Identification and H2O2 sensitivity of the major constitutive MAPK phosphatase from rat brain
55
Citation
30
Reference
10
Related Paper
Citation Trend
Keywords:
Dual-specificity phosphatase
Dithiothreitol
DUSP6
Dual-specificity phosphatase
Dithiothreitol
DUSP6
Cite
Citations (55)
There are multiple regulatory devices of protein phosphatase type 2A (PP2A), known on the biochemical level: association of regulatory subunits, interaction with other proteins, covalent modification by phosphorylation on tyrosyl and threonine residues, methylation of the carboxy terminus (see ref. for a recent review). In this chapter still another device will be described and discussed. Although PP2A is generally known as a phosphatase that specifically dephosphorylates seryl and threonyl residues, it can also operate as a phosphotyrosyl phosphatase, and this activity can be regulated independently. Historically, PP2A was the first phosphatase described that could remove phosphate from phosphotyrosyl residues (,) and is, therefore, a "dual specificity" phosphatase "avant la lettre." The in vitro characterization of the dual specificity of PP2A indicated that the phosphoseryl and phosphotyrosyl phosphatase activities exhibited distinct catalytic properties and thermostability (,); they were either conversely affected by free ATP or pyrophosphate (), or concurrently stimulated by tubulin (). Further observations led to the isolation of a protein that specifically stimulates the phosphotyrosyl phosphatase activity of PP2A without affecting its Ser-P/Thr-P phosphatase activity (,, , ). This phosphotyrosyl phosphatase activator or PTPA was shown to be highly specific for the dimeric form of PP2A (PP2AD) (). PTPA stimulates the PTPase activity of PP2AD in a time-dependent enzyme-like reaction requiring ATP, Mg2+ as essential cofactor that cannot ble ATP analogs ().be replaced by nonhydrolysa
DUSP6
Dual-specificity phosphatase
Cite
Citations (18)
Kinases and phosphatases are key regulators of cellular behavior; their combined activity determines the output of intracellular signaling cascades. To better understand phosphatase regulation, our lab has developed a set of high-throughput, substrate-specific phosphatase activity assays that profile the dynamics of cytoplasmic and nuclear phosphatase activity independently. Protein phosphorylation by kinases has been viewed as an active process requiring precise control while protein dephosphorylation by phosphatases has been thought of as a constitutive and unregulated process. Studies have shown that kinase regulation and specificity is important in determining cellular behavior. Only recently have similar observations been made for phosphatases. We now appreciate that phosphatase activity can be regulated post-translationally and that their phosphosite specificity and subcellular localization are key determinants of intracellular signaling regulation. Previously, our lab has developed a whole cell phosphatase assay to better understand phosphatase regulation and quantified the dynamics of dual specificity phosphatase (DUSP) activities on MAPK substrates. We expanded the existing assay to quantify the activity of subcellularly partitioned phosphatases as well as phosphatases with different specificities including protein tyrosine phosphatases and protein serine/threonine phosphatases. We purified six recombinant phosphosubstrates: ERK2(T202/Y204), p38(T180/Y182), JNK(T183/Y185), MAPKAPK2(T334), CREB(S133), and STAT1(Y701). In a 96-well format, each well is coated with a single phosphosubstrate and incubated with cytoplasmic or nuclear extracts containing active phosphatases. During cellular lysis extracts are ATP-depleted to preclude kinase activity. Phosphatase activity is quantified by immunodetection and ELISA readout of the remaining unreacted phosphoprotein. We developed a lysis method that results in the cytoplasmic and nuclear fractionation of active phosphatases from adherent cell cultures. We validated compartmentalization of known cytoplasmic and nuclear proteins by western blot and showed compartment-specific phosphatase measurements with our assay platform (Fig 1 representative data). The low measurement noise of this assay (CV<20%) renders it sensitive and able to reliably detect phophatase activity with less than 50,000 cell lysates. Thus, we can quantify phosphatase activity toward all 6 substrates from a single extract making our assay amenable to parallelization. We demonstrate the power of our assay by profiling the kinetics of phosphatase activity in response to viral infection and cytokine stimulation. Our results show unique activity profiles depending on subcellular localization and substrate specificity. In this study we developed an assay platform to quantify nuclear and cytoplasmic substrate specific phosphatase activities. These assays are compatible with any adherent cell system and provides researchers with a method to study substrate specific phosphatase activity in a parallelizable and high-throughput manner. Support or Funding Information This work was supported by the UVA Biotechnology Training Program, NIAID, and the American Heart Association. A. Lysis schematic B. MCF10A WB C & D pMK2 Cyto and Nuc PPase Assay
DUSP6
Dephosphorylation
Dual-specificity phosphatase
Cite
Citations (0)
Abstract Protein phosphatases are integrally associated with the regulation of cellular signaling. The mechanisms underlying the specific regulatory roles are likely to be unique to each cell system. Nevertheless, analysis of phosphatase regulation in a number of systems has identified phosphatase targeting through association with a wide range of binding partners to be a fundamental mechanism of regulation. Using protein phosphatase 2A (PP2A) as an example, this snapshot summarizes these fundamental mechanisms of protein phosphatase regulation.
DUSP6
Dual-specificity phosphatase
14-3-3 protein
Cite
Citations (14)
Extracellular signal-regulated kinases 1 and 2 (ERKs) are central regulators of many physiological and pathological processes. Their activity is regulated by phosphorylation on both tyrosine and threonine residues within their activation loops by MAPK / ERK kinases 1 and 2 (MEKs). Removal of phosphate from either the tyrosine, the threonine, or from both residues together can inactivate ERKs. Indeed members of the three groups of protein phosphatases, protein Ser / Thr phosphatase, protein Tyr phosphatase, and dual specificity phosphatases have been implicated in the inactivation of ERKs. In this review, we describe the various mechanisms involved in the inactivation of ERKs during different stages of mitogenic stimulation of quiescent cells. Keywords: erk, mapk, mkp, protein tyr phosphatase, protein ser thr phosphatase
Dual-specificity phosphatase
DUSP6
Cite
Citations (31)
DUSP6
Dephosphorylation
Dual-specificity phosphatase
Cite
Citations (29)
We have described recently the purification and cloning of PP2A (protein phosphatase 2A) leucine carboxylmethyltransferase. We studied the purification of a PP2A-specific methylesterase that co-purifies with PP2A and found that it is tightly associated with an inactive dimeric or trimeric form of PP2A. These inactive enzyme forms could be reactivated as Ser/Thr phosphatase by PTPA (phosphotyrosyl phosphatase activator of PP2A). PTPA was described previously by our group as a protein that stimulates the in vitro phosphotyrosyl phosphatase activity of PP2A; however, PP2A-specific methyltransferase could not bring about the activation. The PTPA activation could be distinguished from the Mn2+ stimulation observed with some inactive forms of PP2A, also found associated with PME-1 (phosphatase methylesterase 1). We discuss a potential new function for PME-1 as an enzyme that stabilizes an inactivated pool of PP2A.
DUSP6
Cite
Citations (101)
DUSP6
Dual-specificity phosphatase
Cite
Citations (50)
Lafora Disease (LD) is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs). LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, and glucan binding assays. Our results demonstrate that laforin prevalently exists as a monomer with a small dimer fraction both in vitro and in vivo. Of mechanistic importance, laforin monomer and dimer possess equal phosphatase activity, and they both associate with malin and bind glucans to a similar extent. However, we found differences between the two states' ability to interact simultaneously with malin and carbohydrates. Furthermore, we tested other members of the glucan phosphatase family. Cumulatively, our data suggest that laforin monomer is the dominant form of the protein and that it contains phosphatase activity.
Lafora Disease
Dual-specificity phosphatase
DUSP6
Cite
Citations (31)
We identified an essential Saccharomyces cerevisiae protein, Tap42, that associates with Sit4, a type 2A-related protein phosphatase, and with the type 2A phosphatase catalytic subunits. The association of Tap42 with the phosphatases does not require the previously identified phosphatase subunits. Genetic analysis suggests that Tap42 functions positively with both phosphatases. Mutations in TAP42 can confer almost complete rapamycin resistance. In addition, Tap42/Sit4 and Tap42/PP2A complex formation is regulated by nutrient growth signals and the rapamycin-sensitive Tor signaling pathway. These findings, combined with the defect in translation of the tap42-11 mutant at the nonpermissive temperature, suggest that Tap42, Sit4, and PP2A are components of the Tor signaling pathway.
TOR signaling
DUSP6
Cite
Citations (500)