title: Phosphorylation at serine 318 is not required for inhibition of T cell activation by ALX.
abstract: The activation of T cells and the initiation of an immune response is tightly controlled by both positive and negative regulators. Two adaptors which function as negative regulators of T cell activation are ALX and LAX. ALX constitutively associates with LAX in T cells, and T cells from mice deficient in ALX and LAX display similar hyper-responsiveness upon T cell receptor (TCR)/CD28 stimulation, including increased production of interleukin-2. During T cell activation, ALX is inducibly phosphorylated, however the site of ALX phosphorylation had not been previously identified and the role of phosphorylation in the inhibitory function of ALX was not known. Here, using mass spectrometry, we demonstrate that ALX is phosphorylated on a serine at position 318. Substitution of alanine for serine at this position (ALX S318A) leads to an abrogation of the mobility shift in ALX induced upon TCR/CD28 stimulation. However, ALX S318A retained the ability to bind to and stimulate tyrosine phosphorylation of LAX. In addition, overexpression of ALX S318A inhibited RE/AP activation upon TCR/CD28 stimulation to a similar extent as wild-type ALX. Therefore, although ALX is inducibly phosphorylated upon TCR/CD28 stimulation, this phosphorylation is not required for ALX to inhibit T cell activation.
authors.affiliation: Department of Immunology, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Shapiro
authors.forename: Lynn
authors.initials: L
authors.lastname: Spruce
authors.forename: Rhianna
authors.initials: R
authors.lastname: Sundsbak
authors.forename: Puspa
authors.initials: P
authors.lastname: Thapa
authors.forename: Virginia Smith
authors.initials: VS
authors.lastname: Shapiro
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D018106:CD28 Antigens / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D013058:Mass Spectrometry; D051379:Mice; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D018106:CD28 Antigens; C442142:HSH2D protein, human; C469997:LAX1 protein, human; D011948:Receptors, Antigen, T-Cell; D012694:Serine
references.citation: J Immunol. 2006 Apr 1;176(7):4163-72
references.pmid: 16547253
references.citation: Nature. 2000 Mar 23;404(6776):402-7
references.pmid: 10746729
references.citation: Anal Chem. 2002 Oct 15;74(20):5383-92
references.pmid: 12403597
references.citation: J Biol Chem. 1997 Jan 10;272(2):952-60
references.pmid: 8995387
references.citation: J Exp Med. 1996 Nov 1;184(5):1663-9
references.pmid: 8920856
references.citation: Cell. 1998 Jan 9;92(1):83-92
references.pmid: 9489702
references.citation: J Immunol. 2008 Nov 15;181(10):7055-61
references.pmid: 18981125
references.citation: Eur J Immunol. 1999 Jan;29(1):132-42
references.pmid: 9933094
references.citation: Mol Cell Biol. 2006 Aug;26(16):6005-15
references.pmid: 16880512
references.citation: J Biol Chem. 2002 Nov 29;277(48):46151-8
references.pmid: 12359715
references.citation: Nat Immunol. 2003 Feb;4(2):117-23
references.pmid: 12514734
references.citation: Immunol Rev. 2009 Nov;232(1):240-54
references.pmid: 19909368
references.citation: J Immunol. 2005 May 1;174(9):5612-9
references.pmid: 15843560
references.citation: Curr Opin Immunol. 1997 Jun;9(3):396-404
references.pmid: 9203422
references.citation: J Biol Chem. 2005 Feb 4;280(5):3507-15
references.pmid: 15569688
references.citation: Anal Chem. 2003 Sep 1;75(17):4646-58
references.pmid: 14632076
references.citation: Mol Cell Biol. 1997 Jul;17(7):4051-8
references.pmid: 9199340
references.citation: J Exp Med. 2002 Dec 16;196(12):1617-26
references.pmid: 12486104
references.citation: Trends Immunol. 2002 Aug;23(8):413-20
references.pmid: 12133805
references.citation: J Biol Chem. 2004 Sep 24;279(39):40647-52
references.pmid: 15284240
references.citation: J Biol Chem. 2003 Nov 14;278(46):45128-34
references.pmid: 12960172