In situ determination of a PKA phosphorylation site in the C-terminal region of filamin
In situ determination of a PKA phosphorylation site in the C-terminal region of filamin
复制标题
DOI：
10.1023/b:mcbi.0000026052.76418.55
复制
发表时间：
2004-05-01
影响因子：
4.3
通讯作者：
Ibarra, MD
中科院分区：
文献类型：
--
作者：
Jay, D;García, EJ;Ibarra, MD
通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹分析，亚克隆了人内皮肌动蛋白结合蛋白-280（ABP-280或ABP，非肌丝蛋白）的C-末端区域，并在哺乳动物细胞系统中有效表达。正如氨基酸序列所预测的那样，该片段是一个79 kD肽（残基1671 - 2361，加上表达质粒中包含的N末端融合肽的3.9 kD），含有两个潜在的cAMP依赖性蛋白激酶（PKA）磷酸化位点（丝氨酸2152和苏氨酸2336）预计存在于该分子区域。在cAMP升高剂的存在下孵育细胞增强了P-32摄取到片段中。定点突变分析表明，丝氨酸2152是内源性激活PKA的ABP C-末端区域的唯一底物。这一残基，它属于丝氨酸-脯氨酸基序的磷酸化的功能的影响，细丝蛋白在细胞骨架重组的作用方面进行了讨论。
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 P-32 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.