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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The microtubule array in neuronal cells undergoes extensive growth, dynamics and rearrangements during neurite outgrowth. While little is known about how these changes are regulated, microtubule-associated proteins (MAPs) including tau protein are likely to perform an important role. Tau is one of the MAPs in mammalian brain. When isolated it is usually a mixture of several isoforms containing between 341 and 441 residues that arise from alternative splicing. Tau can be phosphorylated by several protein kinases. Phosphorylation at certain sites results in major structural and functional changes, as seen by changes in electrophoretic mobility, interaction with microtubules, molecular length and elasticity. Here we show that the sites of phosphorylation by four kinases (PKA, PKC, CK and CaMK) all lie in the C-terminal microtubule-binding half of tau, but only the phosphorylation by CaM kinase shows the pronounced shift in electrophoretic mobility characteristic for tau from Alzheimer neurofibrillary tangles. By using a combination of limited proteolysis, protein sequencing and protein engineering we show that a single phosphorylation site is responsible for this shift, located at Ser 405 in the C-terminal tail of the protein outside the region of internal repeats. Phosphorylation at this site not only reduces the electrophoretic mobility of tau, it also makes the protein long and stiff, as shown earlier. The site is likely to be phosphorylated in tau from Alzheimer neurofibrillary tangles.
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PMID:Phosphorylation of microtubule-associated protein tau: identification of the site for Ca2(+)-calmodulin dependent kinase and relationship with tau phosphorylation in Alzheimer tangles. 212 43

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.
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PMID: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. 215 22

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

Calcium, calmodulin-dependent protein kinase (Ca/CaM kinase) is an important component of calcium signalling mechanisms in the brain, but little is known about the properties of this protein phosphorylation system in astrocytes. Addition of calcium and calmodulin to supernatant or membrane fractions obtained from rat astrocytes in primary culture increased phosphate incorporation into an exogenously added substrate, casein, and into endogenous protein substrates; this increase was greater than that observed with either calcium alone or calmodulin alone. The calcium, calmodulin-stimulated increase was inhibited by trifluoperazine, and this inhibition could be overcome by the addition of excess calmodulin. The major substrates for Ca/CaM kinase activity were proteins with molecular weights of 59 and 53 kDa, which were similar, but not identical, to the subunits of Ca/CaM kinase type II from brain. The specific activity of Ca/CaM kinase and the phosphorylation of 59 kDa were increased in astrocyte cultures treated and maintained in dibutyryl cyclic adenosine monophosphate (dBcAMP). These results indicate that astrocytes contain Ca/CaM kinase activity and suggest an interaction between the cAMP and calcium/calmodulin messenger systems in these cells.
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PMID:Calcium/calmodulin-dependent protein kinase activity in primary astrocyte cultures. 254 59

Long-term potentiation (LTP) of synaptic transmission is a widely studied cellular example of synaptic plasticity. However, the identity, localization, and interplay among the biochemical signals underlying LTP remain unclear. Intracellular microelectrodes have been used to record synaptic potentials and deliver protein kinase inhibitors to postsynaptic CA1 pyramidal cells. Induction of LTP is blocked by intracellular delivery of H-7, a general protein kinase inhibitor, or PKC(19-31), a selective protein kinase C (PKC) inhibitor, or CaMKII(273-302), a selective inhibitor of the multifunctional Ca2+-calmodulin-dependent protein kinase (CaMKII). After its establishment, LTP appears unresponsive to postsynaptic H-7, although it remains sensitive to externally applied H-7. Thus both postsynaptic PKC and CaMKII are required for the induction of LTP and a presynaptic protein kinase appears to be necessary for the expression of LTP.
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PMID:Inhibition of postsynaptic PKC or CaMKII blocks induction but not expression of LTP. 254 38

Calcium- and calmodulin-dependent protein kinase activity was studied in pure neuronal and glial cultures. The addition of calcium and calmodulin stimulated 32P incorporation into several neuronal proteins including two in the 50- and 60-kilodalton (kD) region which comigrated with purified forebrain calmodulin kinase II subunits (CaM kinase II). In mature astrocytes, CaM kinase activity was also present, and was inhibited by trifluoroperazine and diazepam. Again in homogenates of these cells, two phosphoproteins of apparent molecular masses of 50 and 60 kD comigrated with purified CaM kinase. CaM kinase activity was absent in immature mixed glia and oligodendrocytes. The presence of CaM kinase in neurons and mature astrocytes was confirmed using monoclonal antibodies specific for the 50-kD subunit of the enzyme. No immunoreactivity was observed in oligodendrocytes. The presence of CaM kinase in astrocytes suggests a more ubiquitous role of this enzyme in regulating cellular processes than was previously recognized.
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PMID:Calmodulin kinase II in pure cultured astrocytes. 282 89

Calcium/calmodulin-stimulated autophosphorylation of a prominent brain calmodulin-dependent protein kinase (Type II CaM kinase) produces dramatic changes in its enzymatic activity. These changes suggest a mechanism by which the kinase could act as a calcium-triggered molecular switch. Incorporation of 3-12 of a possible total of 30 phosphate groups per holoenzyme causes kinase activity toward exogenous substrates as well as autophosphorylation itself to become independent of calcium. Thus, kinase activity could be prolonged beyond the duration of an initial activating calcium signal. The calcium-independent autophosphorylation could further prolong the active state by opposing dephosphorylation by cellular phosphatases.
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PMID:Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch. 300 21

The function of the parathyroid gland is closely linked to intracellular and extracellular Ca2+ concentrations. As a step toward understanding the mechanism of action of Ca2+ on the parathyroid, we examined hyperplastic human parathyroid tissue for Ca2+ and calmodulin-dependent protein kinase activity. In parathyroid homogenates, Ca2+ stimulates the phosphorylation of substrate protein in the presence of calmodulin or phospholipid. The calmodulin (CaM)-stimulated activity is present in a soluble fraction of parathyroid and can be separated from other protein kinase activities by gel filtration chromatography. The concentration dependence of CaM kinase on Ca2+ and CaM was determined using the gel filtration. The Ka values for CaM and calcium were 100 nM and 5 microM, respectively. The fraction containing the CaM kinase activity had a calculated mol wt of 5.5 X 10(5). It contained a protein with a mol wt of 4.9 X 10(4) whose phosphorylation was Ca2+ CaM dependent and a CaM-binding protein of mol wt 4.9 X 10(4) which we suggest may be the catalytic subunit of a type II Ca2+-CaM dependent protein kinase. Hyperplastic human parathyroid tissue contains a type II Ca2+-CaM dependent protein kinase which may serve an important function in Ca2+-directed metabolism.
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PMID:Calcium-calmodulin-dependent protein kinase in hyperplastic human parathyroid glands. 309 99

Calcium/calmodulin (CaM)-dependent protein kinases isolated from bovine and rat brains phosphorylate the microtubule-associated tau protein in the mode that shifts the mobility of tau in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (mode I). This mode of tau phosphorylation is the one that occurs abnormally in Alzheimer's lesions. Purified tau protein in solution can be phosphorylated by the Ca2+/CaM kinases maximally to about 50% of the total tau protein. Incorporation of one phosphate group per mol of tau is sufficient to shift the protein to a slower migrating electrophoretic band. Additional phosphate incorporation into the shifted tau proteins can occur depending on protein kinase concentration. In the presence of phosphatidylserine, tau proteins were phosphorylated to an extent of 100% at a tau: phosphatidylserine ratio of 20. Phosphatidylethanolamine also stimulated tau phosphorylation by Ca2+/CaM kinase and phosphatidylinositol was found to be a potent inhibitor of tau protein phosphorylation. The direct observation that tau proteins interact with phospholipids such as phosphatidylethanolamine and phosphatidylinositol, resulting in a smearing of the protein band on sodium dodecyl sulfate-gel electrophoresis, supports the possibility that tau protein may interact with phospholipid membranes in vivo and that tau protein phosphorylation could be modulated by the phospholipid composition of the membranes with which tau interacts.
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PMID:Phosphorylation of tau proteins to a state like that in Alzheimer's brain is catalyzed by a calcium/calmodulin-dependent kinase and modulated by phospholipids. 312 1

In rat adrenal glomerulosa cells, endogenous substrate proteins for Ca2+/calmodulin (CaM)-dependent protein kinase (glomerulosa CaM kinase) and Ca2+/phospholipid-dependent protein kinase (protein kinase C) were investigated. In a 105,000 g-supernatant fraction (cytosol), the Mr 100,000 protein was phosphorylated in the presence of calcium (calculated free Ca2+ concentration, 460 microM) alone or calcium and CaM, and the phosphorylation of this protein was completely inhibited by the CaM antagonists pimozide (500 microM) and melittin (5 microM) in the presence of calcium alone, respectively. These results indicate that the Mr 100,000 protein is a major substrate for glomerulosa CaM kinase, and considerable amounts of endogenous CaM might be present in the cytosol. In the presence of phospholipids (the micelles of 8 micrograms of phosphatidyl serine and 1 microgram of diacylglycerol), at least twelve proteins of Mr 127,000, 80,000, 70,000, 36,000, 35,000, 33,000, 32,000, 30,000, 27,000, 22,000, 19,000 and 17,000 were phosphorylated, and the phosphorylation of these proteins was enhanced by the addition of calcium, indicating that these proteins are substrates for protein kinase C. No endogenous protein phosphorylation was found in a 105,000 g-particulate fraction. Thus, these findings demonstrate that adrenal glomerulosa cells have specific substrate proteins for glomerulosa CaM kinase and protein kinase C, respectively.
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PMID:Existence of endogenous substrate proteins for Ca2+/calmodulin-dependent and Ca2+/phospholipid-dependent protein kinases in rat adrenal glomerulosa cells. 335 37


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