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)

A cytoskeletal extract of pure axoplasm, highly enriched with neurofilaments (ANF), was prepared from the giant axon of the squid. This ANF preparation also contained potent kinase activities which phosphorylated the Mr greater than 400,000 (high molecular weight) and Mr 220,000 squid neurofilament protein subunits. High salt (1 M) extraction of this ANF preparation solubilized most of the neurofilament proteins and kinase activities and gel filtration on an AcA 44 column separated these two components. The neurofilaments eluted in the void volume of the column while the kinase activities eluted in the 17-44-kDa range of the column. Two major kinase activities were measured in this peak of activity. One of these strongly phosphorylated the phosphate acceptor peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and was completely inhibited by the selective inhibitor of cAMP-dependent kinase Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- NH2 (Wiptide). Since addition of cAMP did not stimulate activity, this suggested that this kinase was a free catalytic subunit of cAMP-dependent kinase associated with the neurofilaments. The second kinase activity most effectively phosphorylated alpha-casein, and this activity was not affected by Wiptide. The alpha-casein phosphorylating activity (ANF kinase) was the principal activity responsible for neurofilament protein phosphorylation, and was not inhibited by various inhibitors against second messenger regulated kinases, suggesting it was related to the casein kinase family. Four lines of evidence indicate ANF kinase was similar to casein kinase I. These were: 1) the apparent molecular weight determined by gel filtration and the chromatographic elution profile on phosphocellulose column corresponded to casein kinase I; 2) heparin, an inhibitor of casein kinase II at 2-5 micrograms/ml, stimulated both ANF kinase and purified casein kinase I at these concentrations, while CKI-7, a relatively selective inhibitor of casein kinase I, inhibited ANF kinase in a comparable dose-response fashion; 3) purified casein kinase I strongly phosphorylated both ANF protein subunits (like ANF kinase) whereas casein kinase II was relatively ineffective; and 4) tryptic peptide maps of the HMW and Mr 220,000 neurofilament proteins after phosphorylation by ANF kinase or purified casein kinase I showed similar 32P-peptide patterns.
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PMID:Principal neurofilament-associated protein kinase in squid axoplasm is related to casein kinase I. 200 43

Previous studies identified synapsin I as a potential substrate for a newly discovered growth factor-sensitive, proline-directed protein kinase originally isolated from rat pheochromocytoma. The present study describes the site-specific phosphorylation of synapsin I by highly purified preparations of proline-directed protein kinase. The incorporation of [32P]phosphate into bovine brain synapsin I was dependent upon both the amount of kinase present and the time of incubation. The maximum stoichiometry of phosphorylation approached 1 mol of phosphate/mol of synapsin I protein. When analyzed by sodium dodecyl sulfate-gel electrophoresis and autoradiography, [32P]phosphate was found to be incorporated into both synapsin Ia and Ib. Phosphoamino acid analysis demonstrated that serine residues were phosphorylated exclusively. Digestion of phosphorylated synapsin I with trypsin followed by high performance liquid chromatography (HPLC) phosphopeptide analysis indicated that the tryptic peptide containing the major phosphorylation site eluted as a single peak at approximately 17% acetonitrile. The primary structure of this phosphopeptide, determined by gas-phase sequencing, was found to be Gln-Ser-Arg-Pro-Val-Ala-Gly-Gly-Pro-Gly-Ala-Pro-Pro-Ala-Thr-Arg-Pro-Pro- Ala-Ser-Pro-Ser-Pro-Gln-Arg. Sequential Edman degradation of this HPLC-purified tryptic phosphopeptide revealed that serine 20 of this peptide was the major phosphorylated residue. This phosphoacceptor site is immediately flanked by a carboxyl-terminal proline residue, an observation that further verifies the proline-directed nature of this protein kinase. The tryptic phosphopeptide corresponds exactly to a sequence in the collagenase-sensitive, proline-rich "tail" region of bovine synapsin I. This novel phosphorylation site is close to but distinct from phosphorylation sites 2 and 3, which are known to be phosphorylated by calcium/calmodulin-dependent protein kinase II and are considered to be of regulatory importance.
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PMID:Phosphorylation of synapsin I at a novel site by proline-directed protein kinase. 210 63

The product(s) of the gene shaggy (sgg) is required for seemingly unrelated events during the development of Drosophila melanogaster. In embryos, maternal and zygotically derived sgg products are required initially to construct a normal syncytial blastoderm and later for normal segmentation. Furthermore, in mutant animals a process of intercellular communication that is required for the segregation of the neural and epidermal lineage during the formation of the central nervous system and the adult peripheral nervous system is disrupted. Here we describe a transcription unit of approximately 40 kb lying within the cloned chromosomal interval 3B1, and provide evidence that it encodes the sgg+ function. Of seven developmentally regulated transcripts that are partially generated by alternative splicing, two seem to be responsible for early sgg activity. Sequence analysis of corresponding cDNA(s) predicts a protein of 514 amino acids with a canonical catalytic domain found in serine/threonine specific protein kinases, linked to an unusual region rich in Gly, Ala and Ser. A search for homologies as well as a comparative study of the kinase catalytic domain with that of other proteins, revealed that the protein kinase domain of sgg is distantly related to the members of the CDC28/cdc2+ subfamily of protein kinases, all of which play cardinal roles in the regulation of the yeast and mammalian cell cycles. Ubiquitous expression of sgg transcripts was found during embryonic stages. A possible role of the sgg protein in a signal transduction pathway necessary for intercellular communication at different stages of development is discussed.
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PMID:An early embryonic product of the gene shaggy encodes a serine/threonine protein kinase related to the CDC28/cdc2+ subfamily. 211 7

The peptides, Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH2, Leu-Arg-Arg-Gln-Ala-Leu-Gly-NH2, and Leu-Arg-Arg-Asn-Ala-Leu-Gly-NH2, serve as active site-directed inhibitors of the cAMP-dependent protein kinase from bovine cardiac muscle. The Asn-containing peptide is a 10-fold more potent inhibitor than its Ala- and Gln-containing counterparts. All three peptides are linear competitive inhibitors versus a peptide-based substrate and uncompetitive inhibitors versus MgATP. The enhanced inhibitory potency of the Asn-peptide, in conjunction with the observed loss of ATP-ase activity of the enzyme in the presence of the inhibitor, suggests that asparagine may serve as a through-space isostere of serine. The uncompetitive inhibition pattern displayed by amide-capped peptides versus MgATP indicates that these species bind in an ordered fashion to the cAMP-dependent protein kinase, with MgATP binding first.
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PMID:Noncovalent active site interactions enhance the affinity and control the binding order of reversible inhibitors of the cAMP-dependent protein kinase. 214 79

The heavy chain of smooth muscle myosin was found to be phosphorylated following immunoprecipitation from cultured bovine aortic smooth muscle cells. Of a variety of serine/threonine kinases assayed, only casein kinase II and calcium/calmodulin-dependent protein kinase II phosphorylated the smooth muscle myosin heavy chain to a significant extent in vitro. Two-dimensional maps of tryptic peptides derived from heavy chains phosphorylated in cultured cells revealed one major and one minor phosphopeptide. Identical tryptic peptide maps were obtained from heavy chains phosphorylated in vitro with casein kinase II but not with calcium/calmodulin-dependent protein kinase II. Of note, the 204-kDa smooth muscle myosin heavy chain but not the 200-kDa heavy chain isoform was phosphorylated by casein kinase II. Partial sequence of the tryptic phosphopeptides generated following phosphorylation by casein kinase II yielded Val-Ile-Glu-Asn-Ala-Asp-Gly-Ser*-Glu-Glu-Glu-Val. The Ser* represents the Ser(PO4) which is in an acidic environment, as is typical for casein kinase II phosphorylation sites. By comparison with the deduced amino acid sequence for rabbit uterine smooth muscle myosin (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737), we have localized the phosphorylated serine residue to the non-helical tail of the 204-kDa isoform of the smooth muscle myosin heavy chain. The ability of the 204-kDa isoform, but not the 200-kDa isoform, to serve as a substrate for casein kinase II suggests that these two isoforms can be regulated differentially.
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PMID:The 204-kDa smooth muscle myosin heavy chain is phosphorylated in intact cells by casein kinase II on a serine near the carboxyl terminus. 217 Mar 99

Glycine is an important inhibitory transmitter in the brainstem and spinal cord. In the trigeminal subnucleus caudalis (medullary dorsal horn) and in the spinal dorsal horn (the relaying centres for processing pain and sensory information), glycine inhibits the glutamate-evoked depolarization and depresses firing of neurons. The binding of glycine to its receptor produces a large increase in Cl- conductance, which causes membrane hyperpolarization. The selectivity and gating properties of glycine receptor channels have been well characterized; the glycine receptor molecules have also been purified. The amino-acid sequence, deduced from complementary DNA clones encoding one of the peptides (the 48K subunit), shows significant homology with gamma-aminobutyric acid A (GABAA) and nicotinic acetylcholine receptor subunits, suggesting that glycine receptors may belong to a superfamily of chemically gated channel proteins. However, very little is known about the modulation of glycine receptor channels. We have investigated the regulation of strychnine-sensitive glycine receptor channels by cyclic AMP-dependent protein kinase in neurons isolated from spinal trigeminal nucleus of rat and report here that the protein kinase A dramatically increased the glycine-induced Cl- currents by increasing the probability of the channel openings. GS protein, which is sensitive to cholera toxin, was involved in the modulation.
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PMID:Modulation of glycine receptor chloride channels by cAMP-dependent protein kinase in spinal trigeminal neurons. 217 40

Multifunctional protein kinase (MFPK) phosphorylates ATP-citrate lyase on peptide B on two sites, BT and BS, on threonine and serine, respectively, inhibitor 2 on a threonyl residue, and glycogen synthase at sites 2 and 3. The phosphorylation sites BT and BS of ATP-citrate lyase are dependent on prior phosphorylation at site A whereas site A phosphorylation is decreased by prior phosphorylation at sites BT and BS. To study the MFPK recognition sites and the site-site interactions, the amino acid sequences of ATP-citrate lyase peptide B and inhibitor 2 were determined and compared to each other and to glycogen synthase sites 3-5. The sequence of the tryptic peptide containing the two phosphorylation sites of peptide B is -Phe-Leu-Leu-Asn-Ala-Ser-Gly-Ser-Thr-Ser-Thr(P)-Pro-Ala-Pro-Ser(P)-Arg-, and the sequence of the MFPK phosphorylation site of inhibitor 2 is -Ile-Asp-Glu-Pro-Ser-Thr(P)-Pro-Tyr-. This inhibitor 2 site is identical with the site phosphorylated by glycogen synthase kinase 3/FA. These results suggest that at least some of the sites phosphorylated by MFPK (BT of ATP-citrate lyase, Thr 72 of inhibitor 2, and sites 3b and 4 of glycogen synthase) contain a Ser/Thr flanked by a carboxyl-terminal proline. However, as MFPK did not phosphorylate a series of peptides containing the -X-Thr/Ser-Pro-X- sequence, this minimum consensus sequence is not sufficient for phosphorylation by MFPK.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sequence of sites on ATP-citrate lyase and phosphatase inhibitor 2 phosphorylated by multifunctional protein kinase (a glycogen synthase kinase 3 like kinase). 217 22

A peptide affinity inactivator, Ac-Leu-Arg-Arg-Ala-(BrAc)Orn-Leu-Gly, was used as a tool to probe for active site residues in the catalytic subunit of bovine cAMP-dependent protein kinase. The peptide inactivated the catalytic subunit in an active site-directed and monophasic manner with a first-order rate constant of 0.03 min-1 and a dissociation constant of 675 microM. Studies with radioactive peptide indicated that approximately one equivalent of peptide was incorporated into each protein molecule. Protein sequencing identified the modified residue as Cys-199. A possible location for Cys-199 within the active site is suggested.
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PMID:Inactivation of the catalytic subunit of bovine cAMP-dependent protein kinase by a peptide-based affinity inactivator. 232 82

A novel calcium-dependent protein kinase (CDPK) previously reported to be activated by the direct binding of Ca2+, and requiring neither calmodulin nor phospholipids for activity [Harmon, A.C., Putnam-Evans, C.L., & Cormier, M.J. (1987) Plant Physiol. 83, 830-837], was purified to greater than 95% homogeneity from suspension-cultured soybean cells (Glycine max, L. Wayne). Purification was achieved by chromatography on DEAE-cellulose, phenyl-Sepharose, Sephadex G-100, and Blue Sepharose. The purified enzyme (native molecular mass = 52,200 Da) resolved into two immunologically related protein bands of 52 and 55 kDa on 10% SDS gels. Enzyme activity was stimulated 40-100-fold by micromolar amounts of free calcium (K0.5 = 1.5 microM free calcium) and was dependent upon millimolar Mg2+. CDPK phosphorylated lysine-rich histone III-S and chicken gizzard myosin light chains but did not phosphorylate arginine-rich histone, phosvitin, casein, protamine, or Kemptide. Phosphorylation of histone III-S, but not autophosphorylation, was inhibited by KCl. CDPK displayed a broad pH optimum (pH 7-9), and kinetic studies revealed a Km for Mg2(+)-ATP of 8 microM and a Vmax of 1.7 mumol min-1 mg-1 with histone III-S (Km = 0.13 mg/mL) as substrate. Unlike many other protein kinases, CDPK was able to utilize Mg2(+)-GTP, in addition to Mg2(+)-ATP, as phosphate donor. The enzyme phosphorylated histone III-S exclusively on serine; however, CDPK autophosphorylated on both serine and threonine residues. These properties demonstrate that CDPK belongs to a new class of protein kinase.
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PMID:Purification and characterization of a novel calcium-dependent protein kinase from soybean. 233 77

We previously described a novel insulin-stimulated protein kinase activity that phosphorylates Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in cytosolic extracts of adipocytes (Yu, K-T., Khalaf, N., and Czech, M. P. (1987) J. Biol. Chem. 262, 16677-16685). In the present experiments, cytosolic extracts of livers from insulin-treated rats also exhibited a 30-100% increase in this Kemptide kinase activity and served as an abundant source for purification. The Kemptide kinase was purified in parallel from liver extracts of insulin-treated or control rats through five chromatographic steps and one polyethylene glycol precipitation. The chromatographic behavior of the insulin-stimulated Kemptide kinase differed significantly from the control kinase on Mono Q and heparin-Sepharose resins. The purified kinase preparations retain insulin stimulations of 2-10-fold. Analysis of the purified control and insulin-stimulated kinases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed single bands with similar silver staining intensity and apparent molecular masses of 52 kDa. The insulin-stimulated Kemptide phosphorylating activity also coincided with the major silver-stained band following isoelectric focusing in polyacrylamide gels. The stimulation of kinase activity in response to administration of insulin is due to an increase in Vmax, whereas the Km for Kemptide (0.3 mM) is unchanged. The apparent molecular mass of the native kinase determined by gel filtration is approximately 50 kDa, suggesting that it exists as a monomer. Either Mg2+ or Mn2+ serve as cofactors for the kinase which phosphorylates a variety of basic substrates including a number of peptides and histones. The activity of the Kemptide kinase is not changed by several compounds that have been shown to modulate other kinases. Based on these data, we conclude 1) a novel insulin-sensitive Kemptide kinase in liver cytosol has been purified to near homogeneity, and 2) insulin administration acutely modulates the specific activity of this Kemptide kinase in livers of intact rats.
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PMID:Purification of a novel insulin-stimulated protein kinase from rat liver. 240 57


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