RECONSTITUTION OF A HEDGEHOG SIGNALING COMPLEX

描述（由申请人提供）：形态发生素的刺猬（Hh）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信号传导导致许多类型的癌症。当Hh与其受体Patched（Ptc）结合时，Hh信号转导启动，激活七跨膜蛋白Smoothened（Smo）。Smo将其激活信号传递到一个大微管（MT）相关的多蛋白信号复合物，称为刺猬信号复合物（HSC）。HSC至少由转录因子Cubitus interruptus（Ci）、蛋白激酶Fused（Fu）和驱动蛋白相关蛋白（KRP）Costal 2（Cos 2）组成。响应于激活的Smo，Cos 2被磷酸化，从MT释放HSC。这种MT释放被认为改变了Ci的阻遏物（Ci 75）和激活物形式之间的比率，最终决定了各种Hh靶基因的表达水平。Smo激活和向HSC发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Cos 2沟通，以调节HSC的功能。我们提出了一种模型，其中存在两种具有离散亚细胞定位和活性的不同HSC：一种是内体相关的并促进C175产生（HSC-R），一种是Smo相关的并促进C1活化（HSC-A）。响应于Hh，并且通过与Cos 2的直接缔合，Smo介导内体相关的HSC-R的抑制和质膜处的HSC-A的活化。这里提出的实验将测试这个模型。我们将确定三个不同的域Cos 2，与MT，内涵体，或与Smo。然后，我们将证明，这些领域是必要的和足够的本地化的HSC的各种亚细胞位置。然后，我们将显示Fu调节Cos 2重新分配Ci以响应Hh的能力。最后，我们将在体内测试该模型，使用这些不同的Cos 2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.