MPK -1 ERK SUBSTRATES AND THE CONTROL OF GERMLINE DEVELOPMENT

描述（由申请人提供）：ERK（细胞外信号调节激酶）是调节动物发育的一系列信号级联的末端激酶，在癌症中经常被不当激活。ERK通过底物蛋白的磷酸化调节生物过程。然而，在大多数情况下，在给定的生物过程中被磷酸化的底物是未知的，底物实现生物结果的机制也不清楚。实验动物模型秀丽隐杆线虫有一个单一的ERK同源物MPK-1，它在种系发育所必需的9个不同过程中起作用。本研究计划的长期目标是：1)鉴定MPK-1的底物，并利用该系统中的种系发育来了解底物的功能、调控和调控相互作用；2)确定秀丽隐杆线虫MPK-1底物的同源物是否为哺乳动物系统中的ERK底物，重点研究小鼠卵母细胞成熟和减数分裂II （MII）停滞。一个由三部分组成的功能基因组筛选鉴定了25个MPK-1底物，它们包含多个与小鼠/人类同源物保守的ERK对接位点，在线虫种系发育的一个或多个过程中起作用。对于许多底物，产生了磷酸化特异性抗体来验证体内MPK-1依赖性磷酸化。我们建议进行两项后续研究。首先是利用秀丽隐杆线虫种系了解底物磷酸化的时间/空间方面，了解磷酸化如何影响功能，并研究底物介导的MPK-1激活的反馈调节。第二种是使用我们产生的磷酸化特异性抗体来测试哺乳动物细胞培养和小鼠卵母细胞中MPK-1底物的同源物是否为ERK底物。ERK在小鼠卵母细胞减数分裂成熟和MII阻滞的许多过程中起作用；我们所产生的磷酸化特异性抗体可能是卵母细胞发育的有用标记物，并可能识别在成熟和MII阻滞中起作用的新底物。最后，我们建议扩展我们的三部分功能基因组筛选，以使用一套不同的初始标准来鉴定新的MPK-1底物。当上游信号级联成员RAF、RAS和受体酪氨酸激酶（如EGF受体）发生致癌突变时，ERK在癌症中被不当激活。由于最终是底物在这些癌症中被不适当磷酸化导致ERK依赖表型，鉴定底物并揭示其功能、调节和调节后果是理解肿瘤生物学及其治疗方法的重要组成部分。公共卫生相关性：ERK MAP激酶信号在正常发育中很重要，在许多癌症（如结直肠癌、黑色素瘤等）中不适当地发生。正是被ERK磷酸化的底物执行了发育过程并导致癌症中不受控制的生长和转移表型。因此，ERK底物的鉴定及其功能表征对于理解正常发育和癌症至关重要，其中底物是癌症治疗结果和治疗靶点的潜在生物标志物。

DESCRIPTION (provided by applicant): ERK (extracellular signal regulated kinase) is the terminal kinase of a number of signaling cascades that regulate animal development and is often inappropriately activated in cancer. ERK regulates biological processes through phosphorylation of substrate proteins. However, in most cases the substrates that are phosphorylated for a given biological process are not known and the mechanism by which substrates achieve a biological outcome is unclear. The experimental animal model C. elegans has a single ERK ortholog, MPK-1, which functions in nine different processes that are necessary for germline development. The long-term objectives of this research program are to: 1) identify substrates of MPK-1 and to use germline development in this system to understand substrate function, regulation and regulatory interactions, and 2) determine whether the orthologs of C. elegans MPK-1 substrates are ERK substrates in mammalian systems, focusing on mouse oocyte maturation and Meiosis II (MII) arrest. A three-part functional genomics screen identified 25 MPK-1 substrates, which contain multiple ERK docking sites conserved in position with their mouse/ human orthologs, that function in one or more processes in C. elegans germline development. For a number of the substrates, phospho-specific antibodies were generated to verify MPK-1 dependent phosphorylation in vivo. We propose two lines of follow-up studies. The first is to use the C. elegans germline to understand temporal/spatial aspects of substrate phosphorylation, to understand how phosphorylation affects function and to investigate substrate mediated feedback regulation of MPK-1 activation. The second is to use the phospho-specific antibodies we have generated to test if the orthologs of MPK-1 substrates are ERK substrates in mammalian cell culture and in mouse cumulus enclosed oocytes. ERK functions in a number of processes within mouse oocyte meiotic maturation and MII arrest; the phospho-specific antibodies we have generated may be useful markers for oocyte development and may identify new substrates that act in maturation and MII arrest. Finally, we propose to extend our three-part functional genomic screen to identify new MPK-1 substrates using a different set of initial criteria. ERK is inappropriately activated in cancers where oncogenic mutations have occurred in the upstream signaling cascade members RAF, RAS and receptor tyrosine kinases (e.g. the EGF receptor). Since it is ultimately the substrates that are being inappropriately phosphorylated in these cancers that lead to ERK dependent phenotype, identifying the substrates and uncovering their function, regulation and regulatory consequences is essential part of understanding tumor biology and how it may be treated. PUBLIC HEALTH RELEVANCE: ERK MAP Kinase signaling is important in normal development and occurs inappropriately in many cancers (e.g. colorectal, melanoma, etc). It is the substrates that are phosphorylated by ERK that execute developmental processes and lead to uncontrolled growth and metastasis phenotypes in cancer. Identification of ERK substrates and characterization of their function is thus essential for understanding both normal development and cancer, where the substrates are potential biomarkers for outcome and therapeutic targets for cancer treatment.