MPK -1 ERK SUBSTRATES AND THE CONTROL OF GERMLINE DEVELOPMENT

描述(申请人提供)：ERK(细胞外信号调节激酶)是调节动物发育的许多信号级联的末端激酶，经常在癌症中被不适当地激活。ERK通过底物蛋白的磷酸化来调节生物过程。然而，在大多数情况下，对于给定的生物过程，底物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。实验动物模型线虫有一个ERK同源基因MPK-1，它在生殖系发育所必需的九个不同过程中发挥作用。本研究计划的长期目标是：1)鉴定MPK-1的底物，并利用该系统中的生殖系发育来了解底物的功能、调节和调节相互作用；2)确定线虫MPK-1底物的同源物是否为哺乳动物系统中的ERK底物，重点关注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学筛选确定了25个MPK-1底物，其中包含多个ERK对接位点，其位置与其鼠标/人类同源基因的位置保守，在线虫种系发育的一个或多个过程中发挥作用。对于一些底物，产生了磷酸特异性抗体来验证体内MPK-1依赖的磷酸化。我们提出了两条后续研究的路线。首先是利用线虫生殖系了解底物磷酸化的时间和空间方面，了解磷酸化是如何影响功能的，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特异性抗体来测试MPK-1底物的同系物在哺乳动物细胞培养和小鼠卵丘封闭卵母细胞中是否为ERK底物。ERK在小鼠卵母细胞减数分裂成熟和MII停滞的许多过程中发挥作用；我们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，并可能识别在成熟和MII停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，以使用一组不同的初始标准来识别新的MPK-1底物。ERK在癌症中被不适当地激活，其中上游信号级联成员RAF、RAS和受体酪氨酸激酶(例如EGF受体)发生了致癌突变。由于在这些癌症中，最终是底物被不适当的磷酸化导致ERK依赖的表型，识别底物并揭示它们的功能、调节和调节后果是理解肿瘤生物学和如何治疗的关键部分。公共卫生相关性：ERK MAP Kinase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)中不适当地出现。正是被ERK磷酸化的底物执行发育过程，并导致癌症中不受控制的生长和转移表型。因此，鉴定ERK底物及其功能对于了解正常发育和癌症是至关重要的，因为底物是癌症治疗结果和治疗靶点的潜在生物标记物。

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.