title: The SLE variant Ala71Thr of BLK severely decreases protein abundance and binding to BANK1 through impairment of the SH3 domain function.
abstract: The B-lymphocyte kinase (BLK) gene is associated genetically with several human autoimmune diseases including systemic lupus erythematosus. We recently described that the genetic risk is given by two haplotypes: one covering several strongly linked single-nucleotide polymorphisms within the promoter of the gene that correlated with low transcript levels, and a second haplotype that includes a rare nonsynonymous variant (Ala71Thr). Here we show that this variant, located within the BLK SH3 domain, is a major determinant of protein levels. In vitro analyses show that the 71Thr isoform is hyperphosphorylated and promotes kinase activation. As a consequence, BLK is ubiquitinated, its proteasomal degradation enhanced and the average life of the protein is reduced by half. Altogether, these findings suggest that an intrinsic autoregulatory mechanism previously unappreciated in BLK is disrupted by the 71Thr substitution. Because the SH3 domain is also involved in protein interactions, we sought for differences between the two isoforms in trafficking and binding to protein partners. We found that binding of the 71Thr variant to the adaptor protein BANK1 is severely reduced. Our study provides new insights on the intrinsic regulation of BLK activation and highlights the dominant role of its SH3 domain in BANK1 binding.
authors.affiliation: Area of Medical Genomics, Centro de Genómica e Investigación Oncológica (GENYO), Pfizer-Universidad de Granada-Junta de Andalucía, PTS, Granada, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Díaz-Barreiro
authors.affiliation: Area of Medical Genomics, Centro de Genómica e Investigación Oncológica (GENYO), Pfizer-Universidad de Granada-Junta de Andalucía, PTS, Granada, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Bernal-Quirós
authors.affiliation: Area of Medical Genomics, Centro de Genómica e Investigación Oncológica (GENYO), Pfizer-Universidad de Granada-Junta de Andalucía, PTS, Granada, Spain.
authors.forename: I
authors.initials: I
authors.lastname: Georg
authors.affiliation: Area of Medical Genomics, Centro de Genómica e Investigación Oncológica (GENYO), Pfizer-Universidad de Granada-Junta de Andalucía, PTS, Granada, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Marañón
authors.affiliation: Area of Medical Genomics, Centro de Genómica e Investigación Oncológica (GENYO), Pfizer-Universidad de Granada-Junta de Andalucía, PTS, Granada, Spain.|Institute for Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: M E
authors.initials: ME
authors.lastname: Alarcón-Riquelme
authors.affiliation: Department of Medical Sciences, Molecular Epidemiology, Uppsala University, Uppsala, Sweden.
authors.forename: C
authors.initials: C
authors.lastname: Castillejo-López
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001402:B-Lymphocytes / Q000276:immunology / Q000473:pathology; D001665:Binding Sites; D045744:Cell Line, Tumor; D015870:Gene Expression; D006207:Half-Life; D006239:Haplotypes; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics* / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology; D059748:Proteolysis; D016415:Sequence Alignment; D054875:Ubiquitination; D019061:src-Family Kinases / Q000235:genetics* / Q000276:immunology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C447641:BANK1 protein, human; D008565:Membrane Proteins; D020033:Protein Isoforms; C584409:BLK protein, human; D019061:src-Family Kinases; D046988:Proteasome Endopeptidase Complex
references.citation: J Cell Sci. 2010 Aug 1;123(Pt 15):2649-62
references.pmid: 20605918
references.citation: N Engl J Med. 2008 Feb 28;358(9):900-9
references.pmid: 18204098
references.citation: Mol Cell Biol. 1996 Sep;16(9):4735-43
references.pmid: 8756631
references.citation: Nat Genet. 2012 Mar 25;44(5):517-21
references.pmid: 22446962
references.citation: Immunol Cell Biol. 2012 Jul;90(6):620-9
references.pmid: 21894171
references.citation: Oncogene. 2004 Oct 18;23(48):7918-27
references.pmid: 15489910
references.citation: EMBO J. 1993 Jul;12(7):2617-24
references.pmid: 7687536
references.citation: Nat Protoc. 2015 Jun;10(6):845-58
references.pmid: 25950237
references.citation: EMBO J. 2007 Mar 21;26(6):1499-510
references.pmid: 17332756
references.citation: J Rheumatol. 2011 Sep;38(9):1866-70
references.pmid: 21765104
references.citation: Mol Cell Biol. 1993 Sep;13(9):5877-87
references.pmid: 8395016
references.citation: Arthritis Rheumatol. 2015 Nov;67(11):2866-76
references.pmid: 26246128
references.citation: Ann Rheum Dis. 2010 Apr;69(4):700-5
references.pmid: 19815934
references.citation: Science. 1990 Jan 19;247(4940):332-6
references.pmid: 2404338
references.citation: Glycobiology. 2014 Sep;24(9):807-17
references.pmid: 25002414
references.citation: FEBS Lett. 2012 Aug 14;586(17):2609-14
references.pmid: 22710157
references.citation: Arthritis Rheum. 2013 Dec;65(12):3239-47
references.pmid: 23983088
references.citation: J Biol Chem. 1995 Oct 27;270(43):25968-75
references.pmid: 7592787
references.citation: Biophys J. 2000 Oct;79(4):1761-70
references.pmid: 11023884
references.citation: Ann Rheum Dis. 2012 Jan;71(1):136-42
references.pmid: 21978998
references.citation: J Autoimmun. 2010 Mar;34(2):155-62
references.pmid: 19796918
references.citation: Hum Mol Genet. 2012 Sep 1;21(17):3918-25
references.pmid: 22678060
references.citation: Mol Biol Cell. 1999 Nov;10(11):3771-86
references.pmid: 10564270
references.citation: J Immunol. 1999 Nov 15;163(10 ):5453-61
references.pmid: 10553071
references.citation: Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7410-4
references.pmid: 1714601
references.citation: Science. 1994 Nov 18;266(5188):1241-7
references.pmid: 7526465
references.citation: Int J Biol Sci. 2012;8(10):1385-97
references.pmid: 23139636
references.citation: Nature. 2014 Feb 20;506(7488):376-81
references.pmid: 24390342
references.citation: J Biol Chem. 1997 Oct 10;272(41):25429-32
references.pmid: 9325251
references.citation: Nat Genet. 2009 Nov;41(11):1216-22
references.pmid: 19838192
references.citation: J Dermatol Sci. 2013 Aug;71(2):122-9
references.pmid: 23642664
references.citation: J Biol Chem. 1995 Jan 6;270(1):333-9
references.pmid: 7529230
references.citation: Nat Genet. 2009 Jul;41(7):820-3
references.pmid: 19503088
references.citation: Biophys J. 1976 Sep;16(9):1055-69
references.pmid: 786399
references.citation: PLoS One. 2013;8(3):e59842
references.pmid: 23555801
references.citation: Ann Rheum Dis. 2012 Jul;71(7):1219-26
references.pmid: 22696686
references.citation: Exp Cell Res. 2007 Jul 15;313(12):2651-66
references.pmid: 17537435
references.citation: Proc Natl Acad Sci U S A. 1999 Aug 17;96(17 ):9557-62
references.pmid: 10449731
references.citation: Arthritis Rheum. 2011 Apr;63(4):884-93
references.pmid: 21452313
references.citation: Cell. 1994 Mar 11;76(5):933-45
references.pmid: 7510218
references.citation: Blood. 1997 Apr 1;89(7):2384-93
references.pmid: 9116282
references.citation: J Exp Med. 2003 Dec 15;198(12):1863-73
references.pmid: 14662906
references.citation: Biochim Biophys Acta. 2009 Oct;1790(10):1345-52
references.pmid: 19619611
references.citation: Immunol Rev. 2009 Mar;228(1):132-48
references.pmid: 19290925
references.citation: PLoS One. 2013 Apr 30;8(4):e61044
references.pmid: 23646104
references.citation: Science. 1993 Feb 19;259(5098):1157-61
references.pmid: 8438166
references.citation: J Cell Sci. 2009 Apr 1;122(Pt 7):965-75
references.pmid: 19258394
references.citation: Nat Genet. 2008 Feb;40(2):211-6
references.pmid: 18204447
references.citation: EMBO J. 2002 Jan 15;21(1-2):83-92
references.pmid: 11782428
references.citation: J Biol Chem. 1993 Apr 25;268(12):8669-74
references.pmid: 8473310
references.citation: J Biol Chem. 1995 Nov 10;270(45):27072-8
references.pmid: 7592958
references.citation: Nature. 2000 Apr 6;404(6778):604-9
references.pmid: 10766243
references.citation: J Immunol. 2015 Jun 15;194(12):5692-702
references.pmid: 25972485
references.citation: Front Immunol. 2012 Jun 12;3:155
references.pmid: 22701458
references.citation: Nat Genet. 2009 Nov;41(11):1228-33
references.pmid: 19838195
references.citation: Nature. 1994 Nov 24;372(6504):375-9
references.pmid: 7802869
references.citation: Oncotarget. 2015 May 10;6(13):10759-71
references.pmid: 25926555
references.citation: Am J Hum Genet. 2014 Apr 3;94(4):586-98
references.pmid: 24702955
references.citation: Nucleic Acids Res. 2009 Sep;37(17):e115
references.pmid: 19561200
references.citation: Genes Immun. 2011 Mar;12(2):100-9
references.pmid: 20861858
references.citation: J Biol Chem. 2010 Nov 12;285(46):35455-61
references.pmid: 20810664