Chemical genetic dissection of Hipk4-dependent Hedgehog pathway activation

Hipk4 依赖性 Hedgehog 通路激活的化学遗传学剖析

• 批准号: 8611320
• 负责人: JAMES K CHEN
• 金额: $ 24.14万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-22 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8611320
• 关键词: Basal cell carcinoma; Base Sequence; Binding; Biochemical; Biochemical Process; Biological; Biological Assay; Biology; Brain; Cell physiology; Cells; Chemicals; Chronic Myeloid Leukemia; Complementary DNA; Complex; Cultured Cells; Disease; Dissection; Dissociation; Embryo; Embryology; Engineering; Erinaceidae; Event; Family; Family member; Flow Cytometry; Fostering; G-Protein-Coupled Receptors; Gene Expression; Gene Targeting; Genetic Programming; Genetic Transcription; Genome; Goals; Growth; Homeostasis; Human; In Vitro; Individual; Integral Membrane Protein; Investigation; Laboratories; Lead; Length; Libraries; Ligands; Link; Malignant Neoplasms; Malignant neoplasm of brain; Maps; Mass Spectrum Analysis; Measures; Molecular; Mutagenesis; NIH 3T3 Cells; Natural regeneration; Null Lymphocytes; Open Reading Frames; Other Genetics; Pathway interactions; Pattern; Phase; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Post-Translational Protein Processing; Process; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Proteolytic Processing; Proteomics; RNA Interference; Regulation; Reporter; Repression; Research; Role; Scaffolding Protein; Signal Transduction; Signaling Protein; Site-Directed Mutagenesis; Skeleton; Skin; Solid; Spinal Cord; Stem cells; Surveys; Therapeutic; Tissues; Transcription Coactivator; Transcription Repressor/Corepressor; Vertebrates; Zebrafish; analog; base; chemical genetics; gain of function; gastrointestinal; hedgehog signal transduction; homeodomain; human SMO protein; in vivo Model; insight; lung small cell carcinoma; medulloblastoma; member; meningioma; morphogens; mutant; overexpression; paralogous gene; polypeptide; polypeptide C; public health relevance; receptor; response; smoothened signaling pathway; tandem mass spectrometry; thiophosphate; transcription factor; treatment strategy; tumorigenesis

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.

细胞对 Hedgehog (Hh) 形态发生素的反应最终导致 Gli 转录因子激活和 执行与组织模式、稳态和转化相关的遗传程序。为了 例如，Hh 信号传导有助于大脑、脊髓、肌肉组织和骨骼的形成，以及 通路失调与基底细胞癌、髓母细胞瘤、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 因此对于我们理解个体发育和肿瘤发生来说是不可或缺的。 通过诱变或 RNA 干扰已鉴定出几种典型的 Hh 信号蛋白 筛选，包括跨膜蛋白 Patched1 (Ptch1) 和 Smoothened (Smo) 以及 Gli- 融合蛋白 (Sufu) 的相互作用蛋白抑制剂。然而，与相关的生化和细胞事件 Gli 的激活仍然是个谜，特别是那些映射到 Smo 下游的激活。为了获得对此的新见解 过程中，我们完成了第一个 Hh 通路激活剂的基因组规模 cDNA 过表达筛选， 调查了 15,483 个哺乳动物开放阅读框 (ORF)。通过这个功能获得屏幕，细胞 经过生物学研究和生化分析，我们发现Hipk4，一个非典型成员 同源结构域相互作用蛋白激酶家族，作用于 Smo 下游，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 的方式发挥作用，消除 Gli 因子的蛋白水解过程， 转录抑制因子，产生细胞内全长蛋白质池，为 Hh 配体做好准备 依赖激活。因此，Hipk4 过表达细胞对 Hh 刺激极其敏感，并且 通过 RNA 干扰沉默内源性 Hipk4 会抑制 Hh 信号转导。二、Hipk4可以 增强细胞对外源 Gli1 或 Gli2 的反应，并提高组成型 Hh 通路活性 在 Sufu 无效细胞中，表明这种丝氨酸/苏氨酸激酶可以上调全转录活性 长度 Gli 蛋白以最大化 Hh 靶基因表达。 我们的研究结果为了解控制 Gli 功能的机制打开了一个新窗口，并发现了 Hipk4 的底物将揭示该过程中的一些关键分子步骤。我们现在正在追求这个目标 通过整合标记 Hipk4 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础 了解 Hh 信号转导并培育治疗 Gli 依赖性疾病的新策略。