Identification and characterization of AtI-2, an Arabidopsis homologue of an ancient protein phosphatase 1 (PP1) regulatory subunit

2011 ◽  
Vol 435 (1) ◽  
pp. 73-83 ◽  
Author(s):  
George W. Templeton ◽  
Mhairi Nimick ◽  
Nicholas Morrice ◽  
David Campbell ◽  
Marilyn Goudreault ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Biochemical Characterization ◽  
Affinity Purification ◽  
Bioinformatic Analysis ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Identification And Characterization ◽  
Eukaryotic Genomes

PP1 (protein phosphatase 1) is among the most conserved enzymes known, with one or more isoforms present in all sequenced eukaryotic genomes. PP1 dephosphorylates specific serine/threonine phosphoproteins as defined by associated regulatory or targeting subunits. In the present study we performed a PP1-binding screen to find putative PP1 interactors in Arabidopsis thaliana and uncovered a homologue of the ancient PP1 interactor, I-2 (inhibitor-2). Bioinformatic analysis revealed remarkable conservation of three regions of plant I-2 that play key roles in binding to PP1 and regulating its function. The sequence-related properties of plant I-2 were compared across eukaryotes, indicating a lack of I-2 in some species and the emergence points from key motifs during the evolution of this ancient regulator. Biochemical characterization of AtI-2 (Arabidopsis I-2) revealed its ability to inhibit all plant PP1 isoforms and inhibitory dependence requiring the primary interaction motif known as RVXF. Arabidopsis I-2 was shown to be a phosphoprotein in vivo that was enriched in the nucleus. TAP (tandem affinity purification)-tag experiments with plant I-2 showed in vivo association with several Arabidopsis PP1 isoforms and identified other potential I-2 binding proteins.

Identification and Characterization of the Human HCG V Gene Product as a Novel Inhibitor of Protein Phosphatase-1†

Biochemistry ◽  
1998 ◽  
Vol 37 (47) ◽  
pp. 16728-16734 ◽  
Author(s):  
Jun Zhang ◽  
Lifang Zhang ◽  
Sumin Zhao ◽  
Ernest Y. C. Lee
Keyword(s):  
Gene Product ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
V Gene ◽  
Identification And Characterization

scholarly journals Identification and Characterization of Two Bordetella avium Gene Products Required for Hemagglutination

2010 ◽  
Vol 78 (6) ◽  
pp. 2370-2376 ◽  
Author(s):  
Louise M. Temple ◽  
David M. Miyamoto ◽  
Manju Mehta ◽  
Christian M. Capitini ◽  
Stephen Von Stetina ◽  
...  
Keyword(s):  
Whooping Cough ◽  
Bioinformatic Analysis ◽  
Tracheal Ring ◽  
Tracheal Cell ◽  
Bordetella Avium ◽  
Identification And Characterization

ABSTRACT Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.

scholarly journals A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action

2011 ◽  
Vol 22 (1) ◽  
pp. 141-152 ◽  
Author(s):  
Xiao-Wei Chen ◽  
Dara Leto ◽  
Tingting Xiong ◽  
Genggeng Yu ◽  
Alan Cheng ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Small Gtpase ◽  
Regulatory Subunit ◽  
Hydrolysis Of ◽  
Identification And Characterization

Insulin stimulates glucose transport in muscle  and adipose tissue by translocation of glucose transporter 4 (GLUT4) to the plasma membrane. We previously reported that activation of the small GTPase RalA downstream of PI 3-kinase plays a critical role in this process by mobilizing the exocyst complex for GLUT4 vesicle targeting in adipocytes. Here we report the identification and characterization of a Ral GAP complex (RGC) that mediates the activation of RalA downstream of the PI 3-kinase/Akt pathway. The complex is composed of an RGC1 regulatory subunit and an RGC2 catalytic subunit (previously identified as AS250) that directly stimulates the guanosine triphosphate hydrolysis of RalA. Knockdown of RGC proteins leads to increased RalA activity and glucose uptake in adipocytes. Insulin inhibits the GAP complex through Akt2-catalyzed phosphorylation of RGC2 in vitro and in vivo, while activated Akt relieves the inhibitory effect of RGC proteins on RalA activity. The RGC complex thus connects PI 3-kinase/Akt activity to the transport machineries responsible for GLUT4 translocation.

scholarly journals Identification and characterization of phostensin, a protein phosphatase 1 F‐actin cytoskeleton targeting subunit

2009 ◽  
Vol 23 (S1) ◽  
Author(s):  
Tzu‐Fan Wang ◽  
Shu‐Chen Kao ◽  
Shiu‐Lan Wang ◽  
Chun‐Yu Chen ◽  
Ning‐Sheng Lai ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Identification And Characterization

Cloning and identification of MYPT3: a prenylatable myosin targetting subunit of protein phosphatase 1

2001 ◽  
Vol 356 (1) ◽  
pp. 257-267 ◽  
Author(s):  
Jeffrey A. SKINNER ◽  
Alan R. SALTIEL
Keyword(s):  
Molecular Mass ◽  
Protein Phosphatase ◽  
Biochemical Characterization ◽  
Terminal Region ◽  
Protein Phosphatase 1 ◽  
Chromosome 15 ◽  
Glutathione S Transferase ◽  
Interacting Protein

To identify novel protein phosphatase 1 (PP1)-interacting proteins, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the catalytic subunit of PP1 as bait. In the present work, the isolation, identification and initial biochemical characterization of a novel PP1-interacting protein, MYPT3, which is homologous with the myosin phosphatase targetting subunit (MYPT) family, is described. MYPT3 aligns > 99% with a region of mouse genomic DNA clone RP23-156P23 and localizes to chromosome 15, between markers at 44.1–46.5cM, as demonstrated by radiation hybrid mapping. The gene consists of ten exons that encode for a 524-amino acid sequence with a predicted molecular mass of 57529Da. The N-terminal region of MYPT3 consists of a consensus PP1-binding site and multiple ankyrin repeats. MYPT3 is distinguished from related ∼ 110–130kDa MYPT subunits by its molecular mass of 58kDa, and a unique C-terminal region that contains several potential signalling motifs and a CaaX prenylation site. We have shown that affinity-purified glutathione S-transferase (GST)–MYPT3 is prenylated by purified recombinant farnesyltransferase in vitro. Endogenous PP1 from 3T3-L1 lysates specifically interacts with MYPT3. Additionally, purified PP1 activity was inhibited by GST–MYPT3 toward phosphorylase a, myosin light chain and myosin substrate in vitro. Overall, our findings identify a novel prenylatable subunit of PP1 that defines a new subfamily of MYPT.

scholarly journals RIPK1 dephosphorylation and kinase activation by PPP1R3G/PP1γ promote apoptosis and necroptosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jingchun Du ◽  
Yougui Xiang ◽  
Hua Liu ◽  
Shuzhen Liu ◽  
Ashwani Kumar ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Type I ◽  
Interacting Protein ◽  
Kinase Activation ◽  
Necrosis Factor

AbstractReceptor-interacting protein kinase 1 (RIPK1) is a key regulator of inflammation and cell death. Many sites on RIPK1, including serine 25, are phosphorylated to inhibit its kinase activity and cell death. How these inhibitory phosphorylation sites are dephosphorylated is poorly understood. Using a sensitized CRISPR whole-genome knockout screen, we discover that protein phosphatase 1 regulatory subunit 3G (PPP1R3G) is required for RIPK1-dependent apoptosis and type I necroptosis. Mechanistically, PPP1R3G recruits its catalytic subunit protein phosphatase 1 gamma (PP1γ) to complex I to remove inhibitory phosphorylations of RIPK1. A PPP1R3G mutant which does not bind PP1γ fails to rescue RIPK1 activation and cell death. Furthermore, chemical prevention of RIPK1 inhibitory phosphorylations or mutation of serine 25 of RIPK1 to alanine largely restores cell death in PPP1R3G-knockout cells. Finally, Ppp1r3g−/− mice are protected from tumor necrosis factor-induced systemic inflammatory response syndrome, confirming the important role of PPP1R3G in regulating apoptosis and necroptosis in vivo.

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