Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase

Abstract

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)...

Activation of Mitochondrial Protein Phosphatase SLP2 by MIA40 Regulates Seed Germination

“…AtMIA40 immunoprecipitation was performed with 50 mg protein extract isolated from 10-d-old dark-grown Arabidopsis cell culture as in Uhrig et al (2016). Protein extract was pretreated with 20 mM DTT for 45 min at 4°C followed by alkylation with 110 mM NEM for 1 h at 4°C.…”

Structural basis for the preference of the Arabidopsis thaliana phosphatase RLPH2 for tyrosine-phosphorylated substrates

“…The substrate specificity of the RLPH2 enzyme from Arabidopsis was previously defined through a biochemical approach and found to be highly specific for phosphotyrosine, although the sequence of the enzyme revealed a catalytic core that is characteristic of the PPP family (8,10). In addition, AtRLPH2 was found to be sensitive to the tyrosine phosphatase inhibitor vanadate, which typically does not affect the activities of PPP enzymes.…”

“…Our previous work demonstrated that AtRLPH2 displays a clear preference for substrates containing phosphotyrosine compared to substrates containing phosphoserine or phosphothreonine (10). Upon exploring additional peptides as substrates, we discovered that AtRLPH2 readily dephosphorylated peptides derived from mammalian mitogen-activated protein kinases (MAPKs) within their dual phosphorylated pTxpY activation loops and these were some of the best substrate peptides we identified.…”

“…The RLPH2 enzymes have very few eukaryotic representatives outside of photosynthetic organisms and were predicted to localize to the cytosol and nucleus. We characterized Arabidopsis thaliana RLPH2 (AtRLPH2) biochemically, confirmed its cytosolic localization, and unexpectedly found that it is a phosphotyrosine-specific, rather than a phosphoserineor phosphothreonine-specific, protein phosphatase (10). Although the amino acid sequence of RLPH2 contains key motifs shared with phosphoserine-and phosphothreonine-specific phosphatases in the PPP family, RLPH2 has several unique properties, including not only being resistant to the classic serine-threonine phosphatase inhibitors okadaic acid and microcystin but also being sensitive to the PTP phosphatase inhibitor vanadate.…”

“…AtRLPH2 was expressed as reported by Uhrig et al (10). Cells were stored frozen and thawed in resuspension buffer [50 mM Hepes-NaOH (pH 7.5), 150 mM NaCl, 5% (v/v) glycerol, 10 mM imidazole, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride] before lysis using a French press (three rounds at 1000 psi).…”