Expression in Escherichia coli and Phosphorylation with cAMP-Dependent Protein Kinase of the N-Terminal Region of Human Endothelial Actin-Binding Protein

1994 ◽  
Vol 202 (2) ◽  
pp. 764-771 ◽  
Author(s):  
D. Jay ◽  
A. Stracher
Keyword(s):  
Escherichia Coli ◽  
Protein Kinase ◽  
Binding Protein ◽  
Terminal Region ◽  
Actin Binding ◽  
Dependent Protein Kinase ◽  
Actin Binding Protein ◽  
Expression In Escherichia Coli ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals Role of the N-terminal region in covalent modification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: comparison of phosphorylation and ADP-ribosylation

1995 ◽  
Vol 309 (1) ◽  
pp. 119-125 ◽  
Author(s):  
J L Rosa ◽  
J X Pérez ◽  
F Ventura ◽  
A Tauler ◽  
J Gil ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Covalent Modification ◽  
Terminal Region ◽  
Bifunctional Enzyme ◽  
Dependent Protein Kinase ◽  
Adp Ribosylation ◽  
Camp Dependent Protein Kinase ◽  
Arginine Residues ◽  
Dependent Protein

The effect of cyclic AMP (cAMP)-dependent phosphorylation and ADP-ribosylation on the activities of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), was investigated in order to determine the role of the N-terminus in covalent modification of the enzyme. The bifunctional enzyme was demonstrated to be a substrate in vitro for arginine-specific ADP-ribosyltransferase: 2 mol of ADP-ribose was incorporated per mol of subunit. The Km values for NAD+ and PFK-2/FBPase-2 were 14 microM and 0.4 microM respectively. A synthetic peptide (Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln) corresponding to the site phosphorylated by cAMP-dependent protein kinase was ADP-ribosylated on all three arginine residues. Analysis of ADP-ribosylation of analogue peptides containing only two arginine residues, with the third replaced by alanine, revealed that ADP-ribosylation occurred predominantly on the two most C-terminal arginine residues. Sequencing of the ADP-ribosylated native enzyme also demonstrated that the preferred sites were at Arg-29 and Arg-30, which are just N-terminal to Ser-32, whose phosphorylation is catalysed by cAMP-dependent protein kinase (PKA). ADP-ribosylation was independent of the phosphorylation state of the enzyme. Furthermore, ADP-ribosylation of the enzyme decreased its recognition by liver-specific anti-bifunctional-enzyme antibodies directed to its unique N-terminal region. ADP-ribosylation of PFK-2/FBPase-2 blocked its phosphorylation by PKA, and decreased its PFK-2 activity, but did not alter FBPase-2 activity. In contrast, cAMP-dependent phosphorylation inhibited the kinase and activated the bisphosphatase. These results demonstrate that ADP-ribosylation of arginine residues just N-terminal to the site phosphorylated by PKA modulate PFK-2 activity by an electrostatic and/or steric mechanism which does not involved uncoupling of N- and C-terminal interactions as seen with cAMP-dependent phosphorylation.

Regulation of lung epithelial cell morphology by cAMP-dependent protein kinase type I isozyme

2001 ◽  
Vol 280 (6) ◽  
pp. L1282-L1289 ◽  
Author(s):  
Stephanie E. Porter ◽  
Lori D. Dwyer-Nield ◽  
Alvin M. Malkinson
Keyword(s):  
Protein Kinase ◽  
Cell Junctions ◽  
Actin Binding ◽  
Regulatory Subunit ◽  
Type I ◽  
Stable Transfection ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Lung Epithelial ◽  
Dependent Protein

Cell shape is mediated in part by the actin cytoskeleton and the actin-binding protein vinculin. These proteins in turn are regulated by protein phosphorylation. We assessed the contribution of cAMP-dependent protein kinase A isozyme I (PKA I) to lung epithelial morphology using the E10/E9 sibling cell lines. PKA I concentration is high in flattened, nontumorigenic E10 cells but low in their round E9 transformants. PKA I activity was lowered in E10 cells by stable transfection with a dominant negative RIα mutant of the PKA I regulatory subunit and was raised in E9 cells by stable transfection with a wild-type Cα catalytic subunit construct. Reciprocal changes in morphology ensued. E10 cells became rounder and grew in colonies, their actin microfilaments were disrupted, and vinculin localization at cell-cell junctions was diminished. The converse occurred in E9 cells on elevating their PKA I content. Demonstration that PKA I is responsible for the dichotomy in these cellular behaviors suggests that manipulating PKA I concentrations in lung cancer would provide useful adjuvant therapy.

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