Regulation of Protein Phosphatase 2A Activity in Arabidopsis by the RCN1 Protein

Brown University, Providence RI

Investigators

Abstract

Reversible protein phosphorylation is a ubiquitous regulatory mechanism governing biological processes such as the cell cycle, metabolism, transmembrane signalling and gene regulation. Phosphorylating (kinase) and dephosphorylating (phosphatase) enzyme activities are carefully balanced in normal cells, and these activities often are regulated by covalent modifications, association of specific regulatory subunits, and by 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 A and B regulatory subunits to control PP2A activity remain poorly defined. In Arabidopsis, small gene families encode each of the PP2A subunits, and only a few mutations in PP2A genes have been isolated. The ROOTS CURL IN NPA (RCN1) gene encodes one of three regulatory A subunits, and phenotypic analysis of the rcn1 mutant has shown that the RCN1 protein acts as a positive regulator of PP2A activity. Loss of RCN1 function in seedling roots perturbs polar transport of the plant hormone auxin, altering gravity response and growth of lateral roots. 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. This project will extend our analysis of RCN1 function, and will provide key insights into the roles of PP2A and the regulation of its activity in Arabidopsis. The subcellular localization of the RCN1 protein will be determined using reporter gene fusions. Complementation assays will test the ability of other A subunit genes to supply RCN1 function. Isoform-specific antibody reagents will be developed to allow detailed analysis of isoform-specific functions and interactions. PP2A subunits and other proteins that bind to the RCN1 protein will be identified to gain insight into the mechanisms of RCN1 action. Genetic experiments will test for interactions between RCN1 functions and the functions of other genes with roles in auxin transport or signaling. Auxin-responsive reporter genes will be used to determine whether the altered auxin transport activities observed in the rcn1 mutant result in abnormal auxin distribution. These experiments will yield information on the regulation of auxin transport by reversible protein phosphorylation. Our goals in this project are to clarify the role of RCN1 protein in governing PP2A activity, and to elucidate the normal functions of RCN1-containing PP2A enzymes. These studies will lead to a better understanding of the circuitry that regulates PP2A activity in plants.