Multiple forms of phosphatase from human brain: isolation and partial characterization of affi-gel blue nonbinding phosphatase activities.
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ABSTRACT Arpp19 is a potent inhibitor of PP2A-B55 that regulates this phosphatase to ensure the stable phosphorylation of mitotic/meiotic substrates. At G2-M, Arpp19 is phosphorylated by Greatwall on S67. This phosphorylated Arpp19 form displays a high affinity to PP2A-B55 and a slow dephosphorylation rate, acting as an “unfair” competitor of PP2A-B55 substrates. The molecular determinants conferring slow dephosphorylation kinetics to S67 are unknown. PKA also phosphorylates Arpp19. This phosphorylation performed on S109 is essential to maintain prophase I-arrest in Xenopus oocytes although the underlying signaling mechanism is elusive. Here, we characterized the molecular determinants conferring slow dephosphorylation to S67 and controlling PP2A-B55 inhibitory activity of Arpp19. Moreover, we showed that phospho-S109 restricts S67 phosphorylation by increasing its catalysis by PP2A-B55. Finally, we discovered a double feed-back loop between these two phospho-sites which is essential to coordinate the temporal pattern of Arpp19-dependent PP2A-B55 inhibition and Cyclin B/Cdk1 activation during cell division.
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The neuronal tissue-specific protein kinase C (PKC) substrate B-50 can be dephosphorylated by endogenous protein phosphatases (PPs) in synaptic plasma membranes (SPMs). The present study characterizes membrane-associated B-50 phosphatase activity by using okadaic acid (OA) and purified 32P-labeled substrates. At a low concentration of [gamma-32P]ATP, PKC-mediated [32P]phosphate incorporation into B-50 in SPMs reached a maximal value at 30 s, followed by dephosphorylation. OA, added 30 s after the initiation of phosphorylation, partially prevented the dephosphorylation of B-50 at 2 nM, a dose that inhibits PP-2A. At the higher concentration of 1 microM, a dose of OA that inhibits PP-1 as well as PP-2A, a nearly complete blockade of B-50 dephosphorylation was seen. Heat-stable PP inhibitor-2 (I-2) also inhibited dephosphorylation of B-50. The effects of OA and I-2 on B-50 phosphatase activity were additive. Endogenous PP-1- and PP-2A-like activities in SPMs were also demonstrated by their capabilities of dephosphorylating [32P]phosphorylase a and [32P]casein. With these exogenous substrates, sensitivities of the membrane-bound phosphatases to OA and I-2 were found to be similar to those of purified forms of these enzymes. These results indicate that PP-1- and PP-2A-like enzymes are the major B-50 phosphatases in SPMs.
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The deinhibitor protein, responsible for the decreased sensitivity of the ATP, Mg‐dependent protein phosphatase to inhibitor‐1 and the modulator protein, is inactivated by cyclic AMP‐dependent protein kinase and reactivated by dephosphorylation. The specificity of this reaction was tested with the ATP, Mg‐dependent phosphatase in its activated or spontaneously active form, four different forms of polycation‐stimulated phosphatases (PCS H , PCS M , PCS L and PCS C ) and calcineurin. Only the high ‐ M r , polycation‐stimulated protein phosphatase (PCS H ), but not its catalytic subunit (PCS C ), shows a high degree of specificity for the deinhibitor protein. Deinhibitor phosphatase activity of PCS H is affected neither by polycations nor by Mn ions.
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ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNeurofilament-associated protein phosphatase 2A: Its possible role in preserving neurofilaments in filamentous statesTaro Saito, Hiroshi Shima, Yutaka Osawa, Minako Nagao, Brian A. Hemmings, Takeo Kishimoto, and Shin-ichi HisanagaCite this: Biochemistry 1995, 34, 22, 7376–7384Publication Date (Print):June 6, 1995Publication History Published online1 May 2002Published inissue 6 June 1995https://pubs.acs.org/doi/10.1021/bi00022a010https://doi.org/10.1021/bi00022a010research-articleACS PublicationsRequest reuse permissionsArticle Views92Altmetric-Citations67LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
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Protein phosphatase 1
Neurofilament
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Inhibitor-2, purified by an improved procedure, was used to identify protein phosphatases capable of catalysing its dephosphorylation. The results showed that, under our experimental conditions, protein phosphatases-1, 2A and 2B were the only significant protein phosphatases in rabbit skeletal muscle extracts acting on this substrate. Protein phosphatases-1 and 2A accounted for all the inhibitor-2 phosphatase activity in the absence of Ca2+ (resting muscle), and the potential importance of these enzymes in vivo is discussed. Protein phosphatase-2B, a Ca2+-calmodulin-dependent enzyme, could account for up to 30% of the inhibitor-2 phosphatase activity in contracting muscle. The Km of protein phosphatase-1 for inhibitor-2 (40 nM) was 100-fold lower than the Km for phosphorylase a (4.8 microM). This finding, coupled with the failure of inhibitor-2 to inhibit its own dephosphorylation, suggests that inhibitor-2 is dephosphorylated at one of the two sites on protein phosphatase-1 involved in preventing the dephosphorylation of other substrates. The dephosphorylation of inhibitor-2 by protein phosphatase-1 was also unaffected by inhibitor-1, suggesting that the phosphorylation state of inhibitor-2 is unlikely to be controlled by cyclic AMP in vivo.
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Protein phosphatase 1
DUSP6
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Negative regulator
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Together with protein phosphatase 1, protein phosphatase 2A (PP2A) contributes the bulk of Ser/Thr phosphatase activity in most cell types. The predominant form of PP2A is a heterotrimer of catalytic (C), scaffolding (A), and diverse regulatory subunits (B, B', and B''). We have previously shown that N-terminal phosphorylation sites in tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, are specifically dephosphorylated by PP2A holoenzymes containing the B'beta regulatory subunit. Here, we identify a Glu residue conserved in B' regulatory subunits that is critical for dephosphorylation and inactivation of tyrosine hydroxylase in vitro and in PC12 cells. According to the PP2A heterotrimer crystal structure, Glu153 (B'beta numbering) abuts the catalytic site on the C subunit, and we demonstrate that Glu153 substitution inhibits multisite TH dephosphorylation without compromising PP2A/B'beta holoenzyme assembly or in vitro dephosphorylation of model substrates. Apart from its role in modulating TH activity, Glu153 is also necessary for PP2A/B'beta-mediated enhancement of nerve growth factor signaling. Furthermore, global phosphoproteome analysis suggests that Glu153 mediates dephosphorylation of most B'beta substrates in PC12 cells. With regard to selectivity determinants in the substrate, we show that B'beta Glu153 recognizes Arg37 and Arg38 in TH to direct dephosphorylation of both upstream (Ser31) and downstream (Ser40) sites. These results provide evidence of a subunit-spanning substrate docking site on the PP2A/B' holoenzyme, in which negatively charged side chains in the regulatory subunit interact with positive charges proximal to phosphorylated residues to mediate site-specific dephosphorylation.
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