In situ determination of a PKA phosphorylation site in the C-terminal region of filamin.
A C-terminal region of human endothelial actin-binding protein-280 (ABP-280 or ABP, non-muscle filamin) was subcloned and efficiently expressed in a mammalian cells system as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis. As predicted by the aminoacid sequence, the fragment, a 79 kD peptide (residues 1671-2361, plus 3.9 kD from an N-terminal fusion peptide included in the expression plasmid), contained the two potential cAMP-dependent protein kinase (PKA) phosphorylation sites (serine 2152 and threonine 2336) predicted to be present in this region of the molecule. Incubation of cells in the presence of cAMP-elevating agents enhanced 32P uptake into the fragment. Site-directed mutagenesis analysis indicated that serine 2152 is the unique substrate in the C-terminal region of ABP for endogenously activated PKA. The functional implications of phosphorylation of this residue, which belongs to a serine-proline motif, are discussed in terms of the role of filamin in cytoskeleton reorganization.
Jay
David
D
Departamento de Biomedicina Molecular Cardiovascular, Instituto Nacional de Cardiología, Ignacio Chávez, Mexico City, Mexico. jay@sin.conacyt.mx
García
Elizabeth J
EJ
de la Luz Ibarra
María
M
D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases; D004727:Endothelium / Q000737:chemistry; D064448:Filamins; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism
D003285:Contractile Proteins; C578661:FLNC protein, human; D064448:Filamins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases