The long-term objective of my laboratory is a detailed molecular understanding of how cell fates are regulated by division sequence and cell signaling in animal development. During C. elegans embryogenesis, Wnt and MAP kinase signaling pathways specify the endoderm precursor E. We showed that Wnt increases the level of 2-catenin/SYS and MAP kinase reduces the level of TCF/POP-1, together robustly increasing the SYS-1-to-POP ratio and transcriptional activation of endoderm genes in E. In the anterior sister of E, MS, the SYS-1-to-POP-1 ratio is low and endoderm genes are repressed. We observed the asymmetric SYS-1-to-POP-1 ratio in all A-P divisions examined and showed that it is required for all A-P fate differences examined. Studies from us and others have suggested that the MAP kinase pathway also regulates POP-1 activity, in addition to its nuclear levels. The overall objective of this proposal is to understand how phosphorylation by the MAP kinase complex, LIT-1/WRM-1, affects POP-1 activity, how POP-1 sequence itself influence its phosphorylation by LIT-1/WRM-1, and how the information in division sequence is mechanistically linked to the activation of the MAP kinase pathway. The hypotheses to be tested are: (1) LIT-1/WRM-1 regulates POP-1 activity by increasing its binding to SYS-1, in addition to reducing its nuclear levels. (2) LIT-1/WRM-1 regulates POP-1 nuclear levels and transcription activity by phosphorylating two distinct sets of sites whose phosphorylation are mutually exclusive and inhibited by each other. (3) The C-terminal domain of POP-1 promotes LIT-1/WRM-1 phosphorylation by bringing POP-1 to the kinase complex and/or presenting the phosphorylation sites. (4) The upstream regulators for the MAPK pathway also regulate spindle rotation and division axes.
The specific aims are: (1) To investigate the mechanism by which LIT-1/WRM-1 coordinately regulate POP-1 nuclear level and activity. We will identify sites whose phosphorylation enhances POP-1 transcription activity. We will investigate the interactive regulation between phosphorylation sites that lower nuclear levels or increase transcriptional activation. (2) To investigate the mechanism by which the C-terminal 39 amino acids of POP-1 regulate phosphorylation by LIT-1. We will perform biochemical analyses to test models for how the POP-1 C-terminal 39 amino acids might regulate POP- 1 phosphorylation by LIT-1/WRM-1. (3) To identify additional genes in the MOM-4/LIT-1/WRM-1 MAPK pathway. RNAi screens will be performed in a background highly sensitized for defects in this pathway. We will also specifically investigate genes known to regulate cell division axes for their possible role(s) in activating this pathway. Misregulation of division axes or signaling pathways can lead to developmental defects or cancer. These experiments will aid our understanding of these processes.
Colorectal cancer causes more than 50,000 deaths annually in the US, and greater than 90% of these cancers are caused by activating mutations in the Wnt signaling pathway. Our ability to detect, diagnose and treat these cancers (as well as other cancer types and embryological defects caused by deregulated Wnt signaling) will rely heavily upon our understanding of this pathway, its components and their normal regulation. This proposal aims to significantly broaden our understanding of the Wnt signaling pathway, and how it intersects with other signaling pathways during development.
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