Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
A. Gururaj Rao
Plant growth and development, much like in animals, is a highly-coordinated process of cell division and differentiation which generate new cell types and tissues. Receptor kinases respond to extra-cellular cues and mediate appropriate downstream gene expression through auto and trans-phosphorylation events. Arabidopsis CRINKLY4 (ACR4) is a plasma membrane localized receptor-like kinase that plays an important role in formative cell division and stem cell differentiation in root tip and initiates lateral root development in Arabidopsis. The Arabidopsis genome also encodes four homologs of ACR4, referred to as Arabidopsis CRINKLY4 Related (AtCRR) proteins. ACR4 and its homologs have equivalent architecture, comprised of an extracellular ligand binding domain, a transmembrane helix, and an intracellular kinase domain. Cell biology, genetic and in vitro biochemical studies have indicated potential interaction partners within the ACR4 family. However, very little is known of the ACR4 mediated network of interactions and the molecular mechanism of its regulation.
In this dissertation, we report the identification of the catalytic subunit of Protein Phosphatase 2A-3 isoform (PP2A3c) as the first known substrate for the kinase activity of ACR4 and ACR4-homolog CRK1. We provide evidence of a regulatory reciprocal interaction between the catalytic subunit of PP2A3c and ACR4, with PP2A3c serving the dual role as a substrate for ACR4 kinase and as a regulatory phosphatase in modulating kinase activity. To further advance in vitro biochemical and biophysical studies on the regulation of phosphatase activity, we describe expression and purification of MBP-tagged PP2A3c in E. coli. We demonstrate slow activation of phosphatase activity of PP2A3c over time and further demonstrate the ability of the PP2A chaperone, PTPA, to more rapidly activate phosphatase activity in trans, in the presence of ATP. We provide a biochemical context to these results and the usefulness of our system in investigating the role of post-translational modifications. Additionally, we have characterized the biochemical properties of the recombinantly expressed intracellular kinase domain of the ACR4 homolog, CRK1. The in-planta expression of this RLK in the cells of the root tip overlaps the expression pattern of ACR4 and PP2A and we demonstrate that PP2A3c is also a substrate for CRK1. Mass spectrometry analysis confirmed >30 sites of phosphorylation at Serine, Threonine and Tyrosine residues, spanning the entire intracellular domain of CRK1. Mutagenesis experiments identified phosphorylation sites within the activation loop that had both positive and negative effects on the kinase activity of CRK1. Finally, we describe the generation of a highly specific monoclonal antibody to the kinase domain of ACR4.
Sandal, Priyanka, "Characterizing the biochemical interactions between Arabidopsis CRINKLY4 (ACR4), the catalytic subunit of PP2A phosphatase and Arabidopsis CRINKLY4 related kinase (CRK1)" (2017). Graduate Theses and Dissertations. 16747.