scholarly journals Rabbit muscle fructose-1,6-bisphosphatase is phosphorylatedin vivo.

2003 ◽  
Vol 50 (1) ◽  
pp. 115-121 ◽  
Author(s):  
Dariusz Rakus ◽  
Marek Zarzycki ◽  
Andrzej Dzugaj
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Kinase ◽  
Inorganic Phosphate ◽  
Kinetic Properties ◽  
Rabbit Muscle ◽  
Activity Regulation ◽  
Sds Page ◽  
Fructose 1,6 Bisphosphatase ◽  
Amp Inhibition ◽  
Kinase A

Phosphorylated fructose-1,6-bisphosphatase (FBPase) was isolated from rabbit muscle in an SDS/PAGE homogeneous form. Its dephosphorylation with alkaline phosphatase revealed 2.8 moles of inorganic phosphate per mole of FBPase. The phosphorylated FBPase (P-FBPase) differs from the dephosphorylated enzyme in terms of its kinetic properties like K(m) and k(cat), which are two times higher for the phosphorylated FBPase, and in the affinity for aldolase, which is three times lower for the dephosphorylated enzyme. Dephosphorylated FBPase can be a substrate for protein kinase A and the amount of phosphate incorporated per FBPase monomer can reach 2-3 molecules. Since interaction of muscle aldolase with muscle FBPase results in desensitisation of the latter toward AMP inhibition (Rakus & Dzugaj, 2000, Biochem. Biophys. Res. Commun. 275, 611-616), phosphorylation may be considered as a way of muscle FBPase activity regulation.

AtPIP5K1, an Arabidopsis thaliana phosphatidylinositol phosphate kinase, synthesizes PtdIns(3,4)P2 and PtdIns(4,5)P2 in vitro and is inhibited by phosphorylation

2001 ◽  
Vol 359 (3) ◽  
pp. 583-589 ◽  
Author(s):  
Tomas WESTERGREN ◽  
Stephen K. DOVE ◽  
Marianne SOMMARIN ◽  
Christophe PICAL
Keyword(s):  
Arabidopsis Thaliana ◽  
Alkaline Phosphatase ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Substrate Specificities ◽  
Phosphatidylinositol Phosphate ◽  
Dual Substrate Specificity ◽  
Kinase A ◽  
Dual Substrate

PtdIns phosphate kinases (PIPkins), which generate PtdInsP2 isomers, have been classified into three subfamilies that differ in their substrate specificities. We demonstrate here that the previously identified AtPIP5K1 gene from Arabidopsis thaliana encodes a PIPkin with dual substrate specificity in vitro, capable of phosphorylating PtdIns3P and PtdIns4P to PtdIns(3,4)P2 and PtdIns(4,5)P2 respectively. We also show that recombinant AtPIP5K1 is phosphorylated by protein kinase A and a soluble protein kinase from A. thaliana. Phosphorylation of AtPIP5K1 by protein kinase A is accompanied by a 40% inhibition of its catalytic activity. Full activity is recovered by treating phosphorylated AtPIP5K1 with alkaline phosphatase.

scholarly journals Rapid purification and identification of calcyphosine, a Ca2+-binding protein phosphorylated by protein kinase A

1995 ◽  
Vol 306 (1) ◽  
pp. 147-151 ◽  
Author(s):  
R Lecocq ◽  
F Lamy ◽  
C Erneux ◽  
J E Dumont
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Conformational Changes ◽  
Hydrophobic Interaction Chromatography ◽  
Peptide Sequencing ◽  
Native Form ◽  
Sds Page ◽  
Dependent Protein ◽  
Rapid Purification ◽  
Kinase A

A method is presented for the rapid purification of dog thyroid calcyphosine, a protein previously identified as a major substrate for cyclic AMP-dependent protein kinase in dog thyroid slices stimulated by thyrotropin [Lecocq, Lamy and Dumont (1979) Eur. J. Biochem. 102, 147-152]. The protein was previously identified as a spot on two-dimensional gels and is now purified in its native form by a procedure involving three chromatographic steps. Homogeneous calcyphosine identified by SDS/PAGE, immunoblotting and peptide sequencing can be obtained within 7 h. As for calmodulin, Ca(2+)-dependent conformational changes can be shown by Ca(2+)-dependent hydrophobic interaction chromatography using phenyl-Sepharose. Unlike calmodulin, calcyphosine is a substrate for protein kinase A.

Renal brush border membranes from mice with X-linked hypophosphatemia: protein composition, phosphate binding capacity, and protein kinase activity

1984 ◽  
Vol 62 (11) ◽  
pp. 1394-1400 ◽  
Author(s):  
Michèle G. Brunette ◽  
Serge Allard ◽  
Richard Béliveau
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Kinase ◽  
Brush Border ◽  
Inorganic Phosphate ◽  
Kinase Activity ◽  
Protein Composition ◽  
Protein Kinase Activity ◽  
Phosphate Binding ◽  
Phosphate Binding Protein ◽  
Substrate Concentrations

Phosphate uptake by brush-border membrane (BBM) vesicles prepared from hypophosphatemic mice (Hyp) is reduced by half relative to BBM vesicles from normal mice. To investigate this abnormality, we studied the protein composition of BBM, their capacity to bind inorganic phosphate, and their protein kinase activity with and without the addition of exogenous cAMP, in normal and Hyp mice. Gradient polyacrylamide gel electrophoresis of BBM proteins showed 27 bands which were identical in normal and Hyp mice. Incubation of the membranes with ortho[32P]phosphate at 0 °C revealed a phosphate binding protein with an apparent molecular weight (Mr) of 79 000, which has been previously identified in rats as the monomer of alkaline phosphatase. In normal mice, the Scatchard plot of phosphate binding was not linear, suggesting heterogeneity of the binding sites with two major components. At high substrate concentrations, the affinity (K) was 1.42 rnM and maximal binding (Bmax) was 83 pmol/mg protein. At low substrate concentrations, these values were 0.07 mM and 10.9 pmol/mg, respectively. In Hyp mice BBM, only one binding system was found with K and Bmax values of 0.38 mM and 53.8 pmol/mg. Incubation of the membranes with 25 μM[γ-32P]ATP resulted in the phosphorylation of 11 proteins. The major band (Mr: 79 000) corresponded to the inorganic phosphate binding protein, i.e., to the alkaline-phosphatase monomer. The 11 proteins showed maximal phosphorylation at pH 10. The protein of 79 000 Mr showed a second peak of phosphorylation at pH 7.5. When cAMP was added to the incubation medium, another phosphorylated protein clearly appeared at apparent Mr of 85 000. A dose–response curve demonstrated a maximal phosphorylation with 10 μM cAMP. In Hyp mice, the phosphorylation showed a tendency to be decreased for all 11 proteins. However, cAMP-dependent phosphorylation was preserved, suggesting a normal membrane protein kinase activity.

scholarly journals ADP-ribosylation of Rho proteins by Clostridium botulinum exoenzyme C3 is influenced by phosphorylation of Rho-associated factors

1994 ◽  
Vol 300 (1) ◽  
pp. 133-139 ◽  
Author(s):  
G Fritz ◽  
K Aktories
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Clostridium Botulinum ◽  
Rho Proteins ◽  
Cho Cell ◽  
Adp Ribosylation ◽  
Cell Extracts ◽  
Kinase A

Specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study the involvement of phosphorylation in the regulation of the low-molecular-mass GTP-binding protein Rho. Dephosphorylation of CHO cell extracts by alkaline phosphatase treatment resulted in a 80-90% reduction in the C3-catalysed [32P]ADP-ribosylation of Rho proteins in both cytosolic and membrane fractions. Similar results were obtained after dephosphorylation with protein phosphatase type-1 from bovine retina, whereas type-2B and type-2C phosphatases had no effect on the level of subsequent [32P]ADP-ribosylation of Rho by C3. Incubation of CHO cell lysate under phosphorylation conditions increased the subsequent C3-mediated [32P]ADP-ribosylation of Rho proteins. The protein kinase inhibitors H7 and H9 had no effect on [32P]ADP-ribosylation at concentrations which are specific for inhibition of protein kinase A or C. Recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was phosphorylated by protein kinase A; however, the phosphorylation had no stimulatory effect on the ADP-ribosylation of GST-RhoA by C3. An approx. 48 kDa phosphoprotein was identified which bound specifically to recombinant GST-RhoA fusion protein. By gel-permeation chromatography, Rho-containing complexes of approx. 50 kDa and 130-170 kDa were detected. The ADP-ribosylation of Rho in the 130-170 kDa complex was reduced by alkaline phosphatase pretreatment. The data suggest that Rho activity is influenced by phosphorylation of Rho-associated regulatory factors. Phosphorylation/dephosphorylation of these Rho-regulating factors appears to alter the ability of Rho to serve as a substrate for C3-induced [32P]ADP-ribosylation.

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