Chemical genetic dissection of Hipk4-dependent Hedgehog pathway activation

细胞对Hedgehog（Hh）形态发生素的反应在Gli转录因子激活和细胞凋亡中达到顶峰。 执行与组织模式、体内平衡和转化相关的遗传程序。为 例如，Hh信号传导有助于大脑、脊髓、肌肉组织和骨骼的形成， 通路失调与基底细胞癌、成神经管细胞瘤、小细胞肺癌和 慢性粒细胞白血病破译控制Gli活性状态的分子机制是 因此是我们理解个体发生和肿瘤发生的一部分。 几个典型的Hh信号蛋白已通过诱变或RNA干扰鉴定 筛选，包括跨膜蛋白Patched 1（Ptch 1）和Smoothened（Smo）以及Gli- 融合的相互作用蛋白抑制因子（Suppressor of Fused，Sufu）。然而，与之相关的生化和细胞事件 Gli的激活仍然是个谜，特别是那些定位在Smo下游的。为了获得新的见解 在这个过程中，我们完成了第一个基因组规模的Hh通路激活剂的cDNA过表达筛选， 调查了15，483个哺乳动物开放阅读框架（ORF）。通过这个功能获得屏幕，细胞 生物学研究和生化分析，我们发现Hipk 4，一个非典型的成员， 同源结构域相互作用蛋白激酶家族，作用于Smo下游以调节Gli功能。我们 初步研究表明Hipk 4通过至少两种不同的机制调节Gli活性。 首先，Hipk 4以Smo非依赖性方式起作用，以消除Gli因子的蛋白水解加工， 转录阻遏物，产生针对Hh配体引发的全长蛋白质的细胞内池- 依赖性激活因此，Hipk 4过表达细胞对Hh刺激超敏感， 通过RNA干扰沉默内源性Hipk 4抑制Hh信号转导。其次，Hipk 4可以 增强细胞对外源性Gli 1或Gli 2的反应，以及提高组成型Hh途径活性 在Sufu无效细胞中，表明这种丝氨酸/苏氨酸激酶可以上调全- 长度Gli蛋白以最大化Hh靶基因表达。 我们的发现为控制Gli功能的机制打开了一扇新的窗口， Hipk 4的底物将揭示这一过程中的一些关键分子步骤。我们现在正在追求这个目标 通过整合用于标记Hipk 4底物的化学遗传策略，基于质谱的测序， 以及Hh通路状态的各种细胞生物学测量。这些调查将推进我们的基本 Hh信号转导的理解和促进新的策略，用于治疗神经胶质细胞依赖性疾病。

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 this 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.