Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants.Reversible protein phosphorylation, mediated by protein kinases and phosphatases, is a regulatory mechanism key to the functioning of all cell types. With an estimated minimum of 75% of all human proteins controlled by this mechanism (1), connecting the biochemical characteristics of protein kinases and phosphatases to their cellular function is of central importance. Reversible protein phosphorylation can occur on a number of residues, but largely occurs in both plants and animals on serine, threonine, and tyrosine residues at approximate percentages of 86, 12, and 2, respectively (2-6).Protein kinases exist as one large superfamily with over 1050 members encoded in the genome of the model organism Arabidopsis thaliana (6). Conversely, A. thaliana maintains only 150 protein phosphatases, which are categorized into four distinct families conserved across eukaryotes. These include the phospho-protein phosphatases (PPPs), 4 phospho-protein metallo-phosphatases (PPMs), phospho-tyrosine phosphatases (PTPs), and the Asp-based catalysis phosphatases (7). Unlike protein kinases, which employ a single catalytic mechanism, each of the four protein phosphatase families employs differing catalytic mechanisms (7). The PPP, PPM, and Asp-based protein phosphatases coordinate metal ions in their active sites to assist in catalysis, and each has been shown to specifically target phosphorylated serine (pSer) and threonine (pThr) residues on protein substrates (7). The PPM catalytic domains have N-and C-terminal extensions that confer substrate specificity and regulation, whereas the PPP family enzymes all have additional subunits that define function (6)...

Peroxisomes are eukaryotic specific organelles that perform diverse metabolic functions including fatty acid β-oxidation, reactive species metabolism, photorespiration, and responses to stress. However, the potential regulation of these functions by post-translational modifications, including protein phosphorylation, has had limited study. Recently, we identified and catalogued a large number of peroxisomal phosphorylated proteins, implicating the presence of protein kinases in this organelle. Here, we employed available prediction models coupled with sequence conservation analysis to identify 31 protein kinases from the Arabidopsis kinome (all protein kinases) that contain a putative, non-canonical peroxisomal targeting signal type 1 (PTS1). From this, twelve C-terminal domain-PTS1s were demonstrated to be functional in vivo, targeting enhanced yellow fluorescent protein to peroxisomes, increasing the list of presumptive peroxisomal protein kinases to nineteen. Of the twelve protein kinases with functional PTS1s, we obtained full length clones for eight and demonstrated that seven target to peroxisomes in vivo. Screening homozygous mutants of the presumptive nineteen protein kinases revealed one candidate (GPK1) that harbors a sugar-dependence phenotype, suggesting it is involved in regulating peroxisomal fatty acid β-oxidation. These results present new opportunities for investigating the regulation of peroxisome functions.