RECONSTITUTION OF A HEDGEHOG SIGNALING COMPLEX

描述（由申请人提供）：刺猬（Hh）形成因子家族在许多后生动物结构的发育中起着重要的指导作用。与Hh同系物在细胞命运决定中发挥的关键作用一致，异常的Hh信号导致许多类型的癌症。当Hh与其受体补丁（Ptc）结合，激活七跨膜蛋白Smoothened （Smo）时，Hh信号转导开始。Smo将其激活信号传递给大微管（MT）相关的多蛋白信号复合体，称为刺猬信号复合体（HSC）。至少，HSC由转录因子Cubitus interruptus （Ci）、蛋白激酶Fused （Fu）和激酶相关蛋白（KRP） Costal2 （Cos2）组成。Smo激活后，Cos2被磷酸化，从MT中释放HSC。这种MT的释放被认为改变了Ci的抑制因子（Ci75）和激活因子形式之间的比例，最终决定了各种Hh靶基因的表达水平。Smo激活和向HSC发送信号之间的步骤尚未描述。我们提供的证据表明Smo可能直接与Cos2沟通，调节HSC功能。我们提出了一种模型，其中有两种不同的hsc，它们具有离散的亚细胞本地化和活动：一种是内核体相关的，促进Ci75的产生（HSC-R），另一种是Smo相关的，促进Ci激活（HSC-A）。在Hh的作用下，Smo通过与Cos2的直接关联，介导了内核体相关HSC-R的抑制和质膜上HSC-A的激活。本文提出的实验将检验这一模型。我们将在Cos2上确定三个不同的结构域，它们与MT、核内体或Smo相关联。然后，我们将证明这些结构域对于将HSC定位到不同的亚细胞位置是必要和充分的。然后，我们将证明Fu调节Cos2重新分配Ci的能力，以响应Hh。最后，我们将在体内测试该模型，使用这些不同的Cos2突变体选择性地激活HSC-A或抑制HSC-R。

DESCRIPTION (provided by applicant): The hedgehog (Hh) family of morphogens plays important instructional roles in the development of numerous metazoan structures. Consistent with the critical role Hh homologs play in cell fate determination, aberrant Hh signaling results in numerous types of cancer. Hh signal transduction is initiated when Hh binds to its receptor Patched (Ptc), activating the seven-transmembrane protein Smoothened (Smo). Smo transmits its activation signal to a large microtubule (MT) associated multi-protein signaling complex, termed the Hedgehog Signaling Complex (HSC). At a minimum, the HSC consists of the transcription factor Cubitus interruptus (Ci), the protein kinase Fused (Fu) and the kinesin related protein (KRP) Costal2 (Cos2). In response to activated Smo, Cos2 is phosphorylated, releasing the HSC from MTs. This MT release is thought to alter the ratio between repressor (Ci75) and activator forms of Ci, ultimately determining the level of expression of various Hh target genes. The steps between Smo activation and signaling to the HSC have not been described. We have provided evidence that Smo may directly communicate with Cos2 to regulate HSC function. We suggest a model in which there are two distinct HSCs with discrete sub-cellular Iocalizations and activities: one is endosome associated and facilitates Ci75 production (HSC-R), and one is Smo associated and promotes Ci activation (HSC-A). In response to Hh, and through direct association with Cos2, Smo mediates both inhibition of the endosome associated HSC-R, and activation of HSC-A at the plasma membrane. The experiments proposed here will test this model. We will identify three distinct domains on Cos2, which associate with MT, endosomes, or with Smo. We will then demonstrate that these domains are necessary and sufficient for localizing the HSC to various sub-cellular locations. We will then show Fu regulates the ability of Cos2 to redistribute Ci in response to Hh. Finally, we will test this model in vivo, using these various Cos2 mutants to selectively activate HSC-A or inhibit HSC-R.