Chemical genetic dissection of Hipk4-dependent Hedgehog pathway activation

描述（由申请人提供）：细胞对Hedgehog （Hh）形态因子的反应最终导致Gli转录因子激活和与组织模式、稳态和转化相关的遗传程序的执行。例如，Hh信号参与脑、脊髓、肌肉组织和骨骼的形成，通路失调与基底细胞癌、髓母细胞瘤、小细胞肺癌和慢性髓性白血病有关。因此，破译控制Gli活性状态的分子机制对于我们理解个体发生和肿瘤发生是不可或缺的。通过诱变或RNA干扰筛选已经鉴定出几种典型的Hh信号蛋白，包括跨膜蛋白Patched1 （Ptch1）和Smoothened （Smo）以及glii相互作用蛋白Suppressor of Fused （Sufu）。然而，与Gli激活相关的生化和细胞事件仍然是一个谜，特别是那些映射Smo下游的事件。为了获得对这一过程的新见解，我们完成了Hh通路激活因子的第一个基因组级cDNA过表达筛选，调查了15483个哺乳动物的开放阅读框（orf）。通过这种功能获得筛选、细胞生物学研究和生化分析，我们发现Hipk4是同源域相互作用蛋白激酶家族的一个非典型成员，在Smo的下游作用来调节Gli功能。我们的初步研究表明，Hipk4通过至少两种不同的机制调节Gli活性。首先，Hipk4以不依赖于smo的方式消除Gli因子转化为转录抑制因子的蛋白水解过程，在细胞内产生全长蛋白池，为Hh配体依赖性激活做准备。因此，Hipk4过表达的细胞对Hh刺激超敏感，通过RNA干扰沉默内源性Hipk4可抑制Hh信号转导。其次，Hipk4可以增强细胞对外源性Gli1或Gli2的反应，并在Sufu null细胞中提高Hh通路的组成性活性，表明这种丝氨酸/苏氨酸激酶可以上调全长Gli蛋白的转录活性，从而最大化Hh靶基因的表达。我们的发现为控制Gli功能的机制打开了一扇新的窗口，发现Hipk4的底物将揭示这一过程中的一些关键分子步骤。我们现在正在通过整合一种化学遗传策略来标记Hipk4底物，基于质谱的测序和Hh通路状态的各种细胞生物学测量来实现这一目标。这些研究将促进我们对Hh信号转导的基本理解，并为治疗glii依赖性疾病提供新的策略。

DESCRIPTION (provided by applicant): Cellular responses to Hedgehog (Hh) morphogens culminate in Gli transcription factor activation and the execution of genetic programs associated with tissue patterning, homeostasis and transformation. For example, Hh signaling contributes to formation of the brain, spinal cord, musculature, and skeleton, and pathway dysregulation has been linked to basal cell carcinoma, medulloblastoma, small cell lung cancer, and chronic myelogenous leukemia. Deciphering the molecular mechanisms that control Gli activity state is therefore integral to our understanding of ontogeny and oncogenesis. Several canonical Hh signaling proteins have been identified through mutagenesis or RNA interference screens, including the transmembrane proteins Patched1 (Ptch1) and Smoothened (Smo) and the Gli-interacting protein Suppressor of Fused (Sufu). However, the biochemical and cellular events associated with Gli activation remain enigmatic, particularly those that map downstream of Smo. To gain new insights into this process, we have completed the first genome-scale cDNA overexpression screen for Hh pathway activators, surveying 15,483 mammalian open reading frames (ORFs). Through this gain-of-function screen, cell biological studies, and biochemical analyses, we have discovered that Hipk4, an atypical member of the homeodomain-interacting protein kinase family, acts downstream of Smo to modulate Gli function. Our preliminary studies demonstrate that Hipk4 regulates Gli activity through at least two distinct mechanisms. First, Hipk4 acts in a Smo-independent manner to abrogate the proteolytic processing of Gli factors into transcriptional repressors, generating an intracellular pool of full-length protein that is primed for Hh ligand-dependent activation. Accordingly, Hipk4-overexpressing cells are ultrasensitive to Hh stimulation, and silencing of endogenous Hipk4 by RNA interference inhibits Hh signal transduction. Second, Hipk4 can potentiate cellular responses to exogenous Gli1 or Gli2, as well as elevate the constitutive Hh pathway activity in Sufu null cells, indicating that tis serine/threonine kinase can upregulate the transcriptional activity of full-length Gli proteins to maximize Hh target gene expression. Our findings open a new window into the mechanisms that control Gli function, and discovering the substrates of Hipk4 will reveal some of the key molecular steps in this process. We are now pursuing this goal by integrating a chemical genetic strategy for tagging Hipk4 substrates, mass spectrometry-based sequencing, and various cell biological measures of Hh pathway state. These investigations will advance our basic understanding of Hh signal transduction and foster new strategies for the treatment of Gli-dependent diseases.