-
Journal of Neurochemistry Aug 2015The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr(420)) or inhibit (Tyr(531)) Fyn activity. Within the central...
The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr(420)) or inhibit (Tyr(531)) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr(531) and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr(420) and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr(789). Dephosphorylation of Tyr(789) prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction in STEP61 activity increased the phosphorylation of PTPα at Tyr(789), as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol (EtOH) intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by EtOH administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by EtOH leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. STEP61 , PTPα, Fyn, and NMDA receptor (NMDAR) have been implicated in ethanol intake behaviors in the dorsomedial striatum (DMS) in rodents. Here, we report that PTPα is a novel substrate for STEP61. Upon ethanol exposure, STEP61 is phosphorylated and inactivated by protein kinase A (PKA) signaling in the DMS. As a result of STEP61 inhibition, there is an increase in the phosphorylation of PTPα, which translocates to lipid rafts and activates Fyn and subsequent NMDAR signaling. The results demonstrate a synergistic regulation of Fyn-NMDAR signaling by STEP61 and PTPα, which may contribute to the regulation of ethanol-related behaviors. NMDA, N-methyl-D-aspartate; PTPα, receptor-type protein tyrosine phosphatase alpha; STEP, STriatal-Enriched protein tyrosine Phosphatase.
Topics: Animals; Cells, Cultured; Corpus Striatum; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Proto-Oncogene Proteins c-fyn; Rats; Rats, Sprague-Dawley; Receptor-Like Protein Tyrosine Phosphatases, Class 4; Signal Transduction
PubMed: 25951993
DOI: 10.1111/jnc.13160 -
Plant Physiology Mar 2022Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting...
Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg2+- and Mn2+-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H+-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H+-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H+-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H+-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H+-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H+-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H+-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H+-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H+-ATPase in guard cells.
Topics: Arabidopsis; Arabidopsis Proteins; Cell Membrane; Light; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2C; Proton-Translocating ATPases
PubMed: 34894269
DOI: 10.1093/plphys/kiab571 -
The Journal of Biological Chemistry May 2018The integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate...
The integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2α-directed holophosphatase, a therapeutic target in diseases of protein misfolding, is comprised of a regulatory subunit, PPP1R15, and a catalytic subunit, protein phosphatase 1 (PP1). In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in the essential function of eIF2α dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific dephosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the holoenzyme to the [(o-chlorobenzylidene)amino]guanidines Sephin1 or guanabenz, putative small-molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence the requirement for G-actin and sensitivity of the holoenzyme to inhibitors. We found that eIF2α dephosphorylation by PP1 was moderately stimulated by repeat-containing PPP1R15A in an unphysiological low ionic strength buffer, whereas stimulation imparted by the co-presence of PPP1R15A and G-actin was observed under a broad range of conditions, low and physiological ionic strength, regardless of whether the PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether it was paired with native PP1 purified from rabbit muscle or recombinant PP1 purified from bacteria. Furthermore, none of the PPP1R15A-containing holophosphatases tested were inhibited by Sephin1 or guanabenz.
Topics: Actins; Animals; Catalytic Domain; Drug Resistance; Eukaryotic Initiation Factor-2; Gene Expression Regulation; Guanabenz; HeLa Cells; Humans; Phosphorylation; Protein Isoforms; Protein Phosphatase 1; Proteolysis; Rabbits
PubMed: 29618508
DOI: 10.1074/jbc.RA118.002325 -
ELife Oct 2022Protein tyrosine phosphatase receptor-type kappa (PTPRK) is a transmembrane receptor that links extracellular homophilic interactions to intracellular catalytic...
Protein tyrosine phosphatase receptor-type kappa (PTPRK) is a transmembrane receptor that links extracellular homophilic interactions to intracellular catalytic activity. Previously we showed that PTPRK promotes cell-cell adhesion by selectively dephosphorylating several cell junction regulators including the protein Afadin (Fearnley et al, 2019). Here, we demonstrate that Afadin is recruited for dephosphorylation by directly binding to the PTPRK D2 pseudophosphatase domain. We mapped this interaction to a putative coiled coil (CC) domain in Afadin that is separated by more than 100 amino acids from the substrate pTyr residue. We identify the residues that define PTP specificity, explaining how Afadin is selectively dephosphorylated by PTPRK yet not by the closely related receptor tyrosine phosphatase PTPRM. Our work demonstrates that PTP substrate specificity can be determined by protein-protein interactions distal to the active site. This explains how PTPRK and other PTPs achieve substrate specificity despite a lack of specific sequence context at the substrate pTyr. Furthermore, by demonstrating that these interactions are phosphorylation-independent and mediated via binding to a non-catalytic domain, we highlight how receptor PTPs could function as intracellular scaffolds in addition to catalyzing protein dephosphorylation.
Topics: Microfilament Proteins; Phosphorylation; Protein Tyrosine Phosphatases; Substrate Specificity
PubMed: 36264065
DOI: 10.7554/eLife.79855 -
The Biochemical Journal Jun 1992Protein-tyrosine phosphatases (PTPases) play an essential role in the regulation of signal transduction mediated by reversible protein-tyrosine phosphorylation. In order...
Insulin receptor and epidermal growth factor receptor dephosphorylation by three major rat liver protein-tyrosine phosphatases expressed in a recombinant bacterial system.
Protein-tyrosine phosphatases (PTPases) play an essential role in the regulation of signal transduction mediated by reversible protein-tyrosine phosphorylation. In order to characterize individual rat hepatic PTPases that might have specificity for autophosphorylated receptor tyrosine kinases, we isolated cDNA segments encoding three PTPases (PTPase 1B, LAR and LRP) that are expressed in insulin-sensitive liver and skeletal muscle tissue, and evaluated their catalytic activity in vitro. The intrinsic PTPase activities of the full-length PTPase 1B protein and the cytoplasmic domains of LAR and LRP were studied by expression of recombinant cDNA constructs in the inducible bacterial vector pKK233-2 using extracts of a host strain of Escherichia coli that lacks endogenous PTPase activity. Each of the cloned cDNAs dephosphorylated a cognate phosphopeptide derived from the regulatory region of the insulin receptor. Despite having only 30-39% sequence identity in their catalytic domains, LAR and PTPase 1B had similar relative activities between the peptide substrate and intact insulin receptors, and also displayed similar initial rates of simultaneous dephosphorylation of insulin and epidermal growth factor (EGF) receptors. In contrast, LRP exhibited a higher rate of dephosphorylation of both intact receptors relative to the peptide substrate, and also dephosphorylated EGF receptors more rapidly than insulin receptors. These studies indicate that three PTPases with markedly divergent structures have the catalytic potential to dephosphorylate both insulin and EGF receptors in intact cells and that redundant PTPase activity may occur in vivo. For these PTPases to have specific physiological actions in intact cells, they must be influenced by steric effects of the additional protein segments of the native transmembrane enzymes, cellular compartmentalization and/or interactions with regulatory proteins.
Topics: Amino Acid Sequence; Animals; Blotting, Northern; Cloning, Molecular; DNA; ErbB Receptors; Escherichia coli; Liver; Male; Molecular Sequence Data; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases; RNA, Messenger; Rats; Rats, Inbred Strains; Receptor, Insulin; Recombinant Proteins; Sequence Alignment
PubMed: 1599438
DOI: 10.1042/bj2840569 -
IUBMB Life Jan 2002Protein tyrosine phosphatases (PTPs) are a large and structurally diverse family of enzymes that are found in eukaryotes, prokaryotes, viruses, and plants. PTPs catalyse... (Review)
Review
Protein tyrosine phosphatases (PTPs) are a large and structurally diverse family of enzymes that are found in eukaryotes, prokaryotes, viruses, and plants. PTPs catalyse the dephosphorylation of tyrosyl phosphorylated proteins and can either antagonise or potentiate protein tyrosine kinase signalling. PTPs regulate fundamental cellular processes and have been implicated in the etiology and pathogenesis of various human diseases. The epidermal growth factor receptor (EGFR) is a widely distributed protein tyrosine kinase that regulates both normal development and plays a role in pathological conditions such as cancer. This review discusses the structure and function of PTPs and focuses on the PTPs that have been implicated in the dephosphorylation of the EGFR and the consequent suppression of EGFR signalling.
Topics: Animals; ErbB Receptors; Humans; Mice; Mice, Knockout; Mice, Transgenic; Molecular Structure; Phosphorylation; Protein Tyrosine Phosphatases; Signal Transduction
PubMed: 12018405
DOI: 10.1080/15216540210811 -
The Journal of Biological Chemistry Mar 2019Na-H exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma...
Na-H exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser, Ser, Ser, Ser, Ser, Ser, Thr, Ser, and Ser did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser Mutating VPF eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that β-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.
Topics: Amino Acid Sequence; Crystallography, X-Ray; Furans; HEK293 Cells; Humans; Ion Transport; Lipids; Parathyroid Hormone; Phosphates; Phosphoproteins; Phosphorylation; Protein Conformation; Receptors, Neuropeptide Y; Serine; Sodium-Hydrogen Exchangers; Sodium-Phosphate Cotransporter Proteins, Type IIa
PubMed: 30696771
DOI: 10.1074/jbc.RA119.007421 -
Genetics Mar 2016Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic...
Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.
Topics: CDC2 Protein Kinase; Phosphorylation; Protein Phosphatase 2; Protein Tyrosine Phosphatases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tyrosine; ras-GRF1
PubMed: 26715668
DOI: 10.1534/genetics.115.182469 -
The Plant Cell Mar 2020Phosphate (Pi) uptake in plants depends on plasma membrane (PM)-localized phosphate transporters (PTs). OsCK2 phosphorylates PTs and inhibits their trafficking from the...
Phosphate (Pi) uptake in plants depends on plasma membrane (PM)-localized phosphate transporters (PTs). OsCK2 phosphorylates PTs and inhibits their trafficking from the endoplasmic reticulum (ER) to the PM in rice (), but how PTs are dephosphorylated is unknown. We demonstrate that the protein phosphatase type 2C (PP2C) protein phosphatase OsPP95 interacts with OsPT2 and OsPT8 and dephosphorylates OsPT8 at Ser-517. Rice plants overexpressing reduced OsPT8 phosphorylation and promoted OsPT2 and OsPT8 trafficking from the ER to the PM, resulting in Pi accumulation. Under Pi-sufficient conditions, Pi levels were lower in young leaves and higher in old leaves in mutants than in those of the wild type, even though the overall shoot Pi levels were the same in the mutant and the wild type. In the wild type, OsPP95 accumulated under Pi starvation but was rapidly degraded under Pi-sufficient conditions. We show that OsPHO2 interacts with and induces the degradation of OsPP95. We conclude that OsPP95, a protein phosphatase negatively regulated by OsPHO2, positively regulates Pi homeostasis and remobilization by dephosphorylating PTs and affecting their trafficking to the PM, a reversible process required for adaptation to variable Pi conditions.
Topics: Endoplasmic Reticulum; Epistasis, Genetic; Gene Expression Regulation, Plant; Homeostasis; Membrane Transport Proteins; Models, Biological; Mutation; Organ Specificity; Oryza; Phosphates; Phosphorylation; Plant Proteins; Plant Roots; Plant Shoots; Protein Binding; Subcellular Fractions
PubMed: 31919298
DOI: 10.1105/tpc.19.00685 -
FEBS Letters Sep 1993Rat brain microtubule-associated protein MAP1B has been tested as a substrate for Ser/Thr protein phosphatases (PP). The dephosphorylation reactions were followed by...
Rat brain microtubule-associated protein MAP1B has been tested as a substrate for Ser/Thr protein phosphatases (PP). The dephosphorylation reactions were followed by specific antibodies recognizing phosphorylated and phosphorylatable epitopes. One set of phosphorylation sites on MAP1B are referred to as mode I sites, and their phosphorylation is presumably catalyzed by proline-directed protein kinases. These mode I sites are efficiently dephosphorylated by PP2B and 2A but not by PP1. Another set of phosphorylation sites on MAP1B are named mode II sites, and their phosphorylation is possibly due to casein kinase II. These mode II sites are dephosphorylated by PP2A and PP1, the PP2B being ineffective. The selectivity of phosphatases for different sites within the same protein indicates the complexity of the dephosphorylation reactions regulating the functionality of MAP1B in neurons.
Topics: Animals; Brain; Epitopes; Microtubule-Associated Proteins; Phosphoprotein Phosphatases; Phosphorylation; Rats; Substrate Specificity
PubMed: 7690334
DOI: 10.1016/0014-5793(93)80925-k