Reversible protein phosphorylation is a ubiquitous regulatory mechanism governing biological processes such as the cell cycle, metabolism, transmembrane signaling and gene regulation. Phosphorylating and dephosphorylating enzyme activities are carefully balanced in normal cells, and often are regulated by covalent modifications, association of specific regulatory subunits and formation of large multi-component signaling complexes. The protein phosphatase 2A (PP2A) holoenzyme is a heterotrimeric complex containing a catalytic subunit (PP2A-C) bound to one A and one B regulatory subunit. The mechanisms allowing the regulatory subunits to control PP2A activity remain poorly defined. In Arabidopsis, small gene families encode each of the PP2A subunits. The ROOTS CURL IN NPA (RCN1) gene encodes one of three regulatory A subunits, and the RCN1 protein acts as a positive regulator of PP2A activity. Loss of RCN1 function perturbs seedling root growth by altering transport of the growth regulator auxin. Mutations in the RCN1 paralogs PP2AA2 and PP2AA3 have little effect on overall PP2A enzyme activity, indicating that RCN1 plays a cardinal role in regulating PP2A activity. Cryptic functions for the PP2AA2 and PP2AA3 isoforms are unmasked in the rcn1 mutant background. Thus, although the A subunits of Arabidopsis PP2A exhibit very strong sequence similarity, their functions are not fully redundant. The basis for functional specialization of A subunit isoforms is unknown. The proposed experiments focus on defining the protein sequences and/or expression patterns required for the unique biological functions of RCN1, identifying specific binding partners and characterizing conditional and tissue-specific functions. The rcn1 mutant phenotype clearly defines non-redundant aspects of RCN1 A subunit function. The working model is that amino acid sequence determinants and/or unique aspects of the RCN1 expression pattern are required for these non-redundant functions. Furthermore, RCN1-specific functions are mediated by protein complexes that contain different PP2A subunits and accessory factors, targets or regulators. The proposed experiments will identify key determinants of RCN1 function, providing new insight into the regulation and biological functions of PP2A activity. The long-term goals of this project are to define the circuitry that regulates PP2A activity and to elucidate the normal functions of PP2A enzymes in plants. Broader Impacts Protein phosphorylation levels in cells are determined by a highly dynamic balance between the activities of protein kinases and phosphatases, and protein phosphatase activity levels are exquisitely calibrated. The Arabidopsis system provides a powerful model for this analysis because of the molecular and genetic tools that are available for generating and analyzing specific phosphatase mutants and identifying interacting regulatory factors. This systematic approach to the analysis of phosphatase regulation will provide new insights into this fundamental cellular control mechanism. The proposed work will provide training opportunities in the areas of molecular genetics, plant physiology and development, functional genomics and proteomics at the undergraduate, graduate and post-doctoral levels. The PI's laboratory has a strong record of undergraduate authorship on publications. The PI is committed to providing high-quality research opportunities to graduate and undergraduate students, particularly members of under-represented groups, and also is involved in increasing outreach efforts through Brown's MCB Graduate Program to under-represented groups.
