Characterization of the function of a novel protein tyrosine phophatase predicted by the genome sequence of Arabidopsis thaliana

Abstract

Covalent modification of proteins by reversible phosphorylation, catalysed by specific protein kinases and phosphatases, is an important mechanism for enzyme activity regulation in metabolism and signalling pathways. Whereas both groups of proteins are well-characterized in animals, and in microorganisms, our understanding of plant protein phosphatases is limited especially concerning protein tyrosine phosphatases. For this project, genes in the model plant Arabidopsis thaliana potentially coding for protein tyrosine phosphatases were extracted from the genomic sequence database. From it, a dual specific phosphatase was selected. The predicted protein, called AtPTPKISl, appeared to contain a motif corresponding to the kinase interaction sequence (KIS) of the SNFl family. Homologues of this gene are present in other plant species, such as tomato. The ESTs available for AtPTPKISl suggested that this gene could undergo alternative splicing to form mRNAs encoding a frill-length and a truncated arrangement of the protein. The predicted interaction between AtPTPKISl and AKJNll, an Arabidopsis SnRKl, containing the motif KIS, was demonstrated both by assays using yeast two-hybrid system, and by "GST pull-down" assays in vitro. However, attempts to produce functional AtPTPKISl as a recombinant protein in a yeast expression system for further characterisation were inconclusive. The data show that AtPTPKISl can associate with SnRKl, but do not establish whether diis interaction plays a role in planta in regulating the activity of SnRKl, which itself functions as a global metabolic regulator. AtPTPKISl potential function in cells was investigated through establishing its expression pattern. Analysis of EST microarray databases showed opposite pattern of expression between AKIN β subunit and AtPTPKISl. These data strongly suggest that AtPTPKISl is playing a role in modulating the activity of SnRK complexes. However, Arabidopsis mutants containing "knockouts" for this gene and similar genes showed no phenotype, and further work will be necessary to investigate the potential role of AtPTPKISl in metabolic regulation.