Discovering phosphorylation sites on 14-3-3sigma and its binding partners that could be required for Cell-division.
Colorectal cancer is the third leading cause of cancer death in the United States, with a risk of development in 1 out of 20 people. It is expected to take 49,700 lives by 2015 [1]. There is no cure for colorectal cancer, and current treatments involve radiation and chemotherapy. The 14-3-3σ-tumor suppressor gene is associated with colorectal cancer. 14-3-3σ is a p53-dependent, negative regulator of the cell cycle and plays a specific role during translation, as mutations in 14-3-3σ lead to cytokinesis failures [2]. 14-3-3σ is a member of the 14-3-3 superfamily which all regulate cellular signaling by interacting with serine/threonine phosphorylated residues on a variety of proteins [3]. A hyper-methylation in the promoter region renders 14-3-3σ unable to produce a viable protein [3]. Though much is known about the role of 14-3-3σ in translation, its role in cell division as it relates to colorectal cancer is unclear.
The primary goal is to determine the role that 14-3-3σ plays in cell division. To observe its role in cytokinesis, I will use the C. elegans 14-3-3σ homolog, PAR-5, to understanding this function because cell division is easily assayed and imaged in C. elegans embryos [4]. The long-term goal is to determine how 14-3-3σ functions in cell division events.
Hypothesis: 14-3-3σ/PAR-5 and its binding partners regulate the activity proteins necessary for cytokinesis by binding to serine/threonine sites.
Aim 1: Identify and characterize the function of 14-3-3/PAR-5 interacting partners and determine if they play a role in cell division.
Approach: Use STRING to identify binding partners of PAR-5 in C. elegans. Then using Gene Ontology, I will sort proteins based on biological function to determine if these interactions play a role in cell division. Then I will determine if they share similar domains using SMART known to be regulated during cell division.
Rationale: By doing this, I will determine if PAR-5 interacting proteins function in cell division.
Aim 2: To identify conserved serine/threonine phosphorylation sites on PAR-5 binding partners (such as sir-2.1) that function during cell division.
Approach: Using NetPhos 2.0, I will locate the serine/threonine phosphorylation sites on candidate PAR-5 interacting proteins and then use Clustal Omega to determine which of these sites are the most conserved.
Rationale: I will be able to determine where the well-conserved phosphorylation sites are on proteins that function in cell division.
Aim 3: Determine which PAR-5 interacting proteins functions in cell division.
Approach: Using C. elegans and CRISPR-Cas9 technology, I will mutate conserved phosphorylation sites in several of the PAR-5 interacting proteins, create transgenic lines and determine which sites are important for cytokinesis via video microscopy. Once this is determined, I will then tag these proteins with GFP via CRISPR and determine if they localize to cell division structures, such as the spindle or actin ring.
Rationale: By performing this assay, I can identify phosphorylation sites on PAR-5 interacting proteins that are important for cytokinesis and then determine if these proteins localize to structures involved during cell division events.
Outcomes: With this knowledge, I will be able to apply the fundamental aspects of this research to well conserved binding partners of 14-3-3σ to discovery interaction pathway targets in cell division that are necessary for tumor growth. This would allow for treatments/drugs to be created at these sites.
References:
[1] Colon and Rectal Cancer. (n.d.). Retrieved February 19, 2015, from http://www.cancer.gov/cancertopics/types/colon-and-rectal
[2] Wilker, E.W., van Vugt, M., Artim, S.A., Huang, P.H., Petersen, C.P., Reinhardt, H.C., Feng, Y., Sharp, P.A., Sonenberg, N., White, F.M., et al. (2007). 14-3-3 sigma controls mitotic translation to facilitate cytokinesis. Nature 446, 329-332.
[3] Hermeking, H. (2003). The 14-3-3 cancer connection. Nature Reviews Cancer, 3(12), 931-943. doi: 10.1038/nrc1230.
[4] Morton, D., Shakes, D., Nugent, S., Dichoso, D., Wang, W., Golden, A., & Kemphues, K. (n.d.). The Caenorhabditis elegans par-5 Gene Encodes a 14-3-3 Protein Required for Cellular Asymmetry in the Early Embryo. Developmental Biology, 47-58.
[1] Colon and Rectal Cancer. (n.d.). Retrieved February 19, 2015, from http://www.cancer.gov/cancertopics/types/colon-and-rectal
[2] Wilker, E.W., van Vugt, M., Artim, S.A., Huang, P.H., Petersen, C.P., Reinhardt, H.C., Feng, Y., Sharp, P.A., Sonenberg, N., White, F.M., et al. (2007). 14-3-3 sigma controls mitotic translation to facilitate cytokinesis. Nature 446, 329-332.
[3] Hermeking, H. (2003). The 14-3-3 cancer connection. Nature Reviews Cancer, 3(12), 931-943. doi: 10.1038/nrc1230.
[4] Morton, D., Shakes, D., Nugent, S., Dichoso, D., Wang, W., Golden, A., & Kemphues, K. (n.d.). The Caenorhabditis elegans par-5 Gene Encodes a 14-3-3 Protein Required for Cellular Asymmetry in the Early Embryo. Developmental Biology, 47-58.
simonsfinaldraft4.14.2015.pdf | |
File Size: | 76 kb |
File Type: |
simonsfinaldraft4.12.15.docx | |
File Size: | 147 kb |
File Type: | docx |
simonsdraft2saims3.19.15.docx | |
File Size: | 149 kb |
File Type: | docx |
simonsdraft1saims2.26.15.docx | |
File Size: | 158 kb |
File Type: | docx |