Fu Y, Liu H W, Forsythe S M, Kogut P, McConville J F, Halayko A J, Camoretti-Mercado B, Solway J
Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
J Appl Physiol (1985). 2000 Nov;89(5):1985-90. doi: 10.1152/jappl.2000.89.5.1985.
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 NH(2)-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 NH(2)-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.
SM22是一种由201个氨基酸组成的肌动蛋白结合蛋白，在平滑肌细胞中高水平表达。它与钙调蛋白具有结构同源性，但SM22如何与肌动蛋白结合仍不清楚。我们进行了定点诱变，在大肠杆菌中生成了一系列人SM22的氨基末端组氨酸（His）标记突变体，并利用这些突变体分析潜在肌动蛋白结合域的功能重要性。通过共沉降分析表明，纯化的全长重组SM22在体外与肌动蛋白结合。结合不随钙浓度变化。SM22的羧基末端结构域是肌动蛋白亲和力所必需的，因为羧基末端截短的突变体[SM22-(1-186)和SM22-(1-166)]与肌动蛋白的共沉降明显减少，并且未检测到SM22-(1-151)与肌动蛋白的结合。一个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结合，使该区域末端的一对或两对带正电荷残基中和的点突变（KK154LL和/或KR160LL）也有同样效果。氨基酸170-180或170-186的内部缺失也分别部分或几乎完全抑制了肌动蛋白共沉降。在SM22的三个共有蛋白激酶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结合。将Ser-181替换为天冬氨酸（模拟丝氨酸磷酸化）也降低了肌动蛋白结合。对瞬时转染的气道肌细胞的免疫染色显示，全长氨基末端FLAG标记的SM22与肌动蛋白丝共定位，而FLAG-SM22-(1-151)则不然。这些数据证实SM22在体外和体内均与肌动蛋白结合，并且首次证明羧基末端结构域内的多个区域是完全肌动蛋白亲和力所必需的。
