Bioinformatic analysis of the evolution of the plant phosphatome and the biochemical characterization of novel Arabidopsis thaliana Protein Phosphatase 1 complexes.

摘要

In eukaryotes, nearly all cellular processes are regulated in some manner by reversible protein phosphorylation. The opposing phosphorylation and dephosphorylation reactions are catalyzed by protein kinases and phosphatases, respectively. While the protein kinases, with very few exceptions, share the same catalytic domain, the protein phosphatases are much more structurally diverse, with three distinct classes of catalytic domains identified. The Phosphoprotein Phosphatases (PPPs) and Protein Tyrosine Phosphatases (PTPs) have received the majority of research attention, however many of the other phosphatases play key roles in the cell. I examined the protein coding sequences from plant (Arabidopsis thaliana, Oryza sativa and Populus trichocarpa ) and algal (Chlamydomonas reinhardtii and Ostreococcus tauri) genomes for homologs to the less studied phosphatases, as well as those phosphatases that seem to have been lost in the evolution to higher plants. It appears that a set of phosphatases was present in the last common ancestor of plants and animals, and that divergence after that point resulted in two distinct, but overlapping phosphatase sets, with algae at an intermediate position. Notably, it was determined that the Cell Division Cycle (CDC) phosphatases CDC25 and CDC14 are absent in plants, and the CDC14 domain seems to have been adapted as a protein-protein interaction domain. As well, in plants there appears to have been a dramatic increase in the number of the RNA polymerase C-terminal domain phosphatase-like proteins compared to mammals. I then examined more closely one of the PPP family phosphatases, Protein Phosphatase 1 (PP1). PP1 binds to an impressive array of proteins in mammals, and only a single binding partner in plants has been discovered to date. Through affinity chromatography, I identified several putative PP1 interacting proteins, and provide further evidence for the interaction of two of the proteins, AtI-2 (Inhibitor-2) and GEM (GL2 Expression Modulator) with PP1. In the course of characterization, I found AtI-2 to be localized differently than its mammalian counterpart, and GEM to bind a phosphoinositide not previously demonstrated to exist in plants.