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 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.
Jay
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Departamento de Cultivo de Tejidos, Instituto Nacional de Cardiología, Ignacio Chávez, Juan Badiano #1, D. F. México. jay@mailer.main.conacyt.mx
García
E J
EJ
Lara
J E
JE
Medina
M A
MA
de la Luz Ibarra
M
M
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution / Q000235:genetics; D001792:Blood Platelets; D002154:Calpain / Q000378:metabolism; D003285:Contractile Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004727:Endothelium / Q000737:chemistry*; D064448:Filamins; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000235:genetics
D003285:Contractile Proteins; C578661:FLNC protein, human; D064448:Filamins; D007106:Immune Sera; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010768:Phosphoserine; D013912:Threonine; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D002154:Calpain