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Subunit 1 of the Prefoldin Chaperone Complex Is Required for Lymphocyte Development and Function

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Subunit 1 of the Prefoldin Chaperone Complex Is Required for Lymphocyte Development and Function Subunit 1 of the Prefoldin Chaperone Complex Is Required for Lymphocyte Development and Function This information is current as Shang Cao, Gianluca Carlesso, Anna B. Osipovich, Joan of October 1, 2021. Llanes, Qing Lin, Kristen L. Hoek, Wasif N. Khan and H. Earl Ruley J Immunol 2008; 181:476-484; ; doi: 10.4049/jimmunol.181.1.476 http://www.jimmunol.org/content/181/1/476 Downloaded from References This article cites 50 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/181/1/476.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Subunit 1 of the Prefoldin Chaperone Complex Is Required for Lymphocyte Development and Function1 Shang Cao,2 Gianluca Carlesso,2,3 Anna B. Osipovich, Joan Llanes, Qing Lin, Kristen L. Hoek, Wasif N. Khan,4 and H. Earl Ruley4 Prefoldin is a hexameric chaperone that facilitates posttranslational folding of actins and other cytoskeletal proteins by the Tcp1-containing ring complex chaperonin, TriC. The present study characterized mice with a null mutation in Pfdn1, which encodes the first subunit of the Prefoldin complex. Pfdn1-deficient mice displayed phenotypes characteristic of defects in cytoskel- etal function, including manifestations of ciliary dyskinesia, neuronal loss, and defects in B and T cell development and function. B and T cell maturation was markedly impaired at relatively early stages, namely at the transitions from pre-pro-B to pre-B cells ,in the bone marrow and from CD4؊CD8؊ double-negative to CD4؉CD8؉ double-positive T cells in the thymus. In addition mature B and T lymphocytes displayed cell activation defects upon Ag receptor cross-linking accompanied by impaired Ag Downloaded from receptor capping in B cells. These phenotypes illustrate the importance of cytoskeletal function in immune cell development and activation. The Journal of Immunology, 2008, 181: 476–484. ctins and tubulins, as ubiquitous components of the cy- protein substrates directly, Prefoldin significantly enhances the rate toskeleton, play essential roles in fundamental cellular of TRiC-mediated protein folding (13). processes including motility, macromolecular transport, Unlike TRiC, yeast Prefoldin (GimC) is not essential for cell A http://www.jimmunol.org/ signal transduction, and cell division (1). Actin and tubulin sub- viability (9, 10). However, GimC mutations generate cytoskeletal units assemble in a head-to-tail manner by a regulated, energy- defects similar to temperature-conditional TRiC mutations (9, 10), consuming process (2, 3). Fiber asymmetry enables actin and tu- providing further evidence for biochemical cooperation between bulin cytoskeletons to orchestrate rapid and spatially localized the chaperone and chaperonin. Prefoldin subunits 2, 3, and 6 in- changes in cell structure and function involved in diverse pro- fluence Caenorhabditis elegans development (14, 15); thus, de- cesses from axon outgrowth to immune synapse signaling (4, 5). spite evidence of limited promiscuity among chaperones with re- Efficient synthesis of actin and tubulin subunits requires two gard to substrate binding and chaperonin presentation (16), protein complexes, Prefoldin and TRiC/CCT (Tcp1 ring complex/ nonredundant activities of Prefoldin appear to be required for cy- chaperonin-containing Tcp1) that facilitate protein folding (re- toskeletal function in lower eukaryotes. by guest on October 1, 2021 viewed in Ref. 6). Prefoldin is a molecular chaperone consisting of Genetic analysis of mammalian Prefoldin is expected to gener- six radially organized proteins, each forming a coiled-coil tentacle ate phenotypes indicative of biologically important folding sub- that participates in substrate binding (7, 8). Prefoldin was first strates in mammalian cells. In particular, we would like to know identified from genetic interactions implicating the complex in cy- whether Prefoldin mutant phenotypes are consistent with aberrant toskeleton biosynthesis (9) and as a biochemical activity that trans- cytoskeletal function, as in yeast, or possess defects involving a ferred denatured ␤-actin to TRiC (10). TRiC, a cytoplasmic chap- wider range of protein substrates. Mice deficient in Prefoldin chap- eronin, transiently interacts with ϳ10% of cellular proteins and erone functions may also clarify the involvement of the cytoskel- folds several essential proteins, including actins and tubulins, by eton (17, 18) or other misfolded proteins in aging-associated dis- an ATP-dependent mechanism (reviewed in Ref. 11). Nascent eases (19–22). Finally, the availability of Prefoldin-deficient cells ␤-actin binds Prefoldin during translation (12) and is presented to and animals is expected to facilitate functional studies of the chap- the TRiC chaperonin complex (8, 10). Although TRiC can bind erone in different biological processes. In the present study, we characterized a null mutation in the gene (Pfdn1) for Prefoldin subunit 1 induced by gene trapping in Department of Microbiology and Immunology, Vanderbilt University, School of 5 Medicine, Nashville, TN 37232 mouse embryonic stem (ES) cells. We show that Pfdn1-deficient Received for publication January 2, 2008. Accepted for publication April 30, 2008. mice develop to term but display severe abnormalities character- The costs of publication of this article were defrayed in part by the payment of page istic of defects in cytoskeletal function, including ciliary dyskine- charges. This article must therefore be hereby marked advertisement in accordance sia, loss of neuron tracts in the brain, and defects in B and T cell with 18 U.S.C. Section 1734 solely to indicate this fact. development and function similar to but more severe than Wiskott- 1 This work was supported by Public Health Service Grants P01HL68744 (to H.E.R.) Aldrich syndrome protein (WASP) and WASP-interacting protein and AI060729-01 (to W.N.K.). K.L.H. was supported by National Institutes of Health Grant F32-AI069770-01. Additional support was provided by the Department of Mi- (WIP) mutations. These phenotypes appear to reflect processes crobiology and Immunology of Vanderbilt University and by Cancer Center Support such as immune cell signaling and axon guidance that place par- Grant P30CA68485 to the Vanderbilt-Ingram Cancer Center. ticular demands on cytoskeletal synthesis and remodeling. 2 S.C. and G.C. share first authorship and contributed equally to this work. 3 Current address: MedImmune, Inc., One MedImmune Way, Department of Inflam- mation and Autoimmunity, Gaithersburg, MD 20878 5 Abbreviations used in this paper: ES, embryonic stem; BM, bone marrow; MEF, 4 Address correspondence and reprint requests to Dr. Wasif N. Khan and Dr. H. Earl mouse embryonic fibroblast; MZ, marginal zone; WASP, Wiskott-Aldrich syndrome Ruley, Department of Microbiology and Immunology, Vanderbilt University, School protein; WIP, WASP-interacting protein; MFI, mean fluorescent intensity. of Medicine, Medical Center North, 1161 21st Avenue South, Nashville, TN 37232. E-mail addresses: [email protected] and [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 477 Downloaded from FIGURE 2. Growth and survival of Pfdn1 mutant mice. A, Appearance of wild-type (left) and mutant (right) mice at 3 wk of age. B, Average weights of wild-type and heterozygous mice (gray) and homozygous mu- http://www.jimmunol.org/ tant mice (black) at 2 to 4 wk of age. C, Fraction of wild-type and het- erozygous mice (gray) and homozygous mutant mice (black) surviving through 5 wk of age. upstream (U) and downstream (D) primers 5Ј-CAGTCCTCCGATT FIGURE 1. Pfdn1 gene disruption. A, The GTR1.3 gene trap retrovirus GACTGAG-3Ј (Dp) and 5Ј-GGGGTTGTGGGCTCTTTAT-3Ј (Up). The was inserted into the third intron of the Pfdn1 gene as shown. The insert amplification protocol consisted of 35 cycles incubating at 94°C for 1 min, by guest on October 1, 2021 enables upstream Pfdn1 sequences to splice to a 3Ј exon consisting of the 55°C for 1 min, and 72 °C for 1 min. Mice and cells were genotyped by PCR analysis. Twenty to 50 ng of 3Ј end of a puromycin resistance gene (3Ј Puro), a ␤-galactosidase reporter genomic DNA (g) was used as template in a 50-␮l reaction using the Roche (lacZ), and a polyadenylation signal (PA), whereas Neo sequences are PCR mix with the upstream (U) and downstream (D) primers 5Ј-TGG expressed from the promoter of the RNA polymerase 2 gene (Pol2) to GATAATGCCCACAGGTA-3Ј (Dg), 5Ј-AAGCACTCAGAGCAGCA splice to exon 4. Primers complementary to proviral (Dp and Up) and AGTT-3Ј (Ug), and 5Ј-CAGTCCTCCGATTGACTGAG-3Ј (Dp). Samples cellular (Ug and Dg) sequences used
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