The long-term objective of this work is to understand the molecular basis by which cells are polarized. Polarity is a fundamental property of cells that is required for proper development as well as adult physiology. For example, during development cell fate determinants are polarized in dividing cells as a mechanism for generating cell type diversity and the loss of cell polarity is a hallmark of many disease states, including cancer. For spatially and temporally precise establishment of cell polarity to occur, cellular signals must be interpreted and ultimately coupled to the segregation of the relevant cellular components. In diverse cell types, polarity is controlled by the evolutionarily conserved Par complex consisting of Bazooka (Baz;aka Par-3), Par-6, and atypical Protein Kinase C (aPKC). The Rho GTPase Cdc42, which interacts with Par-6, is a primary determinant of Par complex targeting and activation. The first specific aim of the proposal is to determine how Cdc42 alters Par complex activity, by activating the aPKC kinase when it binds to Par-6. We are testing the hypothesis that aPKC regulation arises from activation of a PDZ protein interaction domain present in Par-6. A combined biochemical, cell biological, and structural approach will be utilized for this aim.
The second aim i s to identify the molecular pathways that give rise to activated Cdc42 at specific cellular sites. We are testing the hypothesis that nucleotide exchange factors activate Cdc42 a certain areas of the cell cortex, and other factors inhibit activation elsewhere. For this aim we will utilize the neuroblast as a model system to uncover the molecular pathways that regulate Cdc42 spatial and temporal activation, which is ultimately responsible for Par complex targeting. A combined genetic and biochemical approach will be utilized for this aim. Finally, we are examining how aPKC kinase activity is coupled to the asymmetric segregation of cell fate determinants in neuroblasts with the hypothesis that direct phosphorylation of the basally segregated protein Miranda by aPKC is required for this process. Understanding the molecular events that lead to the coupled recruitment and activation of the Par complex will yield new insight into cell polarity.

Public Health Relevance

Many cells in our body, such as skin cells that provide a physical barrier to the environment, are polarized and loss of polarity is a hallmark of many diseases, including cancer. In this work, we are investigating a set of three proteins, known as the Par complex, that regulate cellular polarities required for proper development and adult physiology. As the loss of polarity is associated with human disease, improving our understanding of the molecules that control this process will contribute to our knowledge of the mechanisms of disease states.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular Signaling and Regulatory Systems Study Section (CSRS)
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Deatherage, James F
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University of Oregon
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Whitney, Dustin S; Peterson, Francis C; Kittell, Aaron W et al. (2016) Binding of Crumbs to the Par-6 CRIB-PDZ Module Is Regulated by Cdc42. Biochemistry 55:1455-61
Whitney, Dustin S; Peterson, Francis C; Kittell, Aaron W et al. (2016) Correction to Binding of Crumbs to the Par-6 CRIB-PDZ Module Is Regulated by Cdc42. Biochemistry 55:2063
Drummond, Michael L; Prehoda, Kenneth E (2016) Molecular Control of Atypical Protein Kinase C: Tipping the Balance between Self-Renewal and Differentiation. J Mol Biol 428:1455-64
Bailey, Matthew J; Prehoda, Kenneth E (2015) Establishment of Par-Polarized Cortical Domains via Phosphoregulated Membrane Motifs. Dev Cell 35:199-210
Graybill, Chiharu; Prehoda, Kenneth E (2014) Ordered multisite phosphorylation of lethal giant larvae by atypical protein kinase C. Biochemistry 53:4931-7
Lu, Michelle S; Mauser, Jonathon F; Prehoda, Kenneth E (2012) Ultrasensitive synthetic protein regulatory networks using mixed decoys. ACS Synth Biol 1:65-72
Graybill, Chiharu; Wee, Brett; Atwood, Scott X et al. (2012) Partitioning-defective protein 6 (Par-6) activates atypical protein kinase C (aPKC) by pseudosubstrate displacement. J Biol Chem 287:21003-11
Mauser, Jonathon F; Prehoda, Kenneth E (2012) Inscuteable regulates the Pins-Mud spindle orientation pathway. PLoS One 7:e29611
Johnston, Christopher A; Whitney, Dustin S; Volkman, Brian F et al. (2011) Conversion of the enzyme guanylate kinase into a mitotic-spindle orienting protein by a single mutation that inhibits GMP-induced closing. Proc Natl Acad Sci U S A 108:E973-8
Smith, Nicholas R; Prehoda, Kenneth E (2011) Robust spindle alignment in Drosophila neuroblasts by ultrasensitive activation of pins. Mol Cell 43:540-9

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