The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor
The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor
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DOI：
10.1074/jbc.m003009200
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发表时间：
2000-11-10
影响因子：
4.8
通讯作者：
Schesser, K
中科院分区：
文献类型：
--
作者：
Dukuzumuremyi, JM;Rosqvist, R;Schesser, K
致病性耶尔森氏菌将蛋白质直接注入真核细胞，干扰许多细胞过程，包括吞噬作用和炎症相关的宿主反应。这些注射的蛋白质之一，耶尔森氏菌蛋白激酶A（YpktA），以前已被证明会影响培养的真核细胞的形态，以及定位到质膜后，其注射到HeLa细胞。在这里，它表明，这些活动介导的YpkA的可分离的结构域。氨基末端，其中包含的激酶结构域，是足够的本地化YpkA的质膜，而YpkA的羧基末端是必需的YpkAs形态的影响。发现YpkAs羧基端区域影响含肌动蛋白的应力纤维的水平，以及阻断耶尔森氏菌感染细胞中GTdR RhoA的活化。我们发现YpkA的羧基末端区域，包含与几种真核RhoA结合激酶的RhoA结合结构域相似的序列，直接与RhoA以及Rac相互作用。（但不是Cdc 42），并显示出对RhoA的GDP结合形式的轻微但可测量的结合偏好。令人惊讶的是，YpkA与RhoA（GDP）的结合既不影响内源性也不影响鸟嘌呤核苷酸交换因子介导的GDP/GTP交换反应表明，YpkA控制激活RhoA水平的机制，而不是简单地阻止鸟嘌呤核苷酸交换因子的活性。我们继续表明，YpkAs激酶活性既不依赖于也不促进其与RhoA和Rac的相互作用，但是，完全依赖于存在于HeLa细胞提取物和胎牛血清中的热敏真核细胞因子。总的来说，我们的数据表明，YpkA具有与真核RhoA/Rac结合激酶的相似性和差异，并表明耶尔森氏菌在与真核细胞相互作用期间利用Rho GTP酶进行独特的活动。
The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpktA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-Containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA, Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.