI 5.2) and 48 kDa with multiple isoforms (pI 5.6-6.2); these proteins generate different peptide maps. The cytosol chiefly contains a 50-kDa (pI 5.2) cAMP binding protein that is similar to the particulate 50-kDa protein on peptide mapping. The flounder cAMP binding proteins have the same pI but lower molecular mass and different peptide profiles than the rat brain RII (54/52 kDa) and RI (50 kDa) cAMP regulatory proteins. The cGMP-specific protein kinase was less prominent, very low levels of cGMP-specific binding proteins being detected either by equilibrium binding or by photoaffinity labeling. A prominent kinase substrate in homogenates is a 50-kDa protein, the phosphorylation of which is increased by Ca and cGMP but decreased by cAMP. When intact tissue was prelabeled with 32Pi and then exposed to cGMP, the phosphorylation of a number of substrates including that of a 50-kDa protein was increased. In summary, the flounder intestine possesses the necessary protein phosphorylation mechanisms to account for the regulation of its ion transport processes by second messengers.
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PMID:Second messenger-specific protein kinases in a salt-absorbing intestinal epithelium. 215 31

To evaluate the role of domain I of calmodulin (CaM) in the activation of target enzymes, a series of CaM mutants was constructed in which domain I (49 amino acids) was substantially deleted, or was exchanged with the homologous region (58 amino acids) of cardiac troponin C (cTnC). The proteins are 1) aM, a mutant CaM in which domain I has been deleted; 2) TaM, first domain of cTnC, last three domains of CaM; 3) TaM-BMI, same as TaM, except the nonfunctional first Ca2(+)-binding domain has been restored by mutagenesis; 4) CaT, first domain of CaM, last three domains of cTnC. These proteins were evaluated for Ca2+ binding properties and as activators of three CaM target enzymes, CaM-dependent phosphodiesterase (PDE), smooth muscle myosin light chain kinase (MLCK), and CaM-dependent multifunctional protein kinase (CaM kinase II). The chimeric proteins containing four domains bound Ca2+ in the manner expected from the number and nature of EF hands. In contrast, aM bound only two Ca2+, suggesting that deletion of domain I may have disrupted binding in one of the remaining three domains, and did not activate the three enzymes. The kinetics of activation of PDE by CaM, TaM, and TaM-BMI were identical. Although cTnC and CaT could maximally activate PDE, the Kact for these mutants were greater than 2000 times than for CaM. All mutated proteins except CaT were poor activators of CaM kinase II and this protein activated the kinase to 65% that of CaM, with a nearly identical Kact. CaT and TaM, were poor agonists of MLCK. Activation of Ca2(+)-binding site I in TaM (TaM-BMI), completely prevented activation of MLCK. In addition, TaM-BMI was a potent competitive inhibitor of MLCK activation by CaM (Ki = 66 nM). We conclude 1) a domain I is necessary to activate these target enzymes, and the substitution of the corresponding region of cTnC into CaM leads to differential effects; 2) an active first Ca2(+)-binding site is not essential for activation of PDE and the primary sequence of the first domain of CaM need not be highly conserved; 3) for CaM kinase II, determinants in the first domain are critical whereas more flexibility exists for the remaining three domains; 4) since TaM-BMI acts as a potent competitive inhibitor of MLCK binding of CaM to a target enzyme and activation can be dissociable events.
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PMID:Chimeric calmodulin-cardiac troponin C proteins differentially activate calmodulin target enzymes. 216 Sep 66

Calponin isolated from chicken gizzard smooth muscle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system composed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phosphorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phosphorylation was unaffected. Furthermore, calponin inhibited the actin-activated MgATPase of fully phosphorylated or thiophosphorylated myosin. Although calponin is a Ca2(+)-binding protein, inhibition did not require Ca2+. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not dependent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, but not by cAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein retained calmodulin and tropomyosin binding capabilities, but actin binding was greatly reduced. The calponin-actin interaction, therefore, appears to be responsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be involved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Calponin function in turn is regulated by Ca2(+)-dependent phosphorylation.
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PMID:Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation. 216 34

Oligonucleotide DNA probes were used to determine the distribution of mRNAs encoding the alpha- and beta-subunits of Ca2+/calmodulin-dependent protein kinase type II (CaM-KII) in developing rat brain. The regional and temporal distribution of these mRNAs closely paralleled the distribution and developmental appearance previously reported for their respective protein subunits. alpha-Subunit mRNA was barely detectable in sagittal sections at 4 d postnatal but increased as much as 10-fold in frontal cortex by day 16. beta-Subunit mRNA, on the other hand, was readily detected at 4 d postnatal and changed only slightly during development. Telencephalic structures exhibited the highest levels of CaM-KII mRNA and the brain stem displayed the least. alpha-Subunit mRNA was not observed in cerebellar granule cells and was barely detectable in Purkinje cells, while the beta-mRNA was easily detected in both neuronal types. mRNAs for both alpha- and beta-subunits were present in many neuronal cell bodies; however, only the alpha-subunit mRNA was localized to molecular layers of the hippocampus and lamina I of the frontal cortex. These layers of neuropil are relatively cell sparse and contain extensive dendritic arborizations and synaptic contacts. Since polyribosomes have been observed near hippocampal dendritic spines, the localization of alpha-subunit mRNA to dendrites of pyramidal and dentate granule cells suggests that this subunit is synthesized in situ at postsynaptic sites. The co-localization of translational machinery and high concentrations of CaM-KII in postsynaptic elements suggests an important relationship between alpha-subunit synthesis and the maintenance and plasticity of postsynaptic structures.
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PMID:In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain. 216 85

It is now well established that autophosphorylation of a threonine residue located next to each calmodulin-binding domain in the subunits of type II Ca2+/calmodulin-dependent protein kinase causes the kinase to remain active, although at a reduced rate, after Ca2+ is removed from the reaction. This autophosphorylated form of the kinase is still sensitive to Ca2+/calmodulin, which is required for a maximum catalytic rate. After removal of Ca2+, new sites are autophosphorylated by the partially active kinase. Autophosphorylation of these sites abolishes sensitivity of the kinase to Ca2+/calmodulin (Hashimoto, Y., Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1987) J. Biol. Chem. 262, 8051-8055). We have identified two pairs of homologous residues, Thr305 and Ser314 in the alpha subunit and Thr306 and Ser315 in the beta subunit, that are autophosphorylated only after removal of Ca2+ from an autophosphorylation reaction. The sites were identified by direct sequencing of labeled tryptic phosphopeptides isolated by reverse-phase high pressure liquid chromatography. Thr305-306 is rapidly dephosphorylated by purified protein phosphatases 1 and 2A, whereas Ser314-315 is resistant to dephosphorylation. We have shown by selective dephosphorylation that the presence of phosphate on Thr305-306 blocks sensitivity of the kinase to Ca2+/calmodulin. In contrast, the presence of phosphate on Ser314-315 is associated with an increase in the Kact for Ca2+/calmodulin of only about 2-fold, producing a relatively small decrease in sensitivity to Ca2+/calmodulin.
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PMID:Activation of type II calcium/calmodulin-dependent protein kinase by Ca2+/calmodulin is inhibited by autophosphorylation of threonine within the calmodulin-binding domain. 216 38

The relationship of the kinase which co-purifies with caldesmon to Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) was investigated by studying the phosphorylation of bovine brain synapsin I, as well-characterized substrate of CaM-kinase II. Synapsin I is a very good substrate (Km = 90 nM) of the co-purifying kinase, which phosphorylates two sites in synapsin I, both of which are distinct from the single site phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of synapsin I is Ca2(+)- and calmodulin-dependent: half-maximal activation occurs at 0.13 microM-Ca2+ and maximal activity at 0.4 microM-Ca2+. Phosphorylation of the co-purifying kinase slightly enhances the rate, but does not alter the stoichiometry, of subsequent synapsin I phosphorylation; it does, however, circumvent the requirement for Ca2+ and calmodulin. The properties of this kinase therefore closely resemble those of CaM-kinase II, and we conclude that it is probably a smooth-muscle isoenzyme of CaM-kinase II.
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PMID:Kinase activity associated with caldesmon is Ca2+/calmodulin-dependent kinase II. 216 10

Two cDNAs, one containing the entire coding region of alpha subunit of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and the other containing only its protein kinase domain, were separately ligated into the bacterial expression vector pET3a and expressed in Escherichia coli. The activity of the recombinant alpha subunit protein was dependent on Ca2+/calmodulin, whereas the activity of the recombinant protein containing only the protein kinase domain (recombinant alpha-I protein) was absolutely independent of Ca2+/calmodulin. These proteins showed similar enzymatic properties to brain CaM kinase II with some minor differences. These results directly demonstrated that the protein kinase domain alone without the rest of the subunit was sufficient to exhibit its activity.
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PMID:Expression of a catalytically active polypeptide of calmodulin-dependent protein kinase II alpha subunit in Escherichia coli. 216 3

The distribution of type II calmodulin kinase (CaM kinase) immunoreactivity was studied in control and septally kindled rat brains. CaM kinase was concentrated in limbic structures, such as the hippocampus, lateral septum and amygdala. Within the hippocampus, the molecular layer of the endal limb of the dentate gyrus, the stratum radiatum, and lacunosum moleculare of CA1 were the most heavily stained regions. The cerebellum was stained only in the molecular and Purkinje cell layers, and very low amounts of immunoreactive protein were present in the brainstem and white matter. Kindling resulted in a significant decrease in CaM kinase immunoreactivity in CA3 and in the dentate of the ventral hippocampus but not in the lateral septum. These data suggest that kindling decreases the number of CaM kinase molecules or alters its antigenic distribution, and provides further evidence that alterations of this enzyme may be important in the kindling phenomenon.
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PMID:Kindling induced changes in calmodulin kinase II immunoreactivity. 216 28


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