Enhanced Photoelectrochemical Method for Sensitive Detection of Protein Kinase A Activity Using TiO2/g-C3N4, PAMAM Dendrimer, and Alkaline Phosphatase

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.

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Okadaic acid enhances prostaglandin E1-induced alkaline phosphatase activity in osteoblast-like cells: regulation at a point downstream from protein kinase A

Prostaglandins Leukotrienes and Essential Fatty Acids ◽

10.1016/s0952-3278(96)90042-3 ◽

1996 ◽

Vol 55 (5) ◽

pp. 357-361 ◽

Cited By ~ 12

Author(s):

Y. Ito ◽

A. Suzuki ◽

Y. Watanabe-Tomita ◽

Y. Oiso ◽

O. Kozawa

Keyword(s):

Alkaline Phosphatase ◽

Protein Kinase ◽

Protein Kinase A ◽

Phosphatase Activity ◽

Okadaic Acid ◽

Alkaline Phosphatase Activity ◽

Prostaglandin E1 ◽

Kinase A

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

Biochemical Journal ◽

10.1042/bj3000133 ◽

1994 ◽

Vol 300 (1) ◽

pp. 133-139 ◽

Cited By ~ 13

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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