EXPRESSION AND PHOSPHORYLATION OF THE LISTERIA-MONOCYTOGENES ACTA PROTEIN IN MAMMALIAN-CELLS
EXPRESSION AND PHOSPHORYLATION OF THE LISTERIA-MONOCYTOGENES ACTA PROTEIN IN MAMMALIAN-CELLS
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DOI：
10.1073/pnas.90.24.11890
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发表时间：
1993-12-15
影响因子：
11.1
通讯作者：
PORTNOY, DA
中科院分区：
文献类型：
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作者：
BRUNDAGE, RA;SMITH, GA;PORTNOY, DA
单核细胞增多性李斯特菌在受感染的真核细胞内的运动提供了一个简单的模型系统来研究非肌肉细胞中基于肌动蛋白的运动机制。单核细胞增生李斯特氏菌的 actA 基因需要诱导宿主肌动蛋白丝的聚合[Kocks, C.、Gouin, E.、Tabouret, M.、Berche, P.、Ohayon, H. & Cossart, P. (1990) Cell 68, 521-531 ； Domann, E.、Wehland, J.、Rohde, M.、Pistor, S.、Hartl, M.、Goebel, W.、Leimeister-Wachter, M.、Wuenscher, M. 和 Chakraborty, T. (1992) EMBO J. 11, 1981-1990]。在本研究中，构建了 actA 基因内的框内缺失突变，并通过等位基因交换将其引入单核细胞增生李斯特氏菌染色体中。这种突变导致小鼠的毒力降低（3 个数量级）。在组织培养细胞中，actA突变体在肌动蛋白丝的成核方面绝对有缺陷，因此细胞间的扩散受到损害。针对包含 ActA 富含脯氨酸重复序列 (DFPPPPTDEEL) 的合成肽产生的抗血清用于表征 ActA 蛋白的表达。来自胞外细菌的 ActA 蛋白在 SDS/PAGE 上迁移为 97-kDa 多肽，而来自受感染细胞的蛋白迁移为三种不同的多肽，一种与 97-kDa 胞外形式和两种稍大的物种共迁移。用冈田酸处理受感染的细胞会导致所有形式的 ActA 数量减少，并出现更大种类的 ActA。对细胞内细菌免疫沉淀的 ActA 进行磷酸酶处理，导致较大的两个物种转化为 97 kDa 形式。用 P-32i 标记受感染的细胞，然后进行免疫沉淀，结果表明 ActA 的最大分子形式被磷酸化。总而言之，这些数据表明 ActA 在细胞内生长过程中被磷酸化。 ActA 细胞内修饰的意义尚不清楚，但我们推测它可能调节 ActA 的细胞内活性。
Movement of Listeria monocytogenes within infected eukaryotic cells provides a simple model system to study the mechanism of actin-based motility in nonmuscle cells. The actA gene of L. monocytogenes is required to induce the polymerization of host actin filaments [Kocks, C., Gouin, E., Tabouret, M., Berche, P., Ohayon, H. & Cossart, P. (1990) Cell 68, 521-531; Domann, E., Wehland, J., Rohde, M., Pistor, S., Hartl, M., Goebel, W., Leimeister-Wachter, M., Wuenscher, M. & Chakraborty, T. (1992) EMBO J. 11, 1981-1990]. In this study, an in-frame deletion mutation within the actA gene was constructed and introduced into the L. monocytogenes chromosome by allelic exchange. This mutation resulted in a decrease (3 orders of magnitude) in virulence for mice. In tissue culture cells, the actA mutant was absolutely defective for the nucleation of actin filaments and consequently was impaired in cell-to-cell spread. Antiserum raised to a synthetic peptide encompassing the proline-rich repeat (DFPPPPTDEEL) of ActA was used to characterize the expression of the ActA protein. The ActA protein derived from extracellular bacteria migrated as a 97-kDa polypeptide upon SDS/PAGE, whereas the protein from infected cells migrated as three distinct polypeptides, one that comigrated with the 97-kDa extracellular form and two slightly larger species. Treatment of infected cells with okadaic acid resulted in decreased amounts of all forms of ActA and the appearance of a larger species of ActA. Phosphatase treatment of ActA immunoprecipitated from intracellular bacteria resulted in conversion of the larger two species to the 97-kDa form. Labeling of infected cells with P-32i followed by immunoprecipitation showed that the largest molecular form of ActA was phosphorylated. Taken together, these data indicate that ActA is phosphorylated during intracellular growth. The significance of the intracellular modification of ActA is not known, but we speculate that it may modulate the intracellular activity of ActA.