Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Purification and characterization of a brain-specific multifunctional calmodulin-dependent protein kinase from rat cerebellum. 130 65

We have previously demonstrated that growth hormone (GH) promotes an increase in tyrosine kinase activity associated with the GH receptor. To gain insight into the role of GH-dependent tyrosine kinase activity in signaling by GH, we investigated the possibility that GH might stimulate MAP kinase, a serine/threonine/tyrosine kinase thought to be a common element in tyrosine kinase-initiated response cascades. Treatment of 3T3-F442A fibroblasts with 100 ng/ml GH results in a 3-6-fold increase in the ability of cell-free extracts to phosphorylate MAP-2 and myelin basic protein. GH-stimulated kinase activity is unaffected by heparin, H7, or cAMP-dependent protein kinase inhibitor peptide, partially reduced by staurosporin and inhibited by fluoride and calcium ions, indicating that the kinase is not protein kinase C or A, casein kinase, or a calcium/calmodulin-dependent protein kinase. Based on gel permeation chromatography, the molecular mass of the GH-stimulated MAP kinase is approximately kDa. Furthermore, anti-phosphotyrosine antibodies revealed the GH-dependent appearance of two phosphotyrosine-containing proteins in cell-free lysates of GH-treated cells that co-migrate with proteins recognized by anti-MAP kinase antibodies. The GH-dependent increase in MAP kinase activity displays a biphasic time course and is dependent on the concentration of GH applied to the cells. GH-dependent MAP kinase activity, partially purified by Mono-Q chromatography, is inactivated by treatment with alkaline phosphatase. Addition of H7 to the cells prior to the addition of GH has no effect, whereas addition of H8 increases MAP kinase activity in control cells with no effect in GH-treated cells, indicating that protein kinase C is unlikely to be an intermediary in the GH-dependent stimulation of MAP kinase activity. These findings indicate that signaling by GH in 3T3-F443A cells may, at least in part, utilize a kinase cascade similar to those that have been proposed for other membrane receptors with associated tyrosine kinase activity.
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PMID:Stimulation by growth hormone of MAP kinase activity in 3T3-F442A fibroblasts. 131 28

Increasing evidence suggests that the postsynaptic density (PSD) plays a critical role in synaptic communication and plasticity. The major PSD protein (mPSDp), a calcium/calmodulin-dependent protein kinase, appears to be central to PSD function. The mPSDp has long been considered identical to the alpha subunit of the soluble calmodulin kinase II (alpha-CKII). However, mPSDp and alpha-CKII do differ in solubility and antigenicity, raising the possibility that the two proteins are distinct. To further define the relationship between the two proteins, we purified the mPSDp to homogeneity from adult rat cerebral cortex and compared the proteins. In contrast to alpha-CKII, the purified mPSDp was insoluble in high concentrations of salt, various detergents, chelators of divalent cations, and the strong denaturant guanidine hydrochloride. The pI value of the mPSDp was 6.2, whereas that of alpha-CKII was 6.7-7.2. The purified mPSDp bound calmodulin in the presence of Ca2+ and was autophosphorylated in a Ca2+/calmodulin-dependent manner. Polyclonal antiserum raised against mPSDp (anti-mPSDp) recognized purified mPSDp or mPSDp in synaptic membrane, indicating immunologic specificity among the synaptic proteins. Anti-mPSDp did not recognize alpha-CKII, whereas anti-alpha-CKII antibodies reacted only weakly with mPSDp, suggesting that the proteins are distinct but structurally similar. Moreover, sequence analysis of protease V8-digested polypeptides revealed that there was at least an 8-amino acid sequence, MLKVPNIS, that is not present in alpha-CKII. Finally, HPLC analysis of V8-digested fragments of mPSDp and alpha-CKII in parallel revealed dissimilar peptide patterns. Thus our observations suggest that mPSDp and alpha-CKII are similar but not identical. The unique physicochemical and structural properties of the mPSDp may provide insights into molecular mechanisms mediating synaptic plasticity.
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PMID:On the identity of the major postsynaptic density protein. 131 76

Transient cerebral ischemia demonstrates an increase in activated oxygen species in the brain that could lead to eventual neuronal cell death. Neuronal cells respond to oxygen free radicals through the restructuring of the cytoskeleton and membranes, mobilization of calcium and gene expression which play a role in cell injury. Ten min of bilateral carotid artery occlusion resulted in a decrease in calcium/calmodulin dependent protein kinase II (CaM kinase II) phosphorylation and activity detected in the brain immediately following ischemia and was partially restored within 24 h of reperfusion. Pretreatment of animals with an anesthetic dose of pentobarbital (40 mg/kg) resulted in partial protection of inactivation of CaM kinase II following ischemia. CaM kinase II activity was maintained following pretreatment of animals with alpha-phenyl N-tert-butyl nitrone (PBN), which traps oxygen free radicals. Infusion of superoxide dismutase or catalase prior to ischemia, blocked CaM kinase II inactivation. Blockage of calcium uptake with bepridil resulted in a marked protection of CaM kinase II inactivation. In addition, trifluoperazine, a calmodulin antagonist also diminished the inhibition of CaM kinase II phosphorylation in our model. These results suggest that ischemia and reperfusion injury results in the generation of activated oxygen and the mobilization of calcium which inactivate CaM kinase II. These results indicate that changes associated with protein kinase activity in the brain following an ischemic insult may have profound effects upon neurodegeneration and neuronal survival.
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PMID:Role of calcium in inactivation of calcium/calmodulin dependent protein kinase II after cerebral ischemia. 133 39

Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by approximately 77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.
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PMID:Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites. 137 33

Calcitonin is a direct inhibitor of osteoclastic activity. Osteoclast retraction is readily induced by calcitonin and it is possible that calcitonin-induced inhibition of bone resorption is in part due to this effect. However, little is known of the mechanisms of this action. In these studies, we have investigated the intracellular signalling pathway of calcitonin-induced osteoclast retraction using cultures of freshly isolated rat osteoclasts. The spread area occupied by single Giemsa-stained rat osteoclasts was measured in vitro by a computer imaging analysis system and used as a quantitative parameter for calculating the degree of osteoclast retraction in response to various agents. Our results show that cAMP may be an important second messenger in the reaction of osteoclasts to calcitonin. Moreover, both protein kinase-A and calcium/calmodulin-dependent protein kinase are involved in the osteoclast retraction induced by this hormone, while cytoskeletal proteins are required for the process to occur.
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PMID:Evidence that protein kinase-A, calcium-calmodulin kinase and cytoskeletal proteins are involved in osteoclast retraction induced by calcitonin. 142 55

We have previously demonstrated that activation of protein kinase C (PKC) by phorbol esters induces selectively IgA synthesis by mouse B cells. In this study, we investigated the effects of a number of protein kinase inhibitors on IgA secretion induced by a recombinant murine IL-5 in LPS-stimulated mouse B cells. The results show that PKC inhibitors, such as sphingosine (SPH), staurosporine (STP) and H-7, blocked IL-5-induced IgA synthesis; the protein kinase A inhibitor HA-1004 and the inhibitor of calcium/calmodulin dependent protein kinase W-7 had no effect on IgA secretion induced by IL-5. The proliferation of the IL-5 sensitive B13 cell line in response to IL-5 was also inhibited by addition of SPH or STP or H-7. The data suggest an involvement of the PKC pathway in IL-5-induced B cell differentiation into IgA secreting cells.
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PMID:IL-5-induced IgA synthesis by LPS-stimulated mouse B cells is prevented by protein kinase C inhibitors. 158 1

The effects of the isoquinolinesulfonamide protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) on CA1 responses in hippocampal slices of the rat were examined to clarify their mode of action, and also to further define the role of Ca(2+) -dependent kinases in long-term potentiation. Initially, the inhibitory potencies of H-7 and HA1004 against both protein kinase C and type II Ca2+/calmodulin-dependent kinase were examined in standard in vitro phosphorylation assays. The apparent Ki values of H-7 and HA1004 for protein kinase C were 9 and 57 microM, respectively. In contrast, the Ki values of H-7 and HA1004 for type II calcium/calmodulin-dependent protein kinase were 156 and 13 microM, respectively. These results indicate that H-7 is a more effective inhibitor of protein kinase C, whereas HA1004 is a more effective inhibitor of type II calcium/calmodulin-dependent protein kinase. Following the induction of long-term potentiation, addition of 50 microM H-7 or HA1004 substantially increased the amplitude of the population spike in a control pathway, while producing no change or a slight increase in the spike amplitude in a previously potentiated long-term potentiation pathway. Moreover, H-7 (50 microM), but not HA1004, produced multiple population spikes in both pathways. Addition of a higher concentration of H-7 (300 microM) reduced the amplitude of the initial population spike but still produced multiple spikes. HA1004 (300 microM) typically produced effects similar to those observed with 50 microM H-7, increasing the amplitude of the control population spike and producing multiple spike activity in both pathways. In contrast to the differential concentration-dependent effects of H-7 on the population spike responses, qualitatively similar effects were observed at both low (50 microM) and high (300 microM) concentrations with regard to synaptic field responses. The initial slope of the population excitatory postsynaptic potential was significantly reduced by H-7, to a similar degree in both pathways. HA1004 produced a modest, but insignificant reduction in both pathways. These results, in conjunction with other reports, suggest that H-7 and HA1004 exert complex concentration-dependent effects with synchronously affect both excitatory and inhibitory synaptic transmission. We hypothesize that reduction of the population excitatory postsynaptic potential and spike (300 microM H-7) is due to reduction of excitatory inputs, whereas enhancement of the population spike amplitude (50 microM H-7) and the production of multiple spikes are due to the reduction of GABA-mediated inhibitory inputs.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Differential effects of isoquinolinesulfonamide protein kinase inhibitors on CA1 responses in hippocampal slices. 165 81

Chicken cardiac C-protein was readily phosphorylated by purified calcium/calmodulin-dependent protein kinase II (CaM-kinase II). Maximum incorporation was about 4 mol of 32P/mol of C-protein subunit. Peptide mapping indicated that some of the sites phosphorylated by CaM-kinase II were located on the same phosphopeptides obtained when C-protein was phosphorylated by the cAMP-dependent protein kinase (peptides T1, T2, and T3). There was a fourth peptide (T3a) which was unique to CaM-kinase II phosphorylation. 32P-Amino acid analysis showed that essentially all of the 32P of peptides T1, T2, and T3a was in phosphoserine. cAMP-dependent protein kinase incorporated 32P only into threonine of peptide T3. Threonine was the preferred site of phosphorylation by CaM-kinase II, but there was significant phosphorylation of a serine in peptide T3. Partially purified C-protein preparations contained an associated calcium/calmodulin-dependent protein kinase. Peptide maps obtained from C-protein phosphorylated by the endogenous kinase were similar to those obtained from C-protein phosphorylated by CaM-kinase II. However, the ratio of phosphothreonine to phosphoserine in peptide T3 was lower. This was due to a contaminating phosphatase in the partially purified C-protein which preferentially dephosphorylated the phosphothreonine of peptide T3. It is suggested that the calcium/calmodulin-dependent protein kinase associated with C-protein is similar or identical to CaM-kinase II and that CaM-kinase II may play a role in the phosphorylation of C-protein in the heart.
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PMID:Phosphorylation of chicken cardiac C-protein by calcium/calmodulin-dependent protein kinase II. 167 69

Recently, a novel type of calcium-dependent protein kinase (CDPK) that requires neither calmodulin nor phospholipids for activation, has been described in plants. We have isolated a cDNA clone for carrot CDPK by probing a library of somatic embryo cDNAs with oligonucleotides corresponding to highly conserved regions of protein kinases. The product of this gene overexpressed in Escherichia coli reacted strongly with monoclonal antibodies to soybean CDPK. The deduced amino acid sequence of carrot CDPK reveals two major functional domains. An N-terminal catalytic domain with greatest homology to calcium/calmodulin-dependent protein kinase type II from rat brain is coupled to a C-terminal calcium-binding domain resembling calmodulin. These features of the primary sequence explain how CDPK binds calcium and suggest a model for CDPK regulation based on similarities to animal calcium/calmodulin-dependent protein kinases.
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PMID:Isolation and sequence analysis of a cDNA clone for a carrot calcium-dependent protein kinase: homology to calcium/calmodulin-dependent protein kinases and to calmodulin. 191 86


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