title: Multipoint binding of the SLP-76 SH2 domain to ADAP is critical for oligomerization of SLP-76 signaling complexes in stimulated T cells.
abstract: The adapter molecules SLP-76 and LAT play central roles in T cell activation by recruiting enzymes and other adapters into multiprotein complexes that coordinate highly regulated signal transduction pathways. While many of the associated proteins have been characterized, less is known concerning the mechanisms of assembly for these dynamic and potentially heterogeneous signaling complexes. Following T cell receptor (TCR) stimulation, SLP-76 is found in structures called microclusters, which contain many signaling complexes. Previous studies showed that a mutation to the SLP-76 C-terminal SH2 domain nearly abolished SLP-76 microclusters, suggesting that the SH2 domain facilitates incorporation of signaling complexes into microclusters. S. C. Bunnell, A. L. Singer, D. I. Hong, B. H. Jacque, M. S. Jordan, M. C. Seminario, V. A. Barr, G. A. Koretzky, and L. E. Samelson, Mol. Cell. Biol., 26:7155-7166, 2006). Using biophysical methods, we demonstrate that the adapter, ADAP, contains three binding sites for SLP-76, and that multipoint binding to ADAP fragments oligomerizes the SLP-76 SH2 domain in vitro. These results were complemented with confocal imaging and functional studies of cells expressing ADAP with various mutations. Our results demonstrate that all three binding sites are critical for SLP-76 microcluster assembly, but any combination of two sites will partially induce microclusters. These data support a model whereby multipoint binding of SLP-76 to ADAP facilitates the assembly of SLP-76 microclusters. This model has implications for the regulation of SLP-76 and LAT microclusters and, as a result, T cell signaling.
authors: Laboratory of Cellular and Molecular Biology.
authors: Nathan P
authors: NP
authors: Coussens
authors: Ryo
authors: R
authors: Hayashi
authors: Patrick H
authors: PH
authors: Brown
authors: Lakshmi
authors: L
authors: Balagopalan
authors: Andrea
authors: A
authors: Balbo
authors: Itoro
authors: I
authors: Akpan
authors: Jon C D
authors: JC
authors: Houtman
authors: Valarie A
authors: VA
authors: Barr
authors: Peter
authors: P
authors: Schuck
authors: Ettore
authors: E
authors: Appella
authors: Lawrence E
authors: LE
authors: Samelson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D020013:Calcium Signaling; D002448:Cell Adhesion; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D059010:Single-Cell Analysis; D013601:T-Lymphocytes / Q000378:metabolism*; D013816:Thermodynamics; D059008:Time-Lapse Imaging; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C106957:FYB1 protein, human; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; C092947:SLP-76 signal Transducing adaptor proteins
references: J Immunol. 2003 Jan 1;170(1):325-33
references: 12496416
references: Biophys J. 2000 Mar;78(3):1606-19
references: 10692345
references: J Biol Chem. 2001 Nov 30;276(48):45207-16
references: 11487585
references: J Biol Chem. 2000 Jul 28;275(30):23355-61
references: 10811803
references: J Cell Biol. 2011 Nov 28;195(5):839-53
references: 22105350
references: Nat Immunol. 2005 Jan;6(1):80-9
references: 15558067
references: PLoS One. 2010 Jul 22;5(7):e11708
references: 20661443
references: J Cell Biol. 2002 Sep 30;158(7):1263-75
references: 12356870
references: Biophys J. 2005 Jul;89(1):619-34
references: 15863475
references: Mol Cell Biol. 2001 Jul;21(13):4208-18
references: 11390650
references: EMBO J. 2010 Jul 21;29(14):2315-28
references: 20562827
references: Proc Natl Acad Sci U S A. 2009 Jul 28;106(30):12436-41
references: 19617540
references: J Biol Chem. 2000 Feb 18;275(7):5143-52
references: 10671560
references: Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7493-8
references: 9207119
references: Biophys J. 2011 May 4;100(9):2309-17
references: 21539801
references: Mol Cell Biol. 2009 Oct;29(20):5578-89
references: 19667077
references: J Exp Med. 2005 Oct 17;202(8):1031-6
references: 16216891
references: J Immunol. 1999 Dec 1;163(11):5753-7
references: 10570256
references: J Biol Chem. 1997 May 2;272(18):11674-7
references: 9115214
references: Curr Protoc Immunol. 2008 May;Chapter 18:Unit 18.15
references: 18491296
references: Biopolymers. 2005;80(5):628-35
references: 15660381
references: Immunity. 2008 Jun;28(6):810-21
references: 18549800
references: Traffic. 2006 Sep;7(9):1143-62
references: 16919152
references: Mol Membr Biol. 2009 Apr;26(3):159-70
references: 19115140
references: Mol Cell Biol. 2011 Jul;31(13):2653-66
references: 21536650
references: Nat Immunol. 2005 Dec;6(12):1253-62
references: 16273097
references: Biochemistry. 2004 Apr 13;43(14):4170-8
references: 15065860
references: Biophys J. 2009 Apr 8;96(7):2604-23
references: 19348745
references: Mol Cell Biol. 2006 Oct;26(19):7155-66
references: 16980618
references: Nat Struct Mol Biol. 2006 Sep;13(9):798-805
references: 16906159
references: Wiley Interdiscip Rev Syst Biol Med. 2013 Jan-Feb;5(1):101-10
references: 23150273
references: Science. 2002 May 3;296(5569):913-6
references: 11988576
references: Science. 1998 Jul 17;281(5375):413-6
references: 9665884
references: Trends Cell Biol. 2008 Oct;18(10):486-93
references: 18760924
references: J Mol Biol. 1999 Nov 5;293(4):971-85
references: 10543978
references: J Immunol. 2011 Nov 1;187(9):4459-66
references: 21949020
references: EMBO J. 2006 Feb 22;25(4):774-84
references: 16467851
references: J Proteome Res. 2010 Aug 6;9(8):4113-22
references: 20568816
references: J Mol Recognit. 2007 Jul-Aug;20(4):245-52
references: 17705331
references: Mol Cell Biol. 2010 Jul;30(14):3421-9
references: 20498282
references: Sci STKE. 2003 Apr 08;2003(177):PL8
references: 12684528
references: Sci Signal. 2011 Mar 08;4(163):ra14
references: 21386095
references: Biochem Biophys Res Commun. 2003 Aug 8;307(4):1038-44
references: 12878216
references: Cold Spring Harb Perspect Biol. 2010 Aug;2(8):a005512
references: 20610546
references: Anal Chem. 2012 Jun 5;84(11):5066-73
references: 22530732
references: Science. 2001 Sep 21;293(5538):2260-3
references: 11567140
references: Bioorg Med Chem Lett. 1999 Aug 2;9(15):2267-72
references: 10465559
references: Science. 2001 Sep 21;293(5538):2263-5
references: 11567141
references: Methods. 2011 May;54(1):4-15
references: 21187153
references: J Exp Med. 2004 Oct 18;200(8):1063-74
references: 15477347
references: Protein Sci. 2007 Jan;16(1):30-42
references: 17192587