Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.17 (CaMKII)
 4,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Synthetic polypeptides were employed as substrates in kinetic analyses of the reaction mechanism for the catalytic subunit of a cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from calf thymus. This enzyme preparation was shown to catalyze the transfer of phosphate from ATP to histone H1 from calf thymus, as well as to two synthetic polypeptides, Arg-Lys-Ala-Ser-Gly-Pro (H1-6) and Arg-Arg-Lys-Ala-Ser-Gly-Pro (H1-7), corresponding to the amino acid sequence about serine-38 in calf H1. A related, basic heptapeptide corresponding to a sequence from pig liver pyruvate kinase, Leu-Arg-Arg-Ala-Ser-Leu-Gly (K), was also a substrate. The stoichiometry of peptide phosphorylation was established in each case as the transfer of 1 mol of phosphate from the gamma position of MgATP to the serine hydroxyl of 1 mol of the peptide. Steady-state, initial-velocity, kinetic parameters were determined for each substrate, using various concentrations of ATP. Under the conditions used, all synthetic peptides reacted with greater maximum velocities than whole histone H1. Nevertheless, the K(m) for H1, 54 muM, was lower than the K(m) values of the synthetic substrates. The most efficient substrate was peptide K, which had a V(max) of 50.6 mumol/min per mg of kinase and a K(m) of 63 muM. In the absence of peptide substrate no ATPase activity was detectable at a sensitivity of 0.05% of the rate of peptide phosphorylation, suggesting that ATP is not cleaved to form an unstable phosphoenzyme complex. The data are consistent with a sequential reaction mechanism involving a ternary complex between enzyme, polypeptide substrate, and ATP.
 ...
PMID:Studies on the mechanism of phosphorylation of synthetic polypeptides by a calf thymus cyclic AMP-dependent protein kinase. 20 Sep 11

Characteristics of the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from the cytosol and in the postsynaptic densities (PSD) of rat brain were investigated. Several proteins were surveyed for their abilities to serve as a substrate for non-autophosphorylated and autophosphorylated CaM kinase IIs from the cytosol and PSD. The tested substrates were separated into two groups. Autophosphorylation of the kinase slightly decreased or did not change its activities towards substrates of the first group: myosin light chain of chicken gizzard, synapsin I, tau factor and microtubule-associated protein 2. In contrast, autophosphorylation of the enzyme increased its activities towards substrates of the second group: syntide-2, histone H1, calcineurin and myelin basic protein. The Ca2+/calmodulin-independent kinase activity increased by autophosphorylation with any of substrates tested. Similar results were obtained with the cytosolic and PSD CaM kinase II. Trifluoperazine and mastoparan, calmodulin binding antagonists, inhibited the activity of the non-autophosphorylated CaM kinase II, but had no effect or only a slight inhibitory effect on the activity of the autophosphorylated CaM kinase II, indicating that the autophosphorylated kinase has no requirement for calmodulin for Ca(2+)-dependent activity and/or a higher affinity for calmodulin The results suggest that the autophosphorylation of CaM kinase II is a subtle mechanism for regulating the interaction between the enzyme and substrate.
 ...
PMID:Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II: effects on interaction between enzyme and substrate. 164 40

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity. Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Small-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone H1 as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.
 ...
PMID:Control of protein phosphatase 2A by simian virus 40 small-t antigen. 170 74

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.
 ...
PMID:Second messenger-specific protein kinases in a salt-absorbing intestinal epithelium. 215 31

When the synaptosomal cytosol fraction from rat brain was chromatographed on a DEAE-cellulose column and assayed for protein phosphatases for tau factor and histone H1, two peaks of activities, termed peak 1 (major) and peak 2 (minor), were separated. Each peak was in a single form 2 (minor), were separated. Each peak was in a single form on Sephacryl S-300 column chromatography. Both peaks 1 and 2 dephosphorylated tau factor phosphorylated by Ca2+/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The Km values were in the range of 0.42-0.84 microM for tau factor. There were no differences in kinetic properties of dephosphorylation between the substrates phosphorylated by the two kinases. The phosphatase activities did not depend on Ca2+, Mn2+ and Mg2+. Immunoprecipitation and immunoblotting analysis using polyclonal antibodies to the catalytic subunit of brain protein phosphatase 2A revealed that both protein phosphatases are the holoenzymic forms of protein phosphatase 2A. Aluminum chloride inhibited the activities of both peaks 1 and 2 with IC50 values of 40-60 microM. These results suggest that dephosphorylation of tau factor in presynaptic nerve terminals is controlled mainly by protein phosphatase 2A and that the neurotoxic effect of aluminum seems to be related mostly to inhibition of dephosphorylation of tau factor.
 ...
PMID:Dephosphorylation of tau factor by protein phosphatase 2A in synaptosomal cytosol fractions, and inhibition by aluminum. 216 75

A protein kinase activity was identified in pig brain that co-purified with microtubules through repeated cycles of temperature-dependent assembly and disassembly. The microtubule-associated protein kinase (MTAK) phosphorylated histone H1; this activity was not stimulated by cyclic nucleotides. Ca2+ plus calmodulin, phospholipids or polyamines. MTAK did not phosphorylate synthetic peptides which are substrates for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase. Ca2+/calmodulin-dependent protein kinase II, protein kinase C or casein kinase II. MTAK activity was inhibited by trifluoperazine [IC50 (median inhibitory concn.) = 600 microM] in a Ca2+-independent fashion. Ca2+ alone was inhibitory [IC50 = 4 mM). MTAK was not inhibited by heparin, a potent inhibitor of casein kinase II, nor a synthetic peptide inhibitor of cyclic AMP-dependent protein kinase. MTAK demonstrated a broad pH maximum (7.5-8.5) and an apparent Km for ATP of 45 microM. Mg2+ was required for enzyme activity and could not be replaced by Mn2+. MTAK phosphorylated serine and threonine residues on histone H1. MTAK is a unique cofactor-independent protein kinase that binds to microtubule structures.
 ...
PMID:Properties of a microtubule-associated cofactor-independent protein kinase from pig brain. 255 23

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.
 ...
PMID:Dephosphorylation of microtubule proteins by brain protein phosphatases 1 and 2A, and its effect on microtubule assembly. 283 18

Poly (ADP-ribose) synthetase from bovine thymus was phosphorylated effectively by protein kinase C in vitro. The phosphorylation was dependent on the activators of this kinase, Ca2+ and phospholipid. The apparent Km for the synthetase was about 8 microM, which was lower than that for histone H1. Though the synthetase was a weak substrate for Ca2+/calmodulin-dependent protein kinase II, other protein kinases, cyclic AMP-dependent and cofactor-independent protein kinases did not phosphorylate the synthetase. Phosphorylation of the synthetase by protein kinase C resulted in appreciable inhibition of the synthetase activity.
 ...
PMID:Poly (ADP-ribose) synthetase is phosphorylated by protein kinase C in vitro. 312 Jul 11

While testing purines related to the non-specific protein kinase inhibitors N6-dimethylaminopurine and N6-(delta 2-isopentenyl)adenine as potential inhibitors of the p34cdc2/cyclin B kinase, we discovered a compound with high specificity, 2-(2-hydroxyethylamino)-6- benzylamino-9-methylpurine (olomoucine). Kinetic analysis of kinase inhibition reveals that olomoucine behaves as a competitive inhibitor for ATP and as a non-competitive inhibitor for histone H1 (linear inhibition for both substrates). The kinase specificity of this inhibition was investigated for 35 highly purified kinases (including p34cdk4/cyclin D1, p40cdk6/cyclin D3, cAMP-dependent and cGMP-dependent kinases, eight protein kinase C isoforms, calmodulin-dependent kinase II, myosin light-chain kinase, mitogen-activated S6 kinase, casein kinase 2, double-stranded RNA-activated protein kinase, AMP-stimulated kinase, eight tyrosine kinases). Most kinases are not significantly inhibited. Only the cell-cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase (and its starfish homologue p44mpk) are substantially inhibited by olomoucine (IC50 values are 7, 7, 7, 3 and 25 microM, respectively). The cdk4/cyclin D1 and cdk6/cyclin D3 kinases are not significantly sensitive to olomoucine (IC50 values greater than 1 mM and 150 microM, respectively). N6-(delta 2-Isopentenyl)adenine is confirmed as a general kinase inhibitor with IC50 values of 50-100 microM for many kinases. The purine specificity of cyclin-dependent kinase inhibition was investigated: among 81 purine derivatives tested, only C2, N6 and N9-substituted purines exert a strong inhibitory effect on the p34cdc2/cyclin B kinase. An essentially similar sensitivity to this olomoucine family of compounds was observed for the brain-specific cdk5/p35 kinase. Structure/activity relationship studies allow speculation on the interactions of olomoucine and its analogues with the kinase catalytic subunit. Olomoucine inhibits in vitro M-phase-promoting factor activity in metaphase-arrested Xenopus egg extracts, inhibits in vitro DNA synthesis in Xenopus interphase egg extracts and inhibits the licensing factor, an essential replication factor ensuring that DNA is replicated only once in each cell cycle. Olomoucine inhibits the starfish oocyte G2/M transition in vivo. Through its unique selectivity olomoucine provides an anti-mitotic reagent that may preferentially inhibit certain steps of the cell cycle.
 ...
PMID:Inhibition of cyclin-dependent kinases by purine analogues. 792 96

Rat vascular smooth muscle cells were synchronized to the quiescent state (G0) by serum deprivation and then stimulated to enter the cell cycle by serum refeeding. At various times of the cell cycle, cells were analyzed for the expression of p34cdc2 and mitogen-activated protein kinase homologues by immunoblotting and for kinase activity toward histone H1, myelin basic protein, and caldesmon. A small amount of p34cdc2 was expressed in the G0/G1 phase (0 to 8 hours). At the G1/S transition (12 hours), the level of p34cdc2 started to accumulate and increased by 60-fold at G2/M (18 hours), accompanied by a more slowly migrating band. Histone H1 kinase activity was undetectable in anti-p34cdc2 immunoprecipitates in the G0/G1 cells but appeared around the G1/S boundary and peaked at G2/M (18 hours). The caldesmon kinase activity exhibited two distinct phases: the first appeared at G0/G1 (0 to 8 hours), and the second appeared at G1/S and continued through G2/M. Two mitogen-activated protein kinase isoforms were expressed throughout the cell cycle. Anti-mitogen-activated protein kinase immunoprecipitates possessed kinase activities toward myelin basic protein and caldesmon, which were activated within 15 minutes after serum stimulation and declined within a few hours. These findings suggest that p34cdc2 and mitogen-activated protein kinase homologues may play significant roles in regulating the progression of the cell cycle of smooth muscle cells, the former at the G2/M transition and the latter at the G0/G1 transition.
 ...
PMID:Smooth muscle cell proliferation. Expression and kinase activities of p34cdc2 and mitogen-activated protein kinase homologues. 838 75


1 2 Next >>