A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium
A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium
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DOI：
10.1091/mbc.10.9.2829
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发表时间：
1999-09-01
影响因子：
3.3
通讯作者：
Firtel, RA
中科院分区：
文献类型：
--
作者：
Lee, S;Parent, CA;Firtel, RA
我们已经确定了一种新的Ras相互作用蛋白，RIP 3，其功能是所需的两个趋化性和合成和中继环AMP（cAMP）的化学引诱信号。rip 3无效细胞不能聚集并且缺乏腺苷酸环化酶的受体活化，但是能够响应cAMP，在悬浮培养物中诱导聚集阶段、聚集后和细胞类型特异性基因表达。此外，rip 3无效细胞不能在cAMP梯度中适当地粘附，并且趋化性高度受损。我们证明，cAMP刺激鸟苷酸环化酶，这是需要的趋化性，减少类似于60%的rip 3无效细胞。这种鸟苷酸环化酶的激活减少可能部分地解释了趋化性的缺陷。当细胞用cAMP脉冲5小时以模拟野生型菌株中发生的内源性cAMP振荡时，细胞将形成聚集体，然而，其中大部分聚集体在丘阶段停止。与野生型菌株中所见的反应不同，在这些实验条件下形成的rip 3无效细胞聚集体非常小，这可能是由于rip 3无效细胞趋化性缺陷。RIP 3无效细胞的许多表型，包括不能响应cAMP激活腺苷酸环化酶和趋化性缺陷，与携带编码推定Ras交换因子AleA的基因破坏的菌株非常相似。我们证明，aleA空细胞也表现出类似的rip 3空细胞的cAMP介导的鸟苷酸环化酶的激活缺陷。一个双敲除突变体（rip 3/aleA空细胞）表现出进一步减少鸟苷酸环化酶的受体激活，这些细胞显示几乎没有细胞极化或运动的cAMP梯度。由于RIP 3优先与活化形式的网骨藻Ras蛋白RasG，这本身是重要的细胞运动相互作用，我们建议，RIP 3和AleA的Ras调节途径的组成部分，参与整合趋化性和信号中继途径，是必不可少的聚集。
We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. Ln addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced similar to 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.