Determination of a cAMP-Dependent Protein Kinase Phosphorylation Site in the C-Terminal Region of Human Endothelial Actin-Binding Protein

• DOI: 10.1006/abbi.2000.1762
• Publication Date: 2002-09-16T13:49:45Z
• Authors: David Jay; Elizabeth J. García; José Enrique Lara; Miguel Angel Medina; María de la Luz I...
• Keywords: Blood Platelets; Threonine; 0301 basic medicine; Calpain; Filamins; Immune Sera; Amino Acid Motifs; Microfilament Proteins; Cyclic AMP-Dependent Protein Kinases; Peptide Fragments; Recombinant Proteins; Molecular Weight; Phosphoserine; 03 medical and health sciences; Contractile Proteins; Amino Acid Substitution; Sequence Analysis, Protein; Mutation; Serine; Humans; Endothelium; Phosphorylation

Three different C-terminal regions of human endothelial actin-binding protein-280 (ABP-280 or ABP; nonmuscle filamin) were subcloned and efficiently expressed in the Escherichia coli BL21 (DE3) system as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As predicted by the aminoacid sequence one of the fragments, a 109-kDa peptide (residues 1671-2647), contained a calpain cleavage site and two potential cAMP-dependent protein kinase (PKA) phosphorylation sites (serine 2152 and threonine 2336). A second fragment, a 74-kDa peptide (residues 1671-2331), contained a calpain cleavage site and one of the three presumptive PKA phosphorylation sites (serine 2152). The third fragment, a 48-kDa peptide (residues 2223-2647), contained only one of the PKA sites (threonine 2336). Phosphorylation of these truncated peptides indicated that only the fragments containing serine 2152 incorporated phosphate after PKA treatment. Site-directed mutagenesis analysis confirmed that serine 2152 is the unique substrate for PKA in the C-terminal region of ABP. The functional significance of phosphorylation of this residue, which belongs to a serine-proline motif, is discussed.