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

基本信息

批准号：
8929276
负责人：
JAMES K CHEN
金额：
$ 19.63万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-22 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8929276
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 Goals Growth Health 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 genetic approach genome-wide 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 receptor response smoothened signaling pathway tandem mass spectrometry thiophosphate transcription factor treatment strategy tumorigenesis

项目摘要

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.

描述（由申请人提供）：对刺猬（HH）形态的细胞反应最终在GLI转录因子激活中，并执行与组织模式，稳态和转化有关的遗传程序。例如，HH信号传导有助于大脑，脊髓，肌肉和骨骼的形成，并且途径失调与基底细胞癌，髓母细胞瘤，小细胞肺癌和慢性粒细胞性白血病有关。因此，破译控制GLI活性状态的分子机制是我们对本体发育和肿瘤发生的理解不可或缺的一部分。已经通过诱变或RNA干扰筛选（包括跨膜蛋白修补1（PTCH1）（PTCH1）和平滑（SMO）和融合融合的Gli相互作用蛋白抑制剂（SUFU）（SUFU），已经鉴定出了几种规范的HH信号蛋白。然而，与Gli激活相关的生化和细胞事件仍然是神秘的，尤其是那些在SMO下游绘制的事件。为了获得有关此过程的新见解，我们已经完成了HH途径激活剂的第一个基因组尺度cDNA筛选，调查了15,483个哺乳动物开放式阅读框（ORFS）。通过这种功能获得的筛选，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非典型成员，在SMO的下游起作用以调节GLI功能。我们的初步研究表明，HIPK4通过至少两种不同的机制来调节GLI活性。首先，HIPK4以SMO独立的方式起作用，将GLI因子的蛋白水解处理中的蛋白水解处理为转录阻遏物，从而产生用于HH配体依赖性激活的全长蛋白的细胞内池。因此，对HIPK4过表达的细胞对HH刺激非常敏感，而RNA干扰对内源性HIPK4的沉默抑制了HH信号转导。其次，HIPK4可以增强对外源GLI1或GLI2的细胞反应，并提高Sufu Null细胞中的组成型HH途径活性，表明TIS丝氨酸/苏氨酸激酶可以上调全长GLI蛋白的转录活性，以最大程度地提高HH靶基因靶基因表达。我们的发现为控制GLI功能的机制打开了一个新窗口，发现HIPK4的底物将揭示此过程中的一些关键分子步骤。现在，我们通过整合用于标记HIPK4底物，基于质谱的测序以及HH途径状态的各种细胞生物学测量的化学遗传策略来追求这一目标。这些研究将提高我们对HH信号转导的基本理解，并促进治疗GLI依赖性疾病的新策略。