Abstract
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.
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Gorlin JB, Yamin R, Egam S, Stewart M, Stossel TP, Kwiatkowski DJ, Hartwig JH: Human endothelial actin-binding protein (ABP-280, non-muscle filamin): A molecular leaf spring. J Cell Biol 111: 1089-1105, 1990
Zhuang QQ, Rosenberg S, Lawrence J, Stracher A: Role of actin binding protein phosphorylation in platelet cytoskeleton assembly. Biochem Biophys Res Commun 118: 508-513, 1984
Chen M, Stracher A: In situ phosphorylation of platelet actin-binding protein by cAMP-dependent protein kinase stabilizes it against proteolysis by calpain. J Biol Chem 264: 14282-14289, 1989
Wu MP, Jay D, Stracher A: Existence of multiple phosphorylated forms of human platelet actin binding protein. Cell Mol Biol Res 40: 351-357, 1994
Jay D, Stracher A: Expression in Escherichia coli and phosphorylation with camp-dependent protein kinase of the N-terminal region of human endothelial actin-binding protein. Biochem Biophys Res Commun 202: 764-771, 1994
Jay D, Stracher A: Expression in Escherichia coli, phosphorylation with camp-dependent protein kinase and proteolysis by calpain of a 71 kD domain of human endothelial actin binding protein. Biochem Biophys Res Commun 232: 555-558, 1997
Jay D, García EJ, Lara JE, Medina MA, Ibarra M de la L: Determination of a cAMP-dependent protein kinase phosphorylation site in the C-terminal region of human endothelial actin-binding protein. Arch Biochem Biophys 377: 80-84, 2000
Tabor S, Richardson CC: A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82: 1074-1078, 1985
Sambrook J, Fritsch EF, Maniatis T: In: Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989
Harlow E, Lane D: Antibodies. In: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, 1988, pp 421-470
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970
Lindberg U, Markey F: In: D.E. MacIntyre, J.L. Gordon (eds), Platelets in Biology and Pathology III. Elsevier Science Publishers B.V., Amsterdam, 1987, pp 220-248
Schutkowski M, Bernhardt A, Zhou XZ, Shen M, Reimer U, Rahfeld JU, Lu KP, Fischer G: Role of phosphorylation in determining the backbone dynamics of the serine/threonine-proline motif and Pin1 substrate recognition. Biochemistry 37: 5566-5575, 1998
Gothel SF, Marahiel MA: Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts. Cell Mol Life Sci 55: 423-436, 1999
Gustke N, Steiner B, Mandelkow EM, Biernat J, Meyer HE, Goedert M, Mandelkow E: The Alzheimer-like phosphorylation of tau protein reduces microtubule binding and involves Ser-Pro and Thr-Pro motifs. FEBS Lett 307: 199-205, 1992
Mandelkow EM, Mandelkow E: Tau as a marker for Alzheimer's disease. Trends Biochem Sci 18: 480-483, 1993
Mandelkow EM, Schweers O, Drewes G, Biernat J, Gustke N, Trinczek B, Mandelkow E: Structure, microtubule interactions, and phosphorylation of tau protein. Ann NY Acad Sci 777: 96-106, 1996
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Jay, D., García, E.J. & de la Luz Ibarra, M. In situ determination of a PKA phosphorylation site in the C-terminal region of filamin. Mol Cell Biochem 260, 49–53 (2004). https://doi.org/10.1023/B:MCBI.0000026052.76418.55
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DOI: https://doi.org/10.1023/B:MCBI.0000026052.76418.55