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 mechanism for synergistic phosphorylation by glycogen synthase kinase 3 (GSK-3) and casein kinase II was studied using a synthetic peptide which contains the sequence of a potentially important proline/serine-rich regulatory region of rabbit muscle glycogen synthase. The peptide, Ac-PRPAS(3a)VPPS(3b)PSLS(3c)RHSS(4)PHQS(5) EDEEEP-amide, has five known phosphorylation sites of the native enzyme designated sites 3a, 3b, 3c, 4, and 5, which are spaced every fourth residue. The peptide was phosphorylated specifically at site 5 by casein kinase II with an apparent Km of 23 microM, but it was not phosphorylated by GSK-3. However, after initial phosphorylation of site 5 by casein kinase II, the peptide became an effective substrate for GSK-3 with an apparent Km of 2 microM. GSK-3 introduced up to four phosphates and appeared to catalyze the sequential modification of sites 4, 3c, 3b, and 3a, respectively. The results can be explained if GSK-3 recognizes the sequence -SXXXS(P). Phosphorylation of site 5 by casein kinase II creates this recognition site. Thereafter, each successive phosphorylation introduced by GSK-3 generates a new recognition site. The results provide a molecular basis to explain the synergistic action of casein kinase II and GSK-3 that is also observed with native glycogen synthase. In addition, this investigation emphasizes how protein recognition sites in some cellular targets may have to be formed post-translationally.
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PMID:Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3. 282 Sep 93

Reconstituted porcine cAMP-dependent protein kinase type I was labeled with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) to study cyclic nucleotide binding and to identify amino acid residues that are either in or in close proximity to the cAMP binding sites. The photoaffinity analogue 8-N3cAMP behaved as cAMP itself with respect to cyclic nucleotide binding. For both cAMP and 8-N3cAMP, 2 mol of nucleotide was bound per mole of type I regulatory subunit monomer (RI), the apparent Kd's observed were approximately 10-17 nM on the basis of either Millipore filtration assays, equilibrium dialysis, or ammonium sulfate precipitation, Scatchard plots showed positive cooperativity, and (4) the Hill coefficients were approximately 1.5-1.6. After photolysis and addition of an excess of cAMP, approximately 1 mol of 8-N3cAMP/mol of RI monomer was covalently incorporated. Tryptic digestion of the labeled protein revealed that two unique tryptic peptides were modified. Proline-271 and tyrosine-371 were identified as the two residues that were covalently modified by 8-N3cAMP in RI. These results contrast with the type II regulatory subunit (RII) where 8-N3cAMP modified covalently a single tyrosine residue [Kerlavage, A. R., & Taylor, S. S. (1980) J. Biol. Chem. 255, 8483-8488]. RI contains two adjacent regions of sequence homology in the COOH-terminal fragment that binds two molecules of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Covalent modification of both cAMP binding sites in cAMP-dependent protein kinase I by 8-azidoadenosine 3',5'-monophosphate. 298 89

The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.
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PMID:Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases. 300 75

The substrate specificity of different forms of polycation-stimulated (PCSH, PCSL, and PCSC) phosphorylase phosphatases and of the catalytic subunit of the MgATP-dependent protein phosphatase from rabbit skeletal muscle was investigated. This was done, with phosphorylase a as the reference substrate, using the synthetic phosphopeptides patterned after the phosphorylated sites of pyruvate kinase (type L) (Arg2-Ala-Ser(32P)-Val-Ala (S2), and its Thr(32P) substitute (T4)), inhibitor-1 (Arg4-Pro-Thr(32P)-Pro-Ala (T5), Arg2-Pro-Thr(32P)-Pro-Ala (T1), and its Ser(32P) substitute (S1)), and some modified phosphopeptides (Arg2-Ala-Thr(32P)-Pro-Ala (T2) and Arg2-Pro-Thr(32P)-Val-Ala (T3)), all phosphorylated by cyclic AMP-dependent protein kinase. In addition, casein(Thr-32P), phosphorylated by casein kinase-2, was also tested. The PCS phosphatases show a striking preference for the T4 configuration, PCSC being the least efficient. The catalytic subunit of the MgATP-dependent phosphatase was almost completely inactive toward all these substrates. As shown for the PCSH phosphatase, and comparing with T4, the two proline residues flanking the Thr(P) in T1 and T5, just as in inhibitor-1, drastically imparied the dephosphorylation by lowering the Vmax and not by affecting the apparent Km. The C-terminal proline (as in T2) by itself represents a highly unfavorable factor in the dephosphorylation. The critical effect of the sequence X-Thr(P)-Pro or Pro-Thr(P)-Pro (T1, T2, T5, and inhibitor-1) can be overcome by manganese ions. The additional finding that this is not the case with the Pro-Ser(P)-Pro sequence (S1) suggests that the effect of Mn2+ is highly substrate specific. These observations show the considerable importance of the primary structure of the substrate in determining the specificity of the protein phosphatases.
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PMID:Dephosphorylation of phosphoproteins and synthetic phosphopeptides. Study of the specificity of the polycation-stimulated and MgATP-dependent phosphorylase phosphatases. 302 75

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.
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PMID:Multiple phosphorylation sites of rat liver glycogen synthase. 309 Oct 84

Glycogen synthase, the rate-limiting enzyme in glycogen biosynthesis, has been postulated to exist as isozymes in rabbit liver and muscle (Camici, M., Ahmad, Z., DePaoli-Roach, A. A., and Roach, P. J. (1984) J. Biol. Chem. 259, 2466-2473). Both isozymes share a number of properties including multiple phosphorylation of the enzyme subunit. In the present study, we determined the amino acid sequences surrounding phosphorylation sites in the rabbit liver isozyme recognized by cyclic AMP-dependent protein kinase. Two dominant phosphopeptides (P-1 and P-2) were generated from tryptic digestion. Amino acid sequences of the purified peptides were determined by automated Edman degradation using a gas-phase sequenator. The locations of phosphorylated residues were identified by measuring 32Pi release during Edman degradation cycles. The NH2-terminal sequence of peptide P-1 is S-L-S(P)-V-T-S-L-G-G-L-P-Q-W-E-V-E-E-L-P-V-D-D-L-L-L-P-E-V. This sequence exhibits a strong homology to the site 2 region in the NH2 terminus of the muscle isozyme. The NH2-terminal sequence of peptide P-2 is M-Y-P-R-P-S(P)-S(P)-V-P-P-S-P-L-G-S-Q-A. This sequence shows strong homology to the site 3 region in the COOH terminus of the muscle isozyme. However, some interesting sequence differences were revealed in this region. For example, substitution of serine for alanine at position 6 of peptide P-2 created a new phosphorylation site for cyclic AMP-dependent protein kinase. Phosphorylation of the proline/serine-rich site 3 region correlated with inactivation of the liver isozyme and suggests an important role for this segment of the molecule in the regulation of glycogen synthase. No phosphorylation sites corresponding to sites 1a and 1b of the muscle isozyme were detected. In addition, the results provide definitive chemical proof that glycogen synthase from rabbit liver and muscle are isozymes encoded by distinct messages.
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PMID:Liver isozyme of rabbit glycogen synthase. Amino acid sequences surrounding phosphorylation sites recognized by cyclic AMP-dependent protein kinase. 309 16

In the eucaryotic nucleus, heterogeneous nuclear RNAs exist in a complex with a specific set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). The C proteins, C1 and C2, are major constituents of hnRNPs and appear to play a role in RNA splicing as suggested by antibody inhibition and immunodepletion experiments. With the use of a previously described partial cDNA clone as a hybridization probe, full-length cDNAs for the human C proteins were isolated. All of the cDNAs isolated hybridized to two poly(A)+ RNAs of 1.9 and 1.4 kilobases (kb). DNA sequencing of a cDNA clone for the 1.9-kb mRNA (pHC12) revealed a single open reading frame of 290 amino acids coding for a protein of 31,931 daltons and two polyadenylation signals, AAUAAA, approximately 400 base pairs apart in the 3' untranslated region of the mRNA. DNA sequencing of a clone corresponding to the 1.4-kb mRNA (pHC5) indicated that the sequence of this mRNA is identical to that of the 1.9-kb mRNA up to the first polyadenylation signal which it uses. Both mRNAs therefore have the same coding capacity and are probably transcribed from a single gene. Translation in vitro of the 1.9-kb mRNA selected by hybridization with a 3'-end subfragment of pHC12 demonstrated that it by itself can direct the synthesis of both C1 and C2. The difference between the C1 and C2 proteins which results in their electrophoretic separation is not known, but most likely one of them is generated from the other posttranslationally. Since several hnRNP proteins appeared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as multiple antigenically related polypeptides, this raises the possibility that some of these other groups of hnRNP proteins are also each produced from a single mRNA. The predicted amino acid sequence of the protein indicates that it is composed of two distinct domains: an amino terminus that contains what we have recently described as a RNP consensus sequence, which is the putative RNA-binding site, and a carboxy terminus that is very negatively charged, contains no aromatic amino acids or prolines, and contains a putative nucleoside triphosphate-binding fold, as well as a phosphorylation site for casein kinase type II. The RNP consensus sequence was also found in the yeast poly(A)-binding protein (PABP), the heterogeneous nuclear RNA-binding proteins A1 and A2, and the pre-rRNA binding protein C23. All of these proteins are also composed of at least two distinct domains: an amino terminus, which possesses one or more RNP consensus sequences, and a carboxy terminus, which is unique to each protein, being very acidic in the C proteins and rich in glycine in A1, and C23 and rich in proline in the poly(A)-binding protein. These findings suggest that the amino terminus of these proteins possesses a highly conserved RNA-binding domain, whereas the carboxy terminus contains a region essential to the unique function and interactions of each of the RNA-binding proteins.
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PMID:Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins. 311 May 98

The amino acid sequence of rabbit skeletal muscle heat-stable inhibitor of the cAMP-dependent protein kinase has been determined by microsequencing techniques. Proof of the structure involved a series of nonoverlapping tryptic fragments for primary identification of 86% of the amino acids. Complementary fragments generated by cleavage with chymotrypsin, Staphylococcus aureus V8 proteinase, and mast cell proteinase II contributed to proof of the structure. The inhibitor is a single polypeptide chain of 75 residues and has a molecular weight of 7829. It lacks tryptophan, proline, and sulfur-containing amino acids. The amino terminus of the inhibitor is blocked by an unidentified group. The amino-terminal region of the molecule contains the kinase inhibitory domain, and synthetic peptides based on the sequence of residues 11-30 are potent competitive inhibitors of the cAMP-dependent protein kinase [Scott, J. D., Fischer, E. H., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383]. Residues 14-22 show considerable homology to the "hinge-regions" of the regulatory subunits of the cAMP-dependent protein kinase. The remainder of the molecule shows no similarity to the known amino acid sequence of any protein.
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PMID:Amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase from rabbit skeletal muscle. 389 70

A non-histone chromatin protein (NHCP), which is specifically phosphorylated by a nuclear cAMP-independent protein kinase from mouse spleen cells in vitro, has been purified from calf thymus chromatin. SDS-polyacrylamide gel electrophoresis shows that the molecular weight of the purified NHCP (single peptide) is 13,000. This peptide consists of 14 amino acid residues and has high serine content (17.9%). However, no methionine, proline, or half-cysteine have been detected. The NHCP is a basic protein (isoelectric point, approximately 9.0), although it has high acidic amino acid content (26.2%). This protein serves as the most effective phosphate acceptor for the kinase in vitro when compared with other chromatin proteins such as histones; the Km value of the kinase for the NHCP is 3.65 x 10(-6) M, whereas that for histone H2a (Mr = 14,000) is 1.08 x 10(-5) M. The present finding that the NHCP phosphorylation by the kinase in vitro is remarkably stimulated by double-stranded DNA but inhibited by whole histone or histone H2a suggests that the phosphorylation may be controlled by both double-stranded DNA and histone H2a.
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PMID:A non-histone chromatin protein that is a specific phosphate acceptor of nuclear cAMP-independent protein kinase from mouse spleen cells. 624 79

A protein that exhibits greater substrate specificity for cGMP-dependent protein kinase than for cAMP-dependent protein kinase has been purified 8,000-fold from cytosol of rabbit cerebellum to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein, termed G-substrate, is a monomer of 23,000 daltons. It is heterogeneous on isoelectric focusing, exhibiting three isoelectric forms over the pH range of 5.2-5.6 cGMP-dependent protein kinase catalyzes the incorporation of 2 mol of phosphate/mol of G-substrate, both into threonine residues. The protein has a high content of aspartate, glutamate, and proline. The hydrodynamic properties, heat stability, and acid solubility of this protein are consistent with an unfolded, nonglobular structure. G-substrate is localized primarily in the cytosol of cerebellum, although low concentrations of a phosphorylated protein with a similar molecular weight are detected in other brain regions.
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PMID:A specific substrate from rabbit cerebellum for guanosine 3':5'-monophosphate-dependent protein kinase. I. Purification and characterization. 625 70


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