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 lactating mammary gland synthesizes and secretes large amounts of phosphoproteins that mainly are associated with the casein fraction of milk. The free amino acids and inorganic phosphate of blood serve as building materials for casein, and the final product appears in milk as a colloidal-sized particle, the casein micelle. According to our present concept, the biosynthesis of casein occurs in two steps: synthesis of the polypeptide chain, followed by phosphate addition. Phosphate groups are transferred to the nascent casein by a protein kinase localized in the Golgi apparatus. The enzyme uses adenosine 5'-triphosphate as the phosphate donor and requires divalent cations. Neighboring amino acids may be important in determining which serine residues in casein are phosphorylated. This review discusses historical and current research on the phosphorylation of casein.
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PMID:Phosphorylation of casein by the lactating mammary gland: a review. 19 45

The cyclic AMP-dependent protein kinase catalyzes the phosphorylation of hydroxyproline present in the heptapeptide, Leu-Arg-Arg-Ala-Hyp-Leu-Gly. The Km value for the reaction with this substrate was high (approximately 18 mM) compared to the Km values reported for the analogous threonine and serine-containing peptides, which were 0.59 mM and 0.016 mM, respectively (Kemp, B.E., Graves, D.J., Benjamini, E., and Krebs, E.G. (1977) J. Biol. Chem. 252, 4888-4894). The Vmax value with the hydroxyproline-containing peptide was 1 mumol . min-1 mg-1 in contrast to Vmax values of 6 mumol . min-1 mg-1 and 20 mumol . min-1 mg-1 for the threonine- and serine-containing peptides, respectively. Phosphate esterified to hydroxyproline present in the peptide was relatively stable in hot alkali, only 10% being released as Pi within 30 min in 0.1 N NaOH at 100 degrees C, whereas all of the phosphate was released from the phosphoserine peptide analogue under these conditions. Phosphohydroxyproline in the peptide was also more stable to acid (5.7 N HCl, 110 degrees C) than phosphoserine, the time for 50% release as Pi being 15 h in contrast to 6 h for the latter.
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PMID:Phosphorylation of hydroxyproline in a synthetic peptide catalyzed by cyclic AMP-dependent protein kinase. 22 52

The phosphoprotein (P) and the large protein (L) constitute the RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV). We show that phosphate-free P protein expressed in bacteria is transcriptionally inactive when reconstituted with L protein and viral N-RNA template free of cellular protein kinase. Phosphorylation of P protein by a cellular kinase(s) was essential for transcription as well as for further phosphorylation by an L-associated kinase, the two kinases acting in a sequential (cascade) manner. Phosphate groups introduced by cell kinase were stable, whereas those due to L kinase underwent a turnover which was coupled to ongoing transcription. We present a model for the phosphorylation pathway of P protein and propose that continued phosphorylation and dephosphorylation of P protein may represent a transcriptional regulatory (on-off) switch of nonsegmented negative-strand RNA viruses.
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PMID:Sequential phosphorylation of the phosphoprotein of vesicular stomatitis virus by cellular and viral protein kinases is essential for transcription activation. 130 93

Both GS alpha-1 and GS alpha-4 were phosphorylated by the purified catalytic sub-unit of protein kinase A. Phosphate incorporation into 220 pmol and 190 pmol of GS alpha-4 and GS alpha-1 after a 1 hour incubation with kinase was 14 pmol and 10 pmol, respectively. These low levels of phosphorylation are due to the thermal lability of purified recombinant GS alpha. However, the phosphorylation was inhibited by guanine nucleotides (GDP-beta-S, GppNHp and GTP) and is, therefore, a specific event. We suggest that, as for GS alpha phosphorylation by protein kinase C (Pyne et al., 1992), the guanine nucleotide-free form of GS alpha is the most likely substrate. Guanine-nucleotides reduce the lifetime and, therefore availability for phosphorylation, of guanine-nucleotide free GS alpha. GS alpha phosphorylation by protein kinase A in vitro provides preliminary evidence that a similar phosphorylation of GS alpha may be an important regulatory event in cells.
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PMID:Phosphorylation of the recombinant spliced variants of the alpha-sub-unit of the stimulatory guanine-nucleotide binding regulatory protein (Gs) by the catalytic sub-unit of protein kinase A. 149 43

Estradiol-17 beta (E2) predetermined protein phosphorylation systems have been identified recently in midpregnant rat corpus luteum. Major type protein kinase activities in these systems were explored here using as probes protein kinase inhibitors. Luteal nuclear, mitochondrial, microsomal and cytosolic fractions were obtained from rats hysterectomized and hypophysectomized on day 12 of pregnancy and then treated for 72 h with E2. In vitro phosphate transfer from [gamma-32P]ATP was monitored by SDS-PAGE followed by autoradiography. Polymyxin B (PMB), 1-200 microM, a PKC inhibitor, completely blocked, in a dose dependent manner, the Ca2+ phospholipid (PL) stimulated radiolabeling of nuclear fraction Mr 79,000 substrate(s) as expected. Similarly, the calmodulin (CaM) antagonist compound 48/80, 1-20 micrograms/ml, inhibited the Ca2+/CaM-dependent phosphorylation of the microsomal fraction Mr 60,000 and Mr 56,000 proteins. The Ca2+ PL-enhanced labeling of mitochondrial fraction Mr 76,000 substrate(s) was only partially susceptible to inhibition by PMB or compound 48/80. Studies of microsomal fraction phosphoprotein bands not stimulated by added cofactors indicated that the radiolabeling of Mr 75,000 protein(s) was partially blocked by compound 48/80 but not by PMB. Phosphate transfer to Mr 41,000 protein(s) was inhibited by the cAMP-dependent kinase protein inhibitor (PKI), while the phosphorylation of Mr 31,000 protein(s) was refractory to all inhibitors employed here. Surprisingly, regardless of hormonal pretreatment, PMB and compound 48/80 activated in every subcellular fraction the cofactor independent appearance of at least one phosphoprotein band, between Mr 87,000-99,000. This novel observation should be instrumental in understanding the actions of these compounds towards living cells.
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PMID:Inhibition and stimulation of rat luteal protein phosphorylation by protein kinase effectors. 204 6

Chromatography of wild-type yeast extracts on DEAE-cellulose columns resolves two populations of glycogen synthase I (glucose-6-P-independent) and D (glucose-6-P-dependent) (Huang, K. P., Cabib, E. (1974) J. Biol. Chem. 249, 3851-3857). Extracts from a glycogen-deficient mutant strain, 22R1 (glc7), yielded only the D form of glycogen synthase. Glycogen synthase D purified from either wild-type yeast or from this glycogen-deficient mutant displayed two polypeptides with molecular masses of 76 and 83 kDa on sodium dodecyl sulfate-gel electrophoresis in a protein ratio of about 4:1. Phosphate analysis showed that glycogen synthase D from either strain of yeast contained approximately 3 phosphates/subunit. The 76- and 83-kDa bands of the mutant strain copurified through a variety of procedures including nondenaturing gel electrophoresis. These two polypeptides showed immunological cross-reactivity and similar peptide maps indicating that they are structurally related. The relative amounts of these two forms remained constant during purification and storage of the enzyme and after treatment with cAMP-dependent protein kinase or with protein phosphatases. The two polypeptides were phosphorylated to similar extent in vitro by the catalytic subunit of mammalian cyclic AMP-dependent protein kinase. Phosphorylation of the enzyme in the presence of labeled ATP followed by tryptic digestion and reversed phase high performance liquid chromatography yielded two labeled peptides from each of the 76- and 83-kDa subunits. Treatment of wild-type yeast with Li+ increased the glycogen synthase activity, measured in the absence of glucose-6-P, by approximately 2-fold, whereas similar treatment of the glc7 mutant had no effect. The results of this study indicate that the GLC7 gene is involved in a pathway that regulates the phosphorylation state of glycogen synthase.
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PMID:Purification and characterization of glycogen synthase from a glycogen-deficient strain of Saccharomyces cerevisiae. 211 10

The relationship between NS protein phosphorylation and RNA polymerase activities was determined in nucleocapsids purified from vesicular stomatitis virus grown in BHK cells. Phosphate incorporation into endogenous NS protein under transcription conditions reached a maximum value of 0.06 mol/mol of NS within 20 to 30 min, while RNA synthesis remained linear for 90 min. Phosphate incorporation into NS increased further upon addition of kinase-free NS protein but not upon addition of nucleocapsid kinase (prepared as described below), indicating that cessation of NS phosphorylation under transcribing conditions was due to substrate exhaustion. When NS was phosphorylated with 32P, less than 8% of the radiolabel was lost during subsequent transcription, indicating that this phosphate did not turn over. Treatment of nucleocapsids with 5'-p-fluorosulfonylbenzoyl adenosine resulted in greater than 90% inhibition of NS phosphorylation but had no effect on RNA polymerase activity. Fast protein liquid (Superose-6) chromatography of a nucleocapsid (L + NS) fraction resulted in complete separation of the viral (L + NS) protein from NS-phosphorylating activity. The addition of this kinase-free (L + NS) fraction to a kinase-deficient N-RNA fraction reconstituted an active RNA polymerase containing less than 20% of the original NS-phosphorylating activity. These results demonstrate that NS-phosphorylating activity is unnecessary during vesicular stomatitis virus RNA synthesis and indicate that all of the protein kinase(s) present in purified nucleocapsids is probably of cellular rather than viral origin.
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PMID:Phosphorylation of NS protein by vesicular stomatitis virus nucleocapsids: lack of effect during RNA synthesis and separation of kinase from L protein. 216 40

Arginine vasopressin (antidiuretic hormone, ADH) stimulation of sodium transport in high electrical resistance epithelia is accompanied by adenylate cyclase stimulation and cAMP accumulation. The hypothesis of direct phosphorylation of the purified amiloride-blockable epithelial Na+ channel protein by cAMP-dependent protein kinase A after ADH treatment of cultured cells was investigated in this study. Phosphate-depleted A6 cells (a cell line derived from toad kidney) were exposed to 32PO4(3-) in the absence or presence of basolateral ADH (100 milliunits/ml). After 20 min (the time needed for ADH to increase maximally Na+ transport), the Na+ channels were extracted from the cells and purified. At every stage of purification, only one subunit of the Na+ channel, namely, the 315-kDa subunit, was specifically phosphorylated as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography or scintillation counting. In addition, a polyclonal antibody raised against purified epithelial Na+ channel protein was able to immunoprecipitate the phosphorylated channel protein from a detergent-solubilized fraction of vasopressin-treated A6 cells. This same subunit was also specifically phosphorylated in vitro when the purified Na+ channel protein was incubated with gamma-[32P]ATP and the purified catalytic subunit of the cAMP-dependent protein kinase. Thus, only a single component, the 315-kDa subunit, of the Na+ channel protein complex (which is composed of six subunits) can be phosphorylated both in vivo and in vitro. This subunit is selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 2-3 mol of 32P/mol of protein.
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PMID:Phosphorylation of a single subunit of the epithelial Na+ channel protein following vasopressin treatment of A6 cells. 245 53

We have developed a simple method for phosphoamino acid analysis of 32P-labeled phosphoproteins using anion-exchange high-performance liquid chromatography (HPLC). Phosphoproteins undergo partial acid hydrolysis and the resulting hydrolysate is injected directly onto a column. The sample is then isocratically eluted from the column by 35 mM phosphoric acid at pH 3.0 with collected fractions analyzed by Cerenkov counting. Phosphoamino acid identification is accomplished by the comparison of the retention times of 32P-labeled peaks to retention times of phosphoamino acid standards which had been monitored at 206 nm. This method has greater sensitivity and is more reliable than cellulose thin-layer electrophoresis and the results obtained by high-efficiency Cerenkov counting can be evaluated immediately, instead of waiting days or weeks for autoradiographic development of cellulose plates. This HPLC protocol is an improvement over other published HPLC protocols in that there is no need for pre- or post-column derivatization and the free [32P]phosphate elutes long after the phosphoamino acids. Thus sensitivity is increased as there is no interference from the free phosphate. Selection of an HPLC anion-exchange column is critical for this separation. Only two of the four columns that we tested performed well. We present data from several phosphoproteins including calcium-calmodulin dependent protein kinase, the beta-subunit of the insulin receptor, and phosphorylated calmodulin to demonstrate the utility of this procedure.
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PMID:Determination of [32P]phosphoamino acids in protein hydrolysates by isocratic anion-exchange high-performance liquid chromatography. 245 72

Phosphate uptake by proximal renal cells derived from the human kidney occurs by a saturable process that is approximately 85% dependent on the presence of sodium. Kinetic analysis is consistent with two distinct transport events with Km of 0.08 and 0.63 mM and Vmax of 3.4 and 11.0 nmol.mg-1.3 min-1, respectively. Parathyroid hormone (PTH), isoproterenol, and prostaglandin E2 (PGE2) increased cellular adenosine 3',5'-cyclic monophosphate (cAMP). PTH-stimulated cAMP prevented binding of the photolabel 8-azido[32P]cAMP with a half-maximal effective concentration (EC50) of 1 nM PTH, 30-fold lower than the EC50 for intracellular cAMP accumulation. These data are qualitatively similar to those observed in OK cells. PTH did not inhibit phosphate uptake in human cells, although it activated cAMP-dependent protein kinase and increased cytosolic calcium. Thus phosphate uptake in human proximal renal cells maintained in short-term culture is unresponsive to PTH in spite of increased cytosolic calcium and activation of the cAMP pathway.
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PMID:Protein kinase A, cytosolic calcium, and phosphate uptake in human proximal renal cells. 247 35


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