Identification of critical functional and regulatory domains in gelsolin.
Identification of critical functional and regulatory domains in gelsolin.
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DOI：
10.1083/jcb.108.5.1717
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发表时间：
1989-05
期刊：
影响因子：
--
通讯作者：
Yin HL
中科院分区：
文献类型：
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作者：
Kwiatkowski DJ;Janmey PA;Yin HL
凝溶胶蛋白可以切断肌动蛋白丝，使肌动蛋白丝组装成核，并覆盖肌动蛋白丝快速生长的末端。这些功能被Ca 2+激活，并被多磷酸肌醇（PPI）抑制。我们在这里报告的研究，旨在划定关键领域内的凝溶胶蛋白的缺失诱变，使用COS细胞分泌截短的血浆凝溶胶蛋白后，DNA转染。删除11%的凝溶胶蛋白从COOH末端导致其能力的主要损失，以促进肌动蛋白丝组装成核步骤，这表明COOH末端结构域是重要的，在这个功能。与此相反，删除79%的凝溶胶蛋白序列的衍生物表现出正常的PPI调节的肌动蛋白酶切割活性。结合以前的结果，使用蛋白水解片段，我们推断，在COOH末端的最小切断凝溶胶蛋白衍生物在这里确定的11个氨基酸序列介导PPI调节的结合的凝溶胶蛋白的肌动蛋白丝的两侧切断前。删除只有3%的凝溶胶蛋白在COOH末端，包括一个二羧酸序列上发现的肌动蛋白的NH 2末端类似，导致在损失的Ca 2+的需求丝切断和单体结合。由于肌动蛋白中的这些残基被认为是凝溶胶蛋白的潜在结合位点，我们的结果提出了凝溶胶蛋白COOH末端的类似序列可能充当Ca 2+调节的假底物的可能性。然而，缺失69-79%的凝溶胶蛋白COOH-末端残基的衍生物表现出正常的Ca 2+调节切割活性，建立了NH 2-末端区域的内在Ca 2+调节。Ca 2+调节的一种或两种机制可能发生在肌动蛋白切断蛋白的凝溶胶蛋白家族的成员中。
Gelsolin can sever actin filaments, nucleate actin filament assembly, and cap the fast-growing end of actin filaments. These functions are activated by Ca2+ and inhibited by polyphosphoinositides (PPI). We report here studies designed to delineate critical domains within gelsolin by deletional mutagenesis, using COS cells to secrete truncated plasma gelsolin after DNA transfection. Deletion of 11% of gelsolin from the COOH terminus resulted in a major loss of its ability to promote the nucleation step in actin filament assembly, suggesting that a COOH-terminal domain is important in this function. In contrast, derivatives with deletion of 79% of the gelsolin sequence exhibited normal PPI-regulated actin filament-severing activity. Combined with previous results using proteolytic fragments, we deduce that an 11- amino acid sequence in the COOH terminus of the smallest severing gelsolin derivative identified here mediates PPI-regulated binding of gelsolin to the sides of actin filaments before severing. Deletion of only 3% of gelsolin at the COOH terminus, including a dicarboxylic acid sequence similar to that found on the NH2 terminus of actin, resulted in a loss of Ca2+-requirement for filament severing and monomer binding. Since these residues in actin have been implicated as potential binding sites for gelsolin, our results raise the possibility that the analogous sequence at the COOH terminus of gelsolin may act as a Ca2+-regulated pseudosubstrate. However, derivatives with deletion of 69-79% of the COOH-terminal residues of gelsolin exhibited normal Ca2+ regulation of severing activity, establishing the intrinsic Ca2+ regulation of the NH2-terminal region. One or both mechanisms of Ca2+ regulation may occur in members of the gelsolin family of actin- severing proteins.