Protein phosphatase type 2C dephosphorylates BAD

Reversible phosphorylation modulates a cells’ susceptibility to apoptosis. The phosphorylation status of BAD, a member of the Bcl-2 protein family, is an important checkpoint governing life-or-death decisions: Phosphorylation of serine residues 112, 136 and 155 on BAD prevents apoptosis. Here we report that BAD is a substrate for PP2C. Ser¹⁵⁵ is involved in heterodimerization with Bcl-X_L. We could demonstrate that PP1, PP2A and PP2C act on this site in vitro. However, only PP2C gives priority to P-Ser¹⁵⁵ compared to P-Ser¹¹² and P-Ser¹³⁶ on BAD. The results indicate that PP2C is an additional factor triggering the pro-apoptotic function of BAD.     

Striatal enriched phosphatase 61 dephosphorylates Fyn at phosphotyrosine 420

A family of protein tyrosine phosphatases enriched within the central nervous system called striatal enriched phosphatase (STEP) has been implicated in the regulation of the N-methyl-d-aspartate receptor. STEP(61), a membrane-associated isoform located in the postsynaptic densities (PSDs) of striatal neurons, contains two transmembrane domains, two proline-rich domains, and a kinase-interacting motif. This study demonstrates that STEP(61) associates with Fyn, a member of the Src family kinases that is also enriched in PSDs. By using human embryonic kidney 293 cells for co-transfection, we determined that a substrate-trapping variant (STEP(61) CS) binds to Fyn but not to other members of the Src family present in PSDs. In a complementary experiment, myc-tagged Fyn immunoprecipitates STEP(61) CS. STEP(61) binds to Fyn through one of its proline-rich domains and the kinase-interacting motif domain, whereas Fyn binds to STEP(61) through its Src homology 2 domain and the unique N-terminal domain. STEP(61) CS pulls down Fyn when the Tyr(420) site is phosphorylated. In vitro, wild-type STEP(61) dephosphorylates Fyn at Tyr(420) but not at Tyr(531). These results suggest that STEP regulates the activity of Fyn by specifically dephosphorylating the regulatory Tyr(420) and may be one mechanism by which Fyn activity is decreased within PSDs.     

Protein phosphatase 1, but not protein phosphatase 2A, dephosphorylates DNA-damaging stress-induced phospho-serine 15 of p53

Okadaic acid (OA) is a protein phosphatase (PP) inhibitor and induces hyperphosphorylation of p53. We investigated whether the inhibition of PP1 by OA promotes the phosphorylation of the serine 15 of p53. In vitro dephosphorylation assay showed that PP1 dephosphorylated ultraviolet C (UVC)-induced phospho-ser15 of p53, and that OA treatment inhibited it. One of the PP1 regulators, growth arrest and DNA damage 34 (GADD34), disturbed PP1 binding with p53, interfered with the dephosphorylation of p53 and increased the amount of phospho-p53 after UVC-treatment. This report provides the first evidence that PP1, but not PP2A, dephosphorylates phospho-serine 15 of p53.     

Protein kinase C-zeta phosphorylates insulin-responsive aminopeptidase in vitro at Ser-80 and Ser-91

Insulin-responsive aminopeptidase (IRAP) colocalizes with glucose transporter type 4 (GLUT4) in adipocytes and is recruited to the plasma membrane in response to insulin. Microinjection of peptides corresponding to the IRAP cytoplasmic domain sequences causes GLUT4 recruitment in adipocytes. Inhibitors of protein kinase C-zeta (PKC-zeta) abolish the insulin-induced GLUT4 recruitment in rat adipocytes. These findings suggest an interesting possibility that PKC-zeta may phosphorylate IRAP, playing a key role in GLUT4/IRAP recruitment. To test this possibility, here we studied the (32)P incorporation into IRAP catalyzed by PKC-zeta in insulin-stimulated cells. There was a small but significant (32)P incorporation into IRAP in rat adipocytes, which was partly abolished upon addition of a PKC-zeta pseudosubstrate, suggesting that PKC-zeta may be responsible in part for the IRAP phosphorylation in adipocytes. PKC-zeta also catalyzed the incorporation of (32)P not only into IRAP in GLUT4 vesicles isolated from rat adipocytes but also into the IRAP cytoplasmic domain inserts in glutathione S-transferase-fusion proteins, demonstrating direct IRAP phosphorylation by PKC-zeta. Reversed-phase HPLC, matrix-assisted laser desorption ionization mass spectrometry, and radiosequencing of the tryptic digests of the (32)P-labeled IRAP fusion proteins identified Ser-80 and Ser-91 as major phosphorylation sites. In GLUT4 vesicles, the (32)P incorporation into IRAP was exclusively localized at a 6.9-kDa tryptic fragment identified as IRAP(76-138) and the (32)P labeling at Ser-80 accounted for 80-90% of the total IRAP labeling, suggesting that Ser-80 is the major phosphorylation site in intact IRAP. These findings are consistent with the possibility that the IRAP cytoplasmic domain phosphorylation by PKC-zeta plays a key role in insulin-induced IRAP or GLUT4 recruitment in adipocytes.     

Subcellular Localization and Ser-137 Phosphorylation Regulate Tumor-suppressive Activity of Profilin-1

The actin-binding protein profilin-1 (Pfn1) inhibits tumor growth and yet is also required for cell proliferation and survival, an apparent paradox. We previously identified Ser-137 of Pfn1 as a phosphorylation site within the poly-l-proline (PLP) binding pocket. Here we confirm that Ser-137 phosphorylation disrupts Pfn1 binding to its PLP-containing ligands with little effect on actin binding. We find in mouse xenografts of breast cancer cells that mimicking Ser-137 phosphorylation abolishes cell cycle arrest and apoptotic sensitization by Pfn1 and confers a growth advantage to tumors. This indicates a previously unrecognized role of PLP binding in Pfn1 antitumor effects. Spatial restriction of Pfn1 to the nucleus or cytoplasm indicates that inhibition of tumor cell growth by Pfn1 requires its nuclear localization, and this activity is abolished by a phosphomimetic mutation on Ser-137. In contrast, cytoplasmic Pfn1 lacks inhibitory effects on tumor cell growth but rescues morphological and proliferative defects of PFN1 null mouse chondrocytes. These results help reconcile seemingly opposed cellular effects of Pfn1, provide new insights into the antitumor mechanism of Pfn1, and implicate Ser-137 phosphorylation as a potential therapeutic target for breast cancer.     

The protein-tyrosine phosphatase SHP-1 binds to and dephosphorylates p120 catenin

A prominent tyrosine-phosphorylated protein of approximately 100 kDa (designated pp100) in epidermal growth factor (EGF)-stimulated A431 cells was found to be a main interaction partner of the protein-tyrosine phosphatase SHP-1 in pull-down experiments with a glutathione S-transferase-SHP-1 fusion protein. Binding was largely mediated by the N-terminal SH2 domain of SHP-1 and apparently direct and independent from the previously described association of SHP-1 with the activated EGF receptor. pp100 was partially purified and identified by mass spectrometric analysis of tryptic fragments, partial amino acid sequencing, and use of authentic antibodies as the 3A isoform of the Armadillo repeat protein superfamily member p120 catenin (p120(ctn)). Different p120(ctn) isoforms expressed in human embryonal kidney 293 cells, exhibited differential binding to SHP-1 that correlated partly with the extent of EGF-dependent p120(ctn) tyrosine phosphorylation. Despite strong phosphorylation, p120(ctn) isoforms 3B and 3AB bound, however, less readily to SHP-1. SHP-1 associated transiently with p120(ctn) in EGF-stimulated A431 cells stably transfected with a tetracycline-responsive SHP-1 expression construct, and p120(ctn) exhibited elevated phosphorylation upon a tetracycline-mediated decrease in the SHP-1 level. Functions of p120(ctn), which are regulated by tyrosine phosphorylation, may be modulated by the described SHP-1-p120(ctn) interaction.     

Protein phosphatase 2B dephosphorylates twitchin, initiating the catch state of invertebrate smooth muscle

"Catch" is the state where some invertebrate muscles sustain high tension for long periods at low ATP hydrolysis rates. Physiological studies using muscle fibers have not yet fully provided the details of the initiation process of the catch state. The process was extensively studied by using an in vitro reconstitution assay with several phosphatase inhibitors. Actin filaments bound to thick filaments pretreated with the soluble protein fraction of muscle homogenate and Ca2+ (catch treatment) in the presence of MgATP at a low free Ca2+ concentration (the catch state). Catch treatment with > 50 microm okadaic acid, > 1 microm microcystin LR, 1 microm cyclosporin A, 1 microm FK506, or 0.2 mm calcineurin autoinhibitory peptide fragment produced almost no binding of the actin filaments, indicating protein phosphatase 2B (PP2B) was involved. Use of bovine calcineurin (PP2B) and its activator calmodulin instead of the soluble protein fraction initiated the catch state, indicating that only PP2B and calmodulin in the soluble protein fraction are essential for the initiation process. The initiation was reproduced with purified actin, myosin, twitchin, PP2B, and calmodulin. 32P autoradiography showed that only twitchin was dephosphorylated during the catch treatment with either the soluble protein fraction or bovine calcineurin and calmodulin. These results indicate that PP2B directly dephosphorylates twitchin and initiates the catch state and that no other component is required for the initiation process of the catch state.     

Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser-74 dephosphorylation

Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn²⁺-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity.     

The catalytic subunit of phosphatase 2A dephosphorylates phosphoopsin

Rod cell outer segments were found to contain a protein phosphatase activity toward phosphoopsin with properties very similar to those of protein phosphatase 1 or 2A. The opsin phosphatase activity was stable to ethanol precipitation, had a Mr of 35,000-38,000 as determined by gel filtration, and was not dependent on divalent cations for activity. The chromatographic properties on DEAE-cellulose of the rod outer segment protein phosphatase were also similar to those reported for protein phosphatase 1 or 2A. In order to distinguish between these two protein phosphatases, we tested homogeneous preparations of protein phosphatases 1 and 2A from skeletal muscle for activity toward phosphoopsin. Protein phosphatase 2A dephosphorylated phosphoopsin at approximately 10% of its rate toward phosphorylase a, whereas protein phosphatase 1 had no activity toward phosphoopsin. We conclude that protein phosphatase 2A is present in the rod cell outer segment and that it is a likely candidate to perform the in vivo dephosphorylation of rhodopsin in the visual cycle.     

Protein phosphatase 1 is essential for Greatwall inactivation at mitotic exit

Entry into mitosis is mediated by the phosphorylation of key cell cycle regulators by cyclin-dependent kinase 1 (Cdk1). In Xenopus embryos, the M-phase-promoting activity of Cdk1 is antagonized by protein phosphatase PP2A-B55. Hence, to ensure robust cell cycle transitions, Cdk1 and PP2A-B55 must be regulated so that their activities are mutually exclusive. The mechanism underlying PP2A-B55 inactivation at mitotic entry is well understood: Cdk1-activated Greatwall (Gwl) kinase phosphorylates Ensa/Arpp19, thereby enabling them to bind to and inhibit PP2A-B55. However, the re-activation of PP2A-B55 during mitotic exit, which is essential for cell cycle progression, is less well understood. Here, we identify protein phosphatase PP1 as an essential component of the PP2A-B55 re-activation pathway in Xenopus embryo extracts. PP1 initiates the re-activation of PP2A-B55 by dephosphorylating Gwl. We provide evidence that PP1 targets the auto-phosphorylation site of Gwl, resulting in efficient Gwl inactivation. This step is necessary to facilitate subsequent complete dephosphorylation of Gwl by PP2A-B55. Thus, by identifying PP1 as the phosphatase initiating Gwl inactivation, our study provides the molecular explanation for how Cdk1 inactivation is coupled to PP2A-B55 re-activation at mitotic exit.     

Protein tyrosine phosphatase receptor type Z dephosphorylates TrkA receptors and attenuates NGF-dependent neurite outgrowth of PC12 cells

Protein tyrosine phosphatase receptor type Z (Ptprz/Ptpzeta / RPTPbeta) is a receptor-like protein tyrosine phosphatase (RPTP) which is predominantly expressed in the central nervous system. Tropomyosin-related kinases (Trks) are single-pass transmembrane molecules that are highly expressed in the developing nervous system. Upon the ligand binding of neurotrophins, Trk receptors are activated through autophosphorylation of tyrosine residues; however, the PTPs responsible for the negative regulation of Trk receptors have not been fully elucidated. Here, we identified Ptprz as a specific PTP that efficiently dephosphorylates TrkA as a substrate. Co-expression of Ptprz with Trk receptors in 293T cells showed that Ptprz suppresses the ligand-independent tyrosine phosphorylation of TrkA, but not of TrkB or TrkC, and that Ptprz attenuates TrkA activation induced by nerve growth factor (NGF). Co-expression analyses with TrkA mutants revealed that Ptprz dephosphorylates phosphotyrosine residues in the activation loop of the kinase domain, which are requisite for activation of the TrkA receptor. Consistent with these findings, forced expression of Ptprz in PC12D cells markedly inhibited neurite extension induced by a low dose of NGF. In addition, an increment in the tyrosine phosphorylation of TrkA was observed in the brain of Ptprz-deficient mice. Ptprz thus appears to be one of the PTPs which regulate the activation and signalling of TrkA receptors.     

The phosphoglucan phosphatase like sex Four2 dephosphorylates starch at the C3-position in Arabidopsis

Starch contains phosphate covalently bound to the C6-position (70 to 80% of total bound phosphate) and the C3-position (20 to 30%) of the glucosyl residues of the amylopectin fraction. In plants, the transient phosphorylation of starch renders the granule surface more accessible to glucan hydrolyzing enzymes and is required for proper starch degradation. Phosphate also confers desired properties to starch-derived pastes for industrial applications. In Arabidopsis thaliana, the removal of phosphate by the glucan phosphatase Starch Excess4 (SEX4) is essential for starch breakdown. We identified a homolog of SEX4, LSF2 (Like Sex Four2), as a novel enzyme involved in starch metabolism in Arabidopsis chloroplasts. Unlike SEX4, LSF2 does not have a carbohydrate binding module. Nevertheless, it binds to starch and specifically hydrolyzes phosphate from the C3-position. As a consequence, lsf2 mutant starch has elevated levels of C3-bound phosphate. SEX4 can release phosphate from both the C6- and the C3-positions, resulting in partial functional overlap with LSF2. However, compared with sex4 single mutants, the lsf2 sex4 double mutants have a more severe starch-excess phenotype, impaired growth, and a further change in the proportion of C3- and C6-bound phosphate. These findings significantly advance our understanding of the metabolism of phosphate in starch and provide innovative options for tailoring novel starches with improved functionality for industry.     

Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin-dependent protein kinases.

Okadaic acid (2 nM) inhibited by 80-90% the protein phosphatase activities in diluted extracts of rat liver, human fibroblasts, and Xenopus eggs acting on three substrates (high mobility group protein-I(Y), caldesmon and histone H1) phosphorylated by a cyclin-dependent protein kinase (CDK) suggesting that a type-2A phosphatase was responsible for dephosphorylating each protein. This result was confirmed by anion exchange chromatography of rat liver and Xenopus extracts, which demonstrated that the phosphatases acting on these substrates coeluted with the two major species of protein phosphatase 2A, termed PP2A1 and PP2A2. When matched for activity toward glycogen phosphorylase, PP2A1 was five- to sevenfold more active than PP2A2 and 35-fold to 70-fold more active than the free catalytic subunit (PP2Ac) toward the three CDK-labeled substrates. Protein phosphatases 1, 2B, and 2C accounted for a negligible proportion of the activity toward each substrate under the assay conditions examined. The results suggest that PP2A1 is the phosphatase that dephosphorylates a number of CDK substrates in vivo and indicate that the A and B subunits that are associated with PP2Ac in PP2A1 accelerate the dephosphorylation of CDK substrates, while suppressing the dephosphorylation of most other proteins. The possibility that PP2A1 activity is regulated during the cell cycle is discussed.     

Laforin, a dual specificity phosphatase that dephosphorylates complex carbohydrates

Laforin is the only phosphatase in the animal kingdom that contains a carbohydrate-binding module. Mutations in the gene encoding laforin result in Lafora disease, a fatal autosomal recessive neurodegenerative disorder, which is diagnosed by the presence of intracellular deposits of insoluble complex carbohydrates known as Lafora bodies. We demonstrate that laforin interacts with proteins known to be involved in glycogen metabolism and rule out several of these proteins as potential substrates. Surprisingly, we find that laforin displays robust phosphatase activity against a phosphorylated complex carbohydrate. Furthermore, this activity is unique to laforin, since several other phosphatases are unable to dephosphorylate polysaccharides. Finally, fusing the carbohydrate-binding module of laforin to the dual specific phosphatase VHR does not result in the ability of this phosphatase to dephosphorylate polysaccharides. Therefore, we hypothesize that laforin is unique in its ability to utilize a phosphorylated complex carbohydrate as a substrate and that this function may be necessary for the maintenance of normal cellular glycogen.     

The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin

Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified beta-catenin, a component of adherens junctions, as a substrate of Pez by a "substrate trapping" approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of beta-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including beta-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro "wound" assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion.     

Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB

The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis.     

An aortic spontaneously active phosphatase dephosphorylates myosin and inhibits actin-myosin interaction

Actin-myosin interaction in aortic actomyosin reportedly requires phosphorylation of the 20,000 dalton myosin light chains. A spontaneously active phosphatase which dephosphorylates phosphorylase $\text{a}$ and isolated phosphorylated cardiac myosin light chains was extracted from bovine aortic smooth muscle. This enzyme, when added to aortic native actomyosin (a) significantly suppressed phosphorylation of the light chains of the native hexameric smooth muscle myosin, (b) accelerated the rate and increased the magnitude of myosin light chain dephosphorylation in actomyosin that had been prephosphorylated, and (c) markedly attenuated the rate of actin-myosin interaction. These results support the hypothesis that myosin phosphorylation and subsequent actin-myosin interactions (contractility) in vascular smooth muscle may be modulated by spontaneously active aortic phosphatase.