Identification and post-translational modification of mitotic regulatory proteins.
EMSL Project ID
12294
Abstract
This project will be conducted as an NCRR collaborative project.This study is designed to identify novel chromosomal passenger (CP) proteins and their post-translational modifications. CPs undergo dramatic changes in their distribution during the cell cycle, localizing to kinetochores, midzone, cleavage furrow and midbody at various stages of mitosis. CPs play critical roles in the proliferation and cell cycle checkpoint signaling pathways and are the subject of intense research due to their importance as mitotic regulators. Using an antibody generated against a phosphorylated peptide, we have observed 9 major proteins that are phosphorylated exclusively during mitosis where they behave as CPs. One of these proteins has been positively identified as Plk3 which is closely related to a known CP, Plk1. Plk1 has been extensively studied and is one of the key regulators of mitosis whose activity is controlled by transcriptional upregulation during G2 and proteasomal degradation at the completion of mitosis. Less is known about Plk3 function but we hypothesize that it plays a similar role as Plk1 and is regulated by post-translational modifications during the cell cycle. There are three main goals of this project:
1. Identify the additional proteins recognized by our antibody using IPs from mitotic HMEC cells as the starting material. For controls, we would use IPs from interphase cells which do not contain the phosphorylated sites recognized by our antibody and from mitotic cells using a non-specific antibody. Identification of novel CPs will significantly advance our knowledge about the proteins involved in mitotic regulation.
2. Map phosphorylation sites on Plk3. There are 2 known phosphorylation sites that are conserved between all Plk family members and a unique site on Plk3 identified by our phosphorylation specific antibody. The goal here is to positively identify these known sites as well as other potential sites. To accomplish this, we will affinity purify Plk3 and identify phosphorylated peptides. Once phosphorylation sites are identified, they will be further characterized to determine the effects of cell cycle progression, manipulation of cell signaling pathways, cell stress, DNA damage, etc. on their phosphorylation status.
3. Examine the composition of complexes containing specifically phosphorylated Plk3. Characterization of the phosphorylation status and composition of intracellular complexes containing Plk3 during various stages of the cell cycle and under conditions that cause cell stress is needed to understand the complex regulation of this multifunctional kinase. A number of proteins physically associate with Plk3 including P53, Chk2, BubR1, Aurora-A, APC/C complex members and BubR1. This project will define the temporal pattern of Plk3 complex formation using isolated protein complexes from cultured HMEC cells during the cell cycle and in the presence of DNA damage.
Project Details
Project type
Exploratory Research
Start Date
2005-02-24
End Date
2007-06-28
Status
Closed
Released Data Link
Team
Principal Investigator
Related Publications
Yang, F., Camp, D.G., Gritsenko, M.A., Luo, Q., Kelly, R.T., Clauss, T.R., Brinkley, W.R., Smith, R.D., and Stenoien, D.L. (2007). Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus. J Cell Sci In Press.