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The functional relevance of low affinity phosphoinositide binding by the PH domain in Dbl family guanine nucleotide exchange factors.

机译：Dbl家族鸟嘌呤核苷酸交换因子中PH结构域与低亲和力磷酸肌醇结合的功能相关性。

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摘要

Low affinity phosphoinositide binding is found in several phosphoinositide recognition domains, including PH domains, and members of the ESCRT complex. In light of the numerous occurrences of these domains in the human proteome, the significance of low affinity phosphoinositide binding has become an important question. We have investigated this question in the PH domain present in Dbl family guanine nucleotide exchange factors (GEFs) and in the ESCRT proteins. We found that while the PH domain in several Dbl family members bound phosphoinositides with low affinity, this property of the PH domain appeared critical for the in vivo GEF activity of the adjacent DH domain. The Dbs DH/PH fragment bound PtdIns(4,5)P2 in vitro with low affinity (∼10 muM), and localized to the cytosol when expressed in vivo as a GFP fusion. However, forced dimerization led to plasma membrane recruitment of the DH/PH fragment, an effect that required the phosphoinositide binding properties of the PH domain. Constitutive plasma membrane targeting of this fragment by a fused PLC-gamma1 PH domain or farnesylation signal, significantly enhanced in vivo GEF activity toward Cdc42 and RhoA, suggesting the PH domain may regulate GEF activity through a cooperative membrane recruiting mechanism. Interestingly, PH domain mutations within a membrane targeted DH/PH fragment led to a significant reduction in Cdc42 and RhoA activation, suggesting an additional regulatory role by the PH domain, such as an allosteric mechanism. In contrast, the Tiam-1 GST-DH/PH fragment bound specifically, though weakly, to PtdIns-3-P in vitro but did not target, when dimerized, to identifiable cellular membranes in vivo. PH domain mutations that abolished PtdIns-3-P binding did not alter bulk localization of Tiam-1, but dramatically impaired in vivo GEF activity, suggesting an non-recruiting regulatory role for the Tiam-1 PH domain, such as an allosteric one. Finally, we investigated the phosphoinositide binding properties of mVps24p and the yeast ESCRT proteins, and found that most bound phosphoinositides promiscuously and in the case of mVps24p, with low affinity, suggesting ESCRT proteins may be recruited to late endosome membranes, by a cooperative mechanism involving weak non-specific phosphoinositide interactions combined with other determinants, such as specific association with ubiquitin.
机译：在几个磷酸肌醇识别结构域（包括PH结构域）和ESCRT复合体成员中发现了低亲和力的磷酸肌醇结合。鉴于人类蛋白质组中这些结构域的大量出现，低亲和力磷酸肌醇结合的重要性已成为一个重要的问题。我们已经在Dbl家族鸟嘌呤核苷酸交换因子（GEFs）和ESCRT蛋白中存在的PH域中研究了这个问题。我们发现，尽管几个Dbl家族成员中的PH结构域以低亲和力结合磷酸肌醇，但是PH结构域的这种性质对于相邻DH结构域的体内GEF活性显得至关重要。 Dbs DH / PH片段在体外以低亲和力（〜10μM）结合PtdIns（4,5）P2，并在体内以GFP融合体表达时定位于胞质溶胶。但是，强制二聚导致DH / PH片段的质膜募集，这种作用需要PH结构域的磷酸肌醇结合特性。通过融合的PLC-gamma1 PH结构域或法尼基化信号对该片段进行本构质膜靶向，可显着增强体内对Cdc42和RhoA的GEF活性，表明PH结构域可通过协同的膜募集机制调节GEF活性。有趣的是，膜靶向的DH / PH片段内的PH结构域突变导致Cdc42和RhoA活化显着降低，这表明PH结构域还具有其他调节作用，例如变构机制。相反，Tiam-1 GST-DH / PH片段虽然微弱地与体外PtdIns-3-P特异性结合，但二聚体化时并未靶向体内可识别的细胞膜。取消PtdIns-3-P结合的PH结构域突变不会改变Tiam-1的整体定位，但会显着削弱体内GEF活性，这表明Tiam-1 PH结构域具有非招募性的调节作用，例如变构结构。最后，我们研究了mVps24p和酵母ESCRT蛋白的磷酸肌醇结合特性，发现大多数结合的磷酸肌醇混杂在一起，在mVps24p的情况下，亲和力很低，这表明ESCRT蛋白可能通过涉及以下机制的协同体被募集到晚期内体膜上弱的非特异性磷酸肌醇相互作用与其他决定因素结合，例如与泛素的特异性结合。

著录项

  • 作者

    Baumeister, Mark A.;

  • 作者单位

    University of Pennsylvania.;

  • 授予单位 University of Pennsylvania.;
  • 学科 Biology Molecular.; Chemistry Biochemistry.; Biophysics General.
  • 学位 Ph.D.
  • 年度 2005
  • 页码 180 p.
  • 总页数 180
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;生物化学;生物物理学;
  • 关键词

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