The Function of Yeast Adenylyl Cyclase-Associated Proteins in Regulation of Cell Growth and Cytoskeletal Structure

酵母腺苷酸环化酶相关蛋白在调节细胞生长和细胞骨架结构中的功能

• 批准号: 06454167
• 负责人: KATAOKA Tohru
• 金额: $ 4.67万
• 依托单位: Kobe University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454167/
• 关键词: Adenylyl Cyclase; Cyclase-Associated Protein; Budding Yeast; Ras protein; CyclicAMP; Posttranslational Modification; Actin-binding Protein

In budding yeast, adenylyl cyclase is regulated by Ras proteins. We found adenylyl cyclase forms a complex with two cyclase-associated proteins ; 70-kDa CAP and 50-kDa p50, and analyzed their modes of interaction with cyclase and their cellular functions.1. CAP is a bifunctional protein ; its N-terminal region is required for association with cyclase and for proper response of cyclase to Ras, while its C-terminal region is presumably involved in cytoskeletal regulation. By mutational analyzes of CAP,we mapped its adenylyl cyclase-binding site to N-terminal 36-amino acid residues. The 36-residues were sufficient for invoking proper cAMP response in yeast lacking CAP.Also, we mapped the CAP-binding site of cyclase to 118 residues near its C terminus. Both of these binding sites contained tandem repetition of LXXLXXX,suggesting the coiled-coil mechanism for their interaction. When the effect of amino acid substitutions of the leucine residues in these sequences was examined, the mutations … More abolished the CAP-cyclase interaction, demonstrating the importance of the coiled-coil mechanism.2. We found that posttranslational modification (especially farnesylation) of Ras is required for activation of adenylyl cyclase, whereas it has no effect on the binding affinity of cyclase for Ras. When cyclase was devoid of its associating CAP,the stimulatory effect of Ras modification on cyclase activation was lost. In this system, overexpression of CAP resumed efficient CAP binding as well as the stimulatory effect of the modification on cyclase. Cyclase mutants lacking the CAP-binding site were insensitive to the stimulatry effect, which was not resumed by CAP overexpression. These results indicate that association with CAP mediates the stimulation of cyclase activation by Ras posttranslational modification.3. By the pyrenyl actin method, we showed that the CAP C-terminal region has G-actin-binding and -sequestering activities. p50 was found not an inherent component of the cyclase complex, and, therefore, was not analyzed any further. Less

在萌芽的酵母中，腺苷酸环化酶受RAS蛋白调节。我们发现腺苷酸环化酶形成具有两个环酶相关蛋白的复合物。 70 kDa CAP和50 kDa P50，并分析了它们与环酶及其细胞功能的相互作用模式。1。帽是双功能蛋白。它的N末端区域与循环酶缔合并适当对RAS的适当响应需要，而其C末端区域可能参与了细胞骨架调节。通过对CAP的突变分析，我们将其腺苷环酶结合位点映射到N末端36-氨基酸保留。 36个分配足以在缺乏帽子的酵母中调用适当的cAMP反应。此外，我们将环酶的盖结合位点映射到其C末端附近的118个保留。这两个结合位点都包含LXXLXXX的串联重复，这表明其相互作用的盘绕螺旋机理。当检查了这些序列中亮氨酸残留物的氨基酸取代的作用时，突变……更多地消除了帽周期酶相互作用，证明了盘绕螺旋机制的重要性。2。我们发现，在激活腺苷酸环化酶的激活需要翻译后修饰（尤其是Farnesylation），而它对Cyclase对RA的结合亲和力没有影响。当循环酶没有其关联帽时，RAS修饰对循环酶激活的刺激作用就会丢失。在该系统中，CAP的过表达恢复了有效的帽结合以及修饰对环化酶的刺激作用。缺乏帽结合位点的环化酶突变体对刺激效果不敏感，而刺激效果并未通过帽过表达恢复。这些结果表明，与CAP的关联介导了通过翻译后修饰的RAS刺激环化酶激活的刺激。3。通过拟南芥肌动蛋白法，我们表明C端C末端区域具有G-肌动蛋白结合和 - 序列活性。发现P50不是循环酶复合物的继承成分，因此没有进一步分析。较少的

项目成果

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T.Minato et al.: "Quantitative analysis of mutually competitive binding of human Raf-1 and yeast adenylyl cyclase to Ras proteins." J.Biol.Chem.269.20845-20851 (1994)

T.Minato 等人：“人 Raf-1 和酵母腺苷酸环化酶与 Ras 蛋白相互竞争结合的定量分析。”

K.Akasaka et al.: "Differential structural requirements for interaction of Ras protein with its distinct downstream effectors." J.Biol.Chem.271. 5353-5360 (1996)

K.Akasaka 等人：“Ras 蛋白与其独特的下游效应子相互作用的不同结构要求。”