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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)

Calmodulin-dependent protein phosphatase purified from bovine cardiac muscle catalyzed the rapid dephosphorylation of Ser-95 of bovine cardiac cAMP-dependent protein kinase regulatory subunit (RII). The kinetic constants determined for the reaction (Km = 20 microM; Vmax = 2 mumol min-1 mg-1) are comparable to those determined for other good substrates of this phosphatase. Because little is known about the determinants of substrate specificity for the calmodulin-dependent phosphatase, various phosphopeptides were used to investigate the structural features important for substrate recognition. Limited proteolysis of phospho-RII with trypsin and chymotrypsin yielded fragments (residues 93-400 and 91-400, respectively) that were poor substrates, whereas digestion with Staphylococcal aureus V8 protease produced three phosphopeptides that were all dephosphorylated as rapidly as intact RII. The sequence of the shortest phosphopeptide produced by S. aureus V8 protease was determined by sequence analysis to be Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Cys-Ala-Glu, corresponding to residues 81-99 of RII. Synthetic phosphopeptides corresponding to residues 81-99, 85-99, 90-99, and 91-99 were prepared to determine the minimum sequence necessary for substrate recognition. Only the 19-residue peptide (81-99) was dephosphorylated with kinetics comparable to RII (Km = 26 microM, Vmax = 1.7 mumol min-1 mg-1). Structural analysis of this peptide indicates that an amphipathic beta-sheet structure may be an important structural determinant for some substrates of the calmodulin-dependent phosphatase.
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PMID:Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity. 301 43

Phosphorylation of the ascarid phosphofructokinase with the catalytic subunit of beef heart cyclic AMP-dependent protein kinase results in the incorporation of 1 mol of P/mol of subunit. Accompanying the phosphorylation there is a 3-4-fold increase in catalytic activity when measured at pH 6.8 with inhibitory levels of ATP. Studies on the effect of phosphorylation on the ATP saturation curve demonstrated that phosphorylation decreased the inhibitory action of ATP. The apparent Km of the catalytic subunit for the phosphofructokinase was 11.2 microM. Chymotryptic or subtilisin digestion of the labeled enzyme released distinct but overlapping phosphopeptides that were purified by high pressure liquid chromatography and sequenced by gas phase peptide sequencing. The sequence of the chymotryptic peptide was Ala-Lys-Gly-Arg-Ser-Asp-Ser(P)-Ile-Val-Pro-Thr. Based on these results and earlier observations, it is proposed that phosphorylation of phosphofructokinase plays an important role in the regulation of energy metabolism in the parasitic helminth.
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PMID:Ascaris suum phosphofructokinase. Phosphorylation by protein kinase and sequence of the phosphopeptide. 302 8

Eukaryotic protein synthesis initiation factor 4E (eIF-4E) was labeled in situ with [32P]orthophosphate in cultured HeLa cells and rabbit reticulocytes and purified by affinity chromatography. Tryptic digestion yielded one labeled peptide which contained predominantly serine and lysine. After treatment of the protein with citraconic anhydride to block epsilon-amino groups of lysyl residues, tryptic digestion yielded a labeled peptide whose composition was consistent with the structure Trp-Ala-Leu-Trp-Phe-Phe-Lys-Asn-Asp-Lys-Ser(P)-Lys-Thr-Trp-Gln-Ala-Asn-L eu-Arg, one of the arginyl peptides predicted from the human eIF-4E cDNA sequence. The only serine in this peptide is located at position 53 of eIF-4E. Thus, it is concluded that eIF-4E contains a single site of phosphorylation for an endogenous protein kinase, which is Ser-53 in the human eIF-4E sequence.
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PMID:Phosphorylation site of eukaryotic initiation factor 4E. 311 45

The amino acid sequences surrounding three major phosphorylation sites in rat and bovine synapsin I have been determined by employing automated gas-phase sequencing and manual Edman degradation of purified phosphopeptide fragments. Site 1 is a serine residue phosphorylated by cAMP-dependent protein kinase and by calcium/calmodulin-dependent protein kinase I. The sequence around site 1 was derived from tryptic/chymotryptic phosphopeptides and overlapping cyanogen bromide cleavage fragments. This sequence, identical in rat and bovine synapsin I, is Asn-Tyr-Leu-Arg-Arg-Arg-Leu-Ser(P)-Asp-Ser-Asn-Phe-Met. Site 1 is located at the NH2 terminus of the protein, within the collagenase-resistant head region. Sites 2 and 3 are serine residues phosphorylated by calcium/calmodulin-dependent protein kinase II. The sequences surrounding bovine site 2 and site 3 were derived from tryptic phosphopeptides and overlapping fragments generated by cleavage with chymotrypsin, collagenase, and endoproteinase Lys-C. The sequence around bovine site 2 is Thr-Arg-Gln-Thr-Ser(P)-Val-Ser-Gly-Gln-Ala-Pro-Pro-Lys, and the sequence around bovine site 3 is Thr-Arg-Gln-Ala-Ser(P)-Gln-Ala-Gly-Pro-Met-Pro-Arg. Sites 2 and 3 are located within the COOH-terminal, collagenase-sensitive tail region of the molecule, separated by 36 amino acids. The sequences surrounding rat site 2 and site 3 were derived from tryptic phosphopeptides. The sequence around rat site 2 is Gln-Ala-Ser(P)-Ile-Ser-Gly-Pro-Ala-Pro-Pro-Lys, and the sequence around rat site 3 is Gln-Ala-Ser(P)-Gln-Ala-Gly-Pro-Gly-Pro-Arg. Thus, the sequences surrounding the four sites that are phosphorylated by calcium/calmodulin-dependent protein kinase II, namely sites 2 and 3 in rat and bovine synapsin I, exhibit a high degree of homology.
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PMID:Amino acid sequences surrounding the cAMP-dependent and calcium/calmodulin-dependent phosphorylation sites in rat and bovine synapsin I. 311 71

Protein kinase C, purified to near homogeneity from the brain, has been tested toward a variety of synthetic peptide substrates including different phosphorylatable residues. While it proved totally inactive toward the tyrosyl peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly, as well as toward several more or less acidic seryl peptides, it phosphorylates with a Ca2+/phospholipid-dependent mechanism, at seryl and/or threonyl residues, many basic peptides, some of which are also good substrates for cAMP-dependent protein kinase (A-kinase). Among the peptides tested, however, the best substrate for protein kinase C, with kinetic constants comparable to those of histones, is the nonapeptide Gly-Ser-Arg6-Tyr, which is not a substrate for A-kinase. Moreover, although the peptide Pro-Arg5-Ser-Ser-Arg-Pro-Val-Arg is a good substrate for both kinases, its derivative with ornitines replacing arginines is phosphorylated only by protein kinase C. Some typical substrates of A-kinase on the other hand, like the peptides Phe-Arg2-Leu-Ser-Ile-Ser-Thr-Glu-Ser and Arg2-Ala-Ser-Val-Ala, are phosphorylated by protein kinase C rather slowly and with unfavourable kinetic constants. It is concluded that, while both protein kinase C and A-kinase need basic groups close to the phosphorylatable residues, their primary structure determinants are quite distinct.
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PMID:Distinct structural requirements of Ca2+/phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase as evidenced by synthetic peptide substrates. 315 99

Calmodulin is specifically phosphorylated by casein kinase 2 (CK 2), but not by casein kinase 1, A kinase, or C kinase. In the present report, the stoichiometry of the phosphorylation of calmodulin by CK 2 in the presence and absence of polylysine and its phosphorylation sites were examined. In the absence of polylysine, the radioactive phosphate incorporated into calmodulin by CK 2 was only 0.01 mol/mol and the phosphorylation occurred at Ser-101. In the presence of polylysine, 1.2 mol of radioactive phosphate was incorporated into 1 mol of calmodulin. In this case, Thr-79 in addition to Ser-101 was phosphorylated, but Ser-81 was not. The sequence around the phosphorylated Thr is Asp-Thr(P)-Asp-Ser-Glu-Glu-Glu-.
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PMID:Determination of the phosphorylation sites of calmodulin catalyzed by casein kinase 2. 324 68

Oligonucleotide-directed mutagenesis was used to produce mutants in the hinge region of the regulatory subunit (R) of the Saccharomyces cerevisiae cAMP-dependent protein kinase. The mutant proteins were expressed in Escherichia coli, purified, urea treated to produce cAMP-free regulatory (R), and analyzed in vitro for catalytic (C) subunit inhibitory activity in the presence and absence of cAMP. When assayed in the absence of cAMP, wild type R dimer inhibited C with an IC50 of 40 nM. Replacement of amino acid residue Ser-145 (the autophosphorylation site of yeast R) with Ala or Gly produced mutants which were 2-10-fold better inhibitors of C, while replacement with Glu, Asp, Lys, or Thr produced mutants which were 2-5-fold worse inhibitors of C relative to wild type R. When assayed in the presence of cAMP, all R subunits had a decreased affinity for C subunit, with Ser-145 and Thr-145 undergoing autophosphorylation. These results suggest that the amino acid at position 145 of R contributes to R-C interaction and therefore influences the equilibrium of yeast protein kinase subunits in vitro.
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PMID:Mutagenesis of the regulatory subunit of yeast cAMP-dependent protein kinase. Isolation of site-directed mutants with altered binding affinity for catalytic subunit. 328 30

Protein kinases represent a diverse family of enzymes that play critical roles in regulation. The simplest and best-understood biochemically is the catalytic (C) subunit of cAMP-dependent protein kinase, which can serve as a framework for the entire family. The amino-terminal portion of the C subunit constitutes a nucleotide binding site based on affinity labeling, labeling of lysines, and a conserved triad of glycines. The region beyond this nucleotide fold also contains essential residues. Modification of Asp 184 with a hydrophobic carbodiimide leads to inactivation, and this residue may function as a general base in catalysis. Despite the diversity of the kinase family, all share a homologous catalytic core, and the residues essential for nucleotide binding or catalysis in the C subunit are invariant in every protein kinase. Affinity labeling and intersubunit cross-linking have localized a portion of the peptide binding site, and this region is variable in the kinase family. The crystal structure of the C subunit also is being solved. The C subunit is maintained in its inactive state by forming a holoenzyme complex with an inhibitory regulatory (R) subunit. This R subunit has a well-defined domain structure that includes two tandem cAMP binding domains at the carboxy-terminus, each of which is homologous to the catabolite gene activator protein in Escherichia coli. Affinity labeling with 8N3 cAMP has identified residues that are in close proximity to the cAMP binding sites and is consistent with models of the cAMP binding sites based on the coordinates of the CAP crystal structure. An expression vector was constructed for the RI subunit and several mutations have been introduced. These mutations address 1) the major site of photoaffinity labeling, 2) a conserved arginine in the cAMP binding site, and 3) the consequences of deleting the entire second cAMP binding domain.
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PMID:CAMP-dependent protein kinase: prototype for a family of enzymes. 329 77

The primary structure of a region on hormone-sensitive lipase was determined to be: Lys-Thr-Glu-Pro-Met-Arg-Arg-Ser- Val-Ser-Glu-Ala-Ala-Leu-Thr-Gln-Pro-Glu-Gly-Pro-Leu-Gly-Thr-Asp-Ser-Leu-Lys. Ser-8 was the only residue in the intact protein phosphorylated by cyclic AMP-dependent protein kinase. However, Ser-10 also appeared to be present in a phosphorylated form, suggesting that it is a target for a distinct protein kinase in vivo.
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PMID:Primary structure of the site on bovine hormone-sensitive lipase phosphorylated by cyclic AMP-dependent protein kinase. 334 39

Four distinct tyrosine protein kinases active on poly(Glu4,Tyr1) and angiotensin II, and operationally termed TPK-I, TPK-IIA, TPK-IIB and TPK-III have been resolved and partially purified from rat spleen particulate fraction by combining DEAE-Sepharose, heparin-Sepharose, phosphocellulose and polylysine-agarose chromatographies. Once partially purified all of them are free of Ser/Thr-specific protein kinase activity as judged using casein, histones, protamine and the peptide Arg-Arg-Ala-Ser-Val-Ala as substrates. TPK-I (apparent molecular mass 64 kDa, by gel filtration) and TPK-IIA (54 kDa) share several properties, including substrate specificity and stimulation by heparin; the latter however is much more responsive to polylysine then the former (10- and 3-fold maximum stimulation, respectively). Conversely TPK-IIB (51 kDa) is markedly inhibited by heparin and it is also characterized by its unique substrate specificity: unlike the other three tyrosine protein kinases it by far prefers the tetrapeptide Glu-Tyr-Ala-Ala over the decapeptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly and readily phosphorylates band-3 protein of red cell membrane. The unusual preference for Mg2+ over Mn2+ as activator and the capability to phosphorylate calmodulin distinguish TPK-III (61 kDa) from the other isoenzymes. Moreover TPK-III is insensitive to heparin and polylysine and is inhibited by quercetin much more efficiently than the other enzymes (I50 = 10 microM). Upon incubation with [gamma-32P]ATP, TPK-I, TPK-IIA and TPK-III give rise to alkali-stable radiolabeled components of 61, 55 and 52 kDa respectively, as evaluated by PAGE/SDS. In every case such a radiolabeling takes place also in the presence of a large excess of phosphorylatable substrate (angiotensin II) while it is readily reversed by isotopic dilution with 10-fold excess unlabeled ATP, supporting the view that it represents an autophosphorylation process. No (auto)phosphorylation product(s) could be detected in TPK-IIB even if its amount, in terms of catalytic activity, was 10-fold higher than that of the others.
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PMID:Characterization of four tyrosine protein kinases from the particulate fraction of rat spleen. 335 7


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