Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 377, Issue 1, Pages 80-84Publisher
ACADEMIC PRESS INC
DOI: 10.1006/abbi.2000.1762
Keywords
actin-binding protein-280; nonmuscle filamin; calpain; protease; cAMP-dependent protein kinase; serine-proline motif
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Three different C-terminal regions of human endothelial actin-binding protein-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 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 moth, is discussed. (C) 2000 Academic Press.
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