Mutagenesis analysis of human SM22: characterization of actin binding
Mutagenesis analysis of human SM22: characterization of actin binding
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DOI：
10.1152/jappl.2000.89.5.1985
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发表时间：
2000-11-01
影响因子：
3.3
通讯作者：
Solway, J
中科院分区：
文献类型：
--
作者：
Fu, YP;Liu, HW;Solway, J
SM22是一种201个氨基酸的肌动蛋白结合蛋白，在平滑肌细胞中高水平表达。它与钙钙蛋白具有结构同源性，但SM22如何与肌动蛋白结合尚不清楚。我们在大肠杆菌中进行了定点诱变，产生了一系列nh2末端组氨酸（His）标记的人SM22突变体，并利用这些突变体分析了潜在肌动蛋白结合域的功能重要性。纯化的全长重组SM22与肌动蛋白结合，通过共沉淀法证实。结合不随钙浓度变化。SM22的COOH末端结构域是肌动蛋白亲和力所必需的，因为COOH末端截断的突变体[SM22-(1-186)和SM22-(1-166)]与肌动蛋白的共沉淀明显减少，并且SM22-(1-151)未检测到肌动蛋白结合。假定的肌动蛋白结合位点（154-KKAQEHKR-161）的内部缺失部分阻止了肌动蛋白的结合，点突变中和了该区域末端的一对或两对带正电的残基（KK154LL和/或KR160LL）。氨基酸170-180和170-186的内部缺失也分别部分或几乎完全抑制肌动蛋白共沉。在SM22中三个公认的蛋白激酶C或酪蛋白激酶II磷酸化位点中，只有Ser-181在体外容易被蛋白激酶C磷酸化，这种磷酸化大大降低了肌动蛋白的结合。Ser-181取代天冬氨酸（模拟丝氨酸磷酸化）也减少了肌动蛋白的结合。瞬时转染的气道肌细胞的免疫染色显示全长nh2末端flag标记的SM22与肌动蛋白丝共定位，而FLAG-SM22-(1-151)不与肌动蛋白丝共定位。这些数据证实了SM22在体外和体内都能与肌动蛋白结合，并首次证明了cooh末端区域内的多个区域是肌动蛋白完全亲和性所必需的。
SM22 is a 201-amino acid actin-binding protein expressed at high levels in smooth muscle cells. It has structural homology to calponin, but how SM22 binds to actin remains unknown. We performed site-directed mutagenesis to generate a series of NH2-terminal histidine (His)-tagged mutants of human SM22 in Escherichia coli and used these to analyze the functional importance of potential actin binding domains. Purified full-length recombinant SM22 bound to actin in vitro, as demonstrated by cosedimentation assay. Binding did not vary with calcium concentration. The COOH-terminal domain of SM22 is required for actin affinity, because COOH terminally truncated mutants [SM22-(1-186) and SM22-(1-166)] exhibited markedly reduced cosedimentation with actin, and no actin binding of SM22-(1-151) could be detected. Internal deletion of a putative actin binding site (154-KKAQEHKR-161) partially prevented actin binding, as did point mutation to neutralize either or both pairs of positively charged residues at the ends of this region (KK154LL and/or KR160LL). Internal deletion of amino acids 170-180 or 170-186 also partially or almost completely inhibited actin cosedimentation, respectively. Of the three consensus protein kinase C or casein kinase II phosphorylation sites in SM22, only Ser-181 was readily phosphorylated by protein kinase C in vitro, and such phosphorylation greatly decreased actin binding. Substitution of Ser-181 to aspartic acid (to mimic serine phosphorylation) also reduced actin binding. Immunostains of transiently transfected airway myocytes revealed that full-length NH2-terminal FLAG-tagged SM22 colocalizes with actin filaments, whereas FLAG-SM22-(1-151) does not. These data confirm that SM22 binds to actin in vitro and in vivo and, for the first time, demonstrate that multiple regions within the COOH-terminal domain are required for full actin affinity.