Determination of a cAMP-dependent protein kinase phosphorylation site in the C-terminal region of human endothelial actin-binding protein

Three different C-terminal regions of human endothelial actin-binding prote in-280 (ABP-280 or ABP; non-muscle 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 ami noacid sequence one of the fragments, a 109-kDa peptide (residues 1671-2647 ), contained a calpain cleavage site and two potential cAMP-dependent prote in kinase (PKA) phosphorylation sites (serine 2152 and threonine 2336). A s econd fragment, a 74-kDa peptide (residues 1671-2331), contained a calpain cleavage site and one of the three presumptive PKA phosphorylation sites (s erine 2152). The third fragment, a 48-kDa peptide (residues 2223-2647), con tained only one of the PKA sites (threonine 2336). Phosphorylation of these truncated peptides indicated that only the fragments containing serine 215 2 incorporated phosphate after PKA treatment. Site-directed mutagenesis ana lysis confirmed that serine 2152 is the unique substrate for PKA in the C-t erminal region of ABP. The functional significance of phosphorylation of th is residue, which belongs to a serine-proline moth, is discussed. (C) 2000 Academic Press.