quantitative-mass-spectrometric-profiling-cancer-cell-proteomes

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Goethe University of Frankfurt

German Cancer Consortium

German Cancer Research Center

Understanding intracellular signal transduction by cell surface receptors requires information about the precise order of relevant modifications on the early transducer elements. Here we introduce the B cell line DT40 and its genetically engineered variants as a model system to determine and functionally characterize post-translational protein modifications in general. This is accomplished by a customized strategy that combines mass spectrometric analyses of protein modifications with subsequent mutational studies. When applied to the B cell receptor (BCR)-proximal effector SLP-65, this approach uncovered a differential and highly dynamic engagement of numerous newly identified phospho-acceptor sites. Some of them serve as kinase substrates in resting cells and undergo rapid dephosphorylation upon BCR ligation. Stimulation-induced phosphorylation of SLP-65 can be early and transient, or early and sustained, or late. Functional elucidation of conspicuous phosphorylation at serine 170 in SLP-65 revealed a BCR-distal checkpoint for some but not all possible B cell responses. Our data show that SLP-65 phosphorylation acts upstream for signal initiation and also downstream during selective processing of the BCR signal. Such a phenomenon defines a receptor-specific signal integrator.

SLP-65 phosphorylation dynamics reveals a functional basis for signal integration by receptor-proximal adaptor proteins.

The growth factor receptor-bound protein 2 (Grb2) is a ubiquitously expressed and evolutionary conserved adapter protein possessing a plethora of described interaction partners for the regulation of signal transduction. In B lymphocytes, the Grb2-mediated scaffolding function controls the assembly and subcellular targeting of activating as well as inhibitory signalosomes in response to ligation of the antigen receptor. Also, integration of simultaneous signals from B-cell coreceptors that amplify or attenuate antigen receptor signal output relies on Grb2. Hence, Grb2 is an essential signal integrator. The key question remains, however, of how pathway specificity can be maintained during signal homeostasis critically required for the balance between immune cell activation and tolerance induction. Here, we summarize the molecular network of Grb2 in B cells and introduce a proteomic approach to elucidate the interactome of Grb2 in vivo.

The B-lymphoid Grb2 interaction code.

B cells require signals transduced by the B cell antigen receptor (BCR) to provide humoral adaptive immunity. These signals are modulated by co-receptors like the Fcγ receptor IIb (FcγRIIb) that prevents activation of B cells after co-ligation with the BCR. Positive and negative effectors need to be precisely organized into signaling complexes, which requires adapter proteins like the growth factor receptor-bound protein 2 (Grb2). Here, we address the question how Grb2-mediated signal integration is affected by FcγRIIb. Our data reveal that concomitant engagement of BCR and FcγRIIb leads to markedly increased Grb2-mediated formation of ternary protein complexes comprising downstream of kinase-3 (Dok-3), Grb2, and the SH2 domain-containing inositol phosphatase (SHIP). Consistently, we found Grb2 to be required for full FcγRIIb-mediated negative regulation. To investigate how FcγRIIb influences the entire Grb2 interactions, we utilized quantitative mass spectrometry to make a differential interactome analysis. This approach revealed a shift of Grb2 interactions towards negative regulators like Dok-3, SHIP and SHP-2 and reduced binding to other proteins like CD19. Hence, we provide evidence that Grb2-mediated signal integration is a dynamic process that is important for the crosstalk between the BCR and its co-receptor FcγRIIb.

Fc gamma receptor IIb modulates the molecular Grb2 interaction network in activated B cells.

Spleen tyrosine kinase Syk provides critical transducer functions for a number of immune cell receptors and has been implicated in the generation of several forms of leukemias. Catalytic activity and the ability of Syk to interact with other signaling elements depend on the phosphorylation status of Syk. We have now identified and quantified the full spectrum of phosphoacceptor sites in human Syk as well as the interactome of Syk in resting and activated B cells by high-resolution mass spectrometry. While the majority of inducible phosphorylations occurred on tyrosine residues, one of the most frequently detected phosphosites encompassed serine 297 located within the linker insert distinguishing the long and short isoforms of Syk. Full-length Syk can associate with more than 25 distinct ligands including the 14-3-3γ adaptor protein, which binds directly to phosphoserine 297. The latter complex attenuates inducible plasma membrane recruitment of Syk, thereby limiting antigen receptor-proximal signaling pathways. Collectively, the established ligand library provides a basis to understand the complexity of the Syk signaling network.

Complex phosphorylation dynamics control the composition of the Syk interactome in B cells.

The B cell receptor (BCR) mediates B cell antigen gathering and acquisition for presentation to T cells. Although the amount of antigen presentation to T cells determines the extent of B cell activation, the molecular mechanisms underlying antigen gathering remain unexplored. Here, through a combination of high-resolution imaging, genetics and quantitative mass spectrometry, we demonstrate that adaptors Grb2 and Dok-3, and ubiquitin ligase Cbl in signaling BCR microclusters mediate association with the microtubule motor dynein. Furthermore, we visualize the localization and movement of these microclusters on the underlying microtubule network. Importantly, disruption of this network or diminished dynein recruitment in Grb2-, Dok-3-, or Cbl-deficient B cells, does not influence microcluster formation or actin-dependent spreading, but abrogates directed movement of microclusters and antigen accumulation. Thus we identify a surprising but pivotal role for dynein and the microtubule network alongside Grb2, Dok-3, and Cbl in antigen gathering during B cell activation.

B cell receptor-mediated antigen gathering requires ubiquitin ligase Cbl and adaptors Grb2 and Dok-3 to recruit dynein to the signaling microcluster.

&#x5408;&#x4F5C;&#x7EC4;&#x88C5;&#x4FE1;&#x53F7;&#x590D;&#x5408;&#x7269;&#xFF0C;&#x7531;&#x9002;&#x914D;&#x5668;&#x86CB;&#x767D;&#x8D28;&#x5FAE;&#x6676;&#x96C6;&#x6210;&#x4FE1;&#x606F;&#x4ECE;&#x8868;&#x9762;&#x7684;&#x53D7;&#x4F53;&#xFF0C;&#x4EE5;&#x786E;&#x5B9A;&#x7EC6;&#x80DE;&#x7684;&#x7ED3;&#x679C;&#x3002;&#x5728; T &#x548C;&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#xFF0C;&#x5728;&#x7531;&#x4E09;&#x4E2A;&#x9002;&#x914D;&#x5668;&#x5FAE;&#x6676;&#x6297;&#x539F;&#x53D7;&#x4F53;&#x4FE1;&#x53F7;&#xFF1A; SLP 76&#x3001; &#x7528;&#x54C1;&#x548C; lat&#x3002;&#x4E09;&#x4E2A;&#x4EBA;&#x4EEC;&#x719F;&#x77E5;&#x7684; SLP 76 &#x916A;&#x6C28;&#x9178;&#x78F7;&#x9178;&#x5316;&#x7F51;&#x7AD9;&#x62DB;&#x8058;&#x5305;&#x62EC; Tec &#x5BB6;&#x5EAD;&#x916A;&#x6C28;&#x9178;&#x6FC0;&#x9176;&#xFF0C;Itk &#x7684;&#x5173;&#x952E;&#x7EC4;&#x4EF6;&#x3002;&#x6211;&#x4EEC;&#x786E;&#x5B9A;&#x4E86;&#x7B2C;&#x56DB;&#x4E2A;&#x8FDB;&#x5316;&#x4FDD;&#x6301; SLP 76 &#x78F7;&#x9178;&#x5316;&#x7F51;&#x7AD9;&#xFF0C;Y173&#xFF0C;&#x8FD9;&#x540E;&#x4E3B;&#x5C0F;&#x9F20;&#x548C; Jurkat T &#x7EC6;&#x80DE; T &#x7EC6;&#x80DE;&#x53D7;&#x4F53;&#x523A;&#x6FC0;&#x78F7;&#x9178;&#x5316;&#x3002;Y173 &#x662F;&#x78F7;&#x8102;&#x9176; C-&gamma;1 (PLC &gamma;1&#xFF09; &#x5728; T &#x548C;&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#x7684;&#x6297;&#x539F;&#x53D7;&#x4F53;&#x8BF1;&#x5BFC;&#x78F7;&#x9178;&#x5316;&#x548C;&#x968F;&#x4E4B;&#x800C;&#x6765;&#x7684;&#x4E0B;&#x6E38;&#x4E8B;&#x4EF6;&#xFF0C;&#x5305;&#x62EC;&#x767D;&#x7EC6;&#x80DE;&#x4ECB;&#x7D20;-2 &#x57FA;&#x56E0;&#x542F;&#x52A8;&#x5B50;&#x7684;&#x6FC0;&#x6D3B; T &#x7EC6;&#x80DE;&#x548C;&#x8131;&#x9897;&#x7C92;&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x767D;&#x7EC6;&#x80DE;&#x4ECB;&#x7D20;-6 &#x751F;&#x4EA7;&#x4E2D;&#x7684;&#x9700;&#x8981;&#x3002;&#x5728;&#x5B8C;&#x6574;&#x7684;&#x5355;&#x5143;&#x683C;&#xFF0C;Y173 &#x78F7;&#x9178;&#x5316;&#x53D6;&#x51B3;&#x4E8E;&#x4E09;&#x4E2A;&#xFF0C;ZAP 70 &#x9776;&#x5411;&#x916A;&#x6C28;&#x9178; SLP 76 &#x603B;&#x7AD9; N &#x62DB;&#x52DF;&#x548C;&#x6FC0;&#x6D3B; Itk&#xFF0C;&#x6709;&#x9009;&#x62E9;&#x6027;&#x5730;&#x78F7;&#x9178;&#x5316; Y173 &#x4F53;&#x5916;&#x7684;&#x6FC0;&#x9176;&#x7684;&#x3002;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x8868;&#x660E; ZAP-70-&#x4F9D;&#x8D56;&#x9884;&#x5145;&#x7684; SLP 76 N &#x7EC8;&#x7AEF;&#x4E09;&#x4E2A;&#x5730;&#x70B9;&#x5373;&#x89E6;&#x53D1;&#x76D1;&#x7BA1;&#x76F8;&#x4E92;&#x4F5C;&#x7528; Itk &#x548C; SLP-76&#xFF0C;&#x5B83;&#x4EEC;&#x6700;&#x7EC8;&#x5FC5;&#x987B;&#x5411;&#x5176;&#x57FA;&#x677F;&#xFF0C;PLC &gamma;1 &#x592B;&#x5987;&#x79EF;&#x6781; Itk &#x987A;&#x5E8F;&#x673A;&#x5236;&#x3002;

&#x5E8F;&#x8D2F;&#x78F7;&#x9178;&#x5316; SLP 76 &#x5728; 173 &#x916A;&#x6C28;&#x9178;&#x662F; T &#x548C;&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#x6FC0;&#x6D3B;&#x6240;&#x9700;&#x3002;

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-Î³1 (PLC-Î³1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-Î³1.

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells.

Assembl&eacute;s en collaboration de signalisation complexes, nucl&eacute;&eacute;es par des prot&eacute;ines de l&#39;adaptateur, int&eacute;grer des informations provenant des r&eacute;cepteurs de surface pour d&eacute;terminer les r&eacute;sultats cellulaires. En T et des mastocytes, signalisation de r&eacute;cepteur de l&#39;antig&egrave;ne est nucl&eacute;&eacute; par trois adaptateurs : SLP-76, Gads et LAT. Trois sites bien caract&eacute;ris&eacute;s de phosphorylation de tyrosine de SLP-76 recrutent des &eacute;l&eacute;ments cl&eacute;s, y compris une kinase de tyrosine Tec-famille, Itk. Nous avons identifi&eacute; un quatri&egrave;me site de phosphorylation &eacute;volutivement conserv&eacute; SLP-76, Y173, qui est phosphoryl&eacute; sur la stimulation des r&eacute;cepteurs lymphocytes primaire murin et cellules T Jurkat. Y173 &eacute;tait n&eacute;cessaire pour la phosphorylation induite par le r&eacute;cepteur de l&#39;antig&egrave;ne de la phospholipase C-&gamma;1 (PLC-&gamma;1) en T et des cellules de m&acirc;t et des &eacute;v&eacute;nements en aval qui en d&eacute;coule, y compris l&#39;activation du promoteur IL-2 dans les cellules T et d&eacute;granulation et production d&#39;IL-6 dans les cellules de m&acirc;t. Dans les cellules intactes, la phosphorylation de Y173 d&eacute;pendait de trois, tyrosines cibl&eacute;es ZAP-70 &agrave; l&#39;extr&eacute;mit&eacute; N-terminale SLP-76 qui recrutent et activer Itk, une kinase qui phosphoryl&eacute; s&eacute;lectivement les Y173 in vitro. Ces donn&eacute;es sugg&egrave;rent un m&eacute;canisme s&eacute;quentiel par lequel un amor&ccedil;age ZAP-70-dependent des SLP-76 sur trois sites de N-terminal d&eacute;clenche des interactions r&eacute;glementaires r&eacute;ciproques entre l&#39;Itk et SLP-76, qui sont finalement tenus d&#39;atteler Itk active &agrave; son substrat, PLC-&gamma;1.

Phosphorylation s&eacute;quentielle de SLP-76 &agrave; tyrosine 173 est requise pour l&#39;activation de T et des mastocytes.

Assemblati in modo cooperativo segnalazione complessi, nucleate da proteine adattatore, integrare le informazioni da recettori di superfici per determinare i risultati cellulari. In T e mastociti, antigene recettore segnalazione &egrave; nucleate da tre adattatori: SLP-76, Gads e LAT. Tre siti di fosforilazione di tirosina di SLP-76 ben caratterizzati reclutano componenti chiave, tra cui una Tec-famiglia tirosina chinasi, Itk. Abbiamo identificato un quarto, evolutivamente conservato sito di fosforilazione di SLP-76, Y173, che &egrave; stato fosforilato dopo stimolazione del recettore delle cellule T in murino primario e cellule Jurkat T. Y173 &egrave; stato richiesto per la fosforilazione del recettore-indotta dell&#39;antigene della fosfolipasi C-&gamma;1 (PLC-&gamma;1) in T e mastociti e per conseguenti eventi a valle, inclusa l&#39;attivazione del promotore IL-2 a cellule T e degranulazione e produzione di IL-6 nei mastociti. Nelle cellule intatte, Y173 fosforilazione dipendeva da tre, targeting per ZAP-70 tirosine presso il N-terminus di SLP-76 che Reclutano e attivare Itk, una chinasi che selettivamente fosforilato Y173 in vitro. Questi dati suggeriscono un meccanismo sequenza per cui innesco ZAP-70-dipendente di SLP-76 a tre siti di N-terminale innesca reciproche interazioni normative tra Itk e SLP-76, che in definitiva sono tenuti a coppia Itk attivo al suo substrato, PLC-&gamma;1.

Fosforilazione sequenza di SLP-76 a tirosina 173 &egrave; richiesta per l&#39;attivazione di T e mastociti.

&#x9023;&#x643A;&#x30A2;&#x30C0;&#x30D7;&#x30BF;&#x30FC;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306B;&#x3088;&#x308B;&#x6838;&#x932F;&#x4F53;&#x3092;&#x4FE1;&#x53F7;&#x7D44;&#x307F;&#x7ACB;&#x3066;&#x7D30;&#x80DE;&#x306E;&#x7D50;&#x679C;&#x3092;&#x5224;&#x65AD;&#x3059;&#x308B;&#x8868;&#x9762;&#x306E;&#x53D7;&#x5BB9;&#x5668;&#x304B;&#x3089;&#x306E;&#x60C5;&#x5831;&#x3092;&#x7D71;&#x5408;&#x3057;&#x307E;&#x3059;&#x3002;T &#x3068;&#x30DE;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x306E;&#x6297;&#x539F;&#x53D7;&#x5BB9;&#x4F53;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x3068;&#x3059;&#x308B;&#x3068;&#x3001;3 &#x3064;&#x306E;&#x30A2;&#x30C0;&#x30D7;&#x30BF;&#x30FC;&#x306B;&#x3088;&#x3063;&#x3066;&#x6838;&#x3067;&#x3059;: SLP 76&#x3001;Gads&#x3001;&#x7DEF;&#x5EA6;3 &#x3064;&#x306E;&#x3088;&#x304F;&#x7279;&#x5FB4;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F; SLP 76 &#x30C1;&#x30ED;&#x30B7;&#x30F3; &#x30EA;&#x30F3;&#x9178;&#x5316;&#x306E;&#x30B5;&#x30A4;&#x30C8; &#x30C6;&#x30C3;&#x30AF;&#x5BB6;&#x65CF;&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x30AD;&#x30CA;&#x30FC;&#x30BC; Itk &#x3092;&#x542B;&#x3080;&#x4E3B;&#x8981;&#x306A;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x3001;&#x52DF;&#x96C6;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x3001;4 &#x756A;&#x76EE;&#x3001;SLP 76 &#x30EA;&#x30F3;&#x9178;&#x5316;&#x306E;&#x30B5;&#x30A4;&#x30C8;&#x3092;&#x9032;&#x5316;&#x7684;&#x306B;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x305F;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA; &#x30DE;&#x30A6;&#x30B9;&#x3068;&#x4F53;&#x306E; T &#x7D30;&#x80DE;&#x306E; T &#x7D30;&#x80DE;&#x53D7;&#x5BB9;&#x4F53;&#x523A;&#x6FC0;&#x306B;&#x3088;&#x308B;&#x30EA;&#x30F3;&#x9178;&#x5316; Y173 &#x8B58;&#x5225;&#x3002;Y173 &#x306F;&#x3001;&#x30DB;&#x30B9;&#x30DB;&#x30EA;&#x30D1;&#x30FC;&#x30BC; C-&gamma;1 (PLC &gamma;1) T &#x3068;&#x30DE;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x306E;&#x6297;&#x539F;&#x53D7;&#x5BB9;&#x4F53;&#x306B;&#x3088;&#x308B;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x304A;&#x3088;&#x3073; IL-2 &#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x306E;&#x6D3B;&#x6027;&#x5316; T &#x7D30;&#x80DE;&#x3068;&#x9846;&#x7C92;&#x3068;&#x30DE;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B; il-6 &#x7523;&#x751F;&#x306A;&#x3069;&#x3092;&#x542B;&#x3080;&#x3001;&#x7D50;&#x679C;&#x306E;&#x30C0;&#x30A6;&#x30F3; &#x30B9;&#x30C8;&#x30EA;&#x30FC;&#x30E0; &#x30A4;&#x30D9;&#x30F3;&#x30C8;&#x306B;&#x5FC5;&#x8981;&#x3060;&#x3063;&#x305F;&#x3002;&#x305D;&#x306E;&#x307E;&#x307E;&#x306A;&#x30BB;&#x30EB;&#x3067;&#x3001;Y173 &#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3068;&#x52DF;&#x96C6;&#x30A4;&#x30E0;&#x30CE;&#x30AF;&#x30ED;&#x30DE;&#x30C8; Y173 in vitro &#x9078;&#x629E;&#x7684;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x3092;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x3059;&#x308B; 3&#x3001;&#x30B6;&#x30C3;&#x30D7; 70 &#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8; &#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x306B; SLP 76 &#x306E; N &#x672B;&#x7AEF;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x3068;&#x3044;&#x3046; ZAP-70 &#x4F9D;&#x5B58;&#x30D7;&#x30E9;&#x30A4;&#x30DF;&#x30F3;&#x30B0; SLP-76 &#x3067; 3 &#x3064;&#x306E; N &#x672B;&#x7AEF;&#x30B5;&#x30A4;&#x30C8;&#x306E;&#x898F;&#x5236;&#x306E;&#x76F8;&#x4E92; Itk &#x3068; SLP 76 &#x6700;&#x7D42;&#x7684;&#x306B;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306A; Itk &#x305D;&#x306E;&#x57FA;&#x677F; PLC &gamma;1 &#x30AB;&#x30C3;&#x30D7;&#x30EB;&#x306E;&#x305F;&#x3081;&#x306B;&#x5FC5;&#x8981;&#x306A;&#x30C8;&#x30EA;&#x30AC;&#x30FC;&#x9010;&#x6B21;&#x6A5F;&#x69CB;&#x3092;&#x63D0;&#x6848;&#x3057;&#x307E;&#x3059;&#x3002;

SLP 76 &#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B7;&#x30E3;&#x30EB; &#x30EA;&#x30F3;&#x9178;&#x5316;&#x30C1;&#x30ED;&#x30B7;&#x30F3; 173 &#x3067;&#x306F; T &#x3068;&#x30DE;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;

&#xD611;&#xB825; &#xB2E8;&#xC9C0;, &#xC5B4;&#xB311;&#xD130; &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0; &#xC758;&#xD574; nucleated &#xC2E0;&#xD638; &#xC870;&#xB9BD; &#xD45C;&#xBA74; &#xC218;&#xC6A9; &#xCCB4; &#xC138;&#xD3EC; &#xACB0;&#xACFC; &#xD655;&#xC778;&#xC5D0;&#xC11C; &#xC815;&#xBCF4;&#xB97C; &#xD1B5;&#xD569; &#xD569;&#xB2C8;&#xB2E4;. T&#xC640; &#xB9C8;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; 3 &#xAC1C;&#xC758; &#xC5B4;&#xB311;&#xD130;&#xC5D0; &#xC758;&#xD574; nucleated&#xB294; &#xD56D; &#xC6D0; &#xC218;&#xC6A9; &#xCCB4; &#xC2E0;&#xD638; &#xC804;&#xB2EC;: SLP-76, Gads &#xBC0F; &#xC704;&#xB3C4; 3 &#xAC1C;&#xC758; well-characterized SLP 76 &#xD2F0;&#xB85C;&#xC2E0; &#xC778; &#xC0B0;&#xD654; &#xC0AC;&#xC774;&#xD2B8; &#xBAA8;&#xC9D1; Tec &#xAC00;&#xC871; tyrosine kinase Itk&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xC8FC;&#xC694; &#xAD6C;&#xC131; &#xC694;&#xC18C;. &#xB124; &#xBC88;&#xC9F8;, &#xC9C4;&#xD654; &#xC801;&#xC73C;&#xB85C; &#xBCF4;&#xC874;&#xB41C; SLP 76 &#xC778; &#xC0B0;&#xD654; &#xC0AC;&#xC774;&#xD2B8; &#xAE30;&#xBCF8; murine Jurkat T &#xC138;&#xD3EC;&#xC5D0;&#xC11C; T &#xC138;&#xD3EC; &#xC218;&#xC6A9; &#xCCB4; &#xC790;&#xADF9;&#xC5D0; &#xB530;&#xB77C; phosphorylated&#xB294; Y173 &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;. Y173 phospholipase C-&gamma;1 (PLC &gamma;1) T&#xC640; &#xB9C8;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC;&#xC5D0;&#xC11C;&#xC758; &#xD56D; &#xC6D0; &#xC218;&#xC6A9; &#xCCB4;&#xB97C; &#xC774;&#xC6A9;&#xD55C; &#xC778; &#xC0B0;&#xD654; &#xBC0F; T &#xC138;&#xD3EC;&#xC640; degranulation &#xB9C8;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xC0DD;&#xC0B0; &#xC77C;&#xB9AC;&#xB178;&#xC774;-6 &#xC77C;&#xB9AC;&#xB178;&#xC774;-2 &#xBC1C;&#xAE30;&#xC778;&#xC758; &#xD65C;&#xC131;&#xD654;&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xACB0;&#xACFC; &#xB2E4;&#xC6B4;&#xC2A4;&#xD2B8;&#xB9BC; &#xC774;&#xBCA4;&#xD2B8;&#xAC00; &#xD544;&#xC694; &#xD588;&#xB2E4;. &#xADF8;&#xB300;&#xB85C; &#xC140;&#xC5D0; Y173 &#xC778; &#xC0B0;&#xD654; 3, ZAP 70 &#xD0C0;&#xAC9F; tyrosines SLP-76&#xC758; N &#xB9D0;&#xB2E8;&#xC5D0;&#xC11C; &#xBAA8;&#xC9D1; &#xACE0; Itk, &#xC120;&#xD0DD;&#xC801;&#xC73C;&#xB85C; Y173 &#xCCB4; &#xC678; phosphorylated &#xD0A4; &#xB2C8; &#xC544; &#xC81C; &#xD65C;&#xC131;&#xD654;&#xC5D0; &#xC758;&#xC874; &#xD55C;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; 3 N &#xD130;&#xBBF8;&#xB110; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0;&#xC11C; SLP-76&#xC758; ZAP 70 &#xC885;&#xC18D; &#xBABB;&#xC4F0;&#xAC8C; Itk&#xC640; SLP-76, &#xAD81;&#xADF9;&#xC801;&#xC73C;&#xB85C; &#xAE30;&#xD310;, PLC-&gamma;1&#xC744; &#xD65C;&#xC131; Itk &#xBD80;&#xBD80; &#xD558;&#xB294; &#xB370; &#xD544;&#xC694;&#xD55C; &#xAC04;&#xC758; &#xC0C1;&#xD638; &#xADDC;&#xC81C; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xC720;&#xBC1C; &#xD558;&#xB294; &#xADF8;&#xAC83;&#xC5D0; &#xC758;&#xD558;&#xC5EC; &#xC21C;&#xCC28;&#xC801; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xC81C;&#xC548; &#xD569;&#xB2C8;&#xB2E4;.

&#xD2F0;&#xB85C;&#xC2E0; 173&#xC5D0;&#xC11C; SLP-76&#xC758; &#xC5F0;&#xC18D; &#xC778; &#xC0B0;&#xD654;&#xB294; T&#xC640; &#xB9C8;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC;&#xC758; &#xD65C;&#xC131;&#xD654;&#xB97C; &#xC704;&#xD574; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;.

Cooperativamente montado complejos, nucleados por prote&iacute;nas adaptadoras de se&ntilde;alizaci&oacute;n integrar informaci&oacute;n de receptores de superficie para determinar los resultados celulares. En T y mastocitos, se&ntilde;alizaci&oacute;n de receptor de ant&iacute;geno es nucleada por tres adaptadores: SLP-76, Gads y LAT. Bien caracterizados SLP-76 tres sitios de tirosina fosforilaci&oacute;n reclutan componentes clave, incluyendo una tirosincinasa Tec-familia, Itk. Identificamos un cuarto, el sitio de fosforilaci&oacute;n de SLP-76 evolutivamente conservado, Y173, que se fosforila sobre estimulaci&oacute;n de receptor de c&eacute;lulas T en primaria murinas y c&eacute;lulas Jurkat T. Y173 fue requerido para la fosforilaci&oacute;n del receptor-inducida de ant&iacute;geno de la fosfolipasa C-&gamma;1 (PLC-&gamma;1) en T y mastocitos y por consiguientes eventos posteriores, incluida la activaci&oacute;n del promotor IL-2 en las c&eacute;lulas T y degranulaci&oacute;n y producci&oacute;n de IL-6 en las c&eacute;lulas del m&aacute;stil. En las c&eacute;lulas intactas, fosforilaci&oacute;n Y173 dependi&oacute; de tres tirosinas orientada a ZAP-70 en el N-terminal de SLP-76 que reclutan y activan Itk, una quinasa que fosforila selectivamente Y173 in vitro. Estos datos sugieren un mecanismo secuencial por el cebado de ZAP-70-dependiente de SLP-76 en tres sitios de N-terminal provoca interacciones reguladoras rec&iacute;procas entre Itk y SLP-76, que en &uacute;ltima instancia deben acoplar Itk activo a su sustrato, PLC-&gamma;1.

Fosforilaci&oacute;n secuencial de SLP-76 en tirosina 173 se requiere para la activaci&oacute;n de T y mastocitos.

Spleen tyrosine kinase Syk and its substrate SLP65 (also called BLNK) are proximal signal transducer elements of the B-cell antigen receptor (BCR). Yet, our understanding of signal initiation and processing is limited owing to the incomplete list of SLP65 interaction partners and our ignorance of their association kinetics. We have now determined and quantified the in vivo interactomes of SLP65 in resting and stimulated B cells by mass spectrometry. SLP65 orchestrated a complex signal network of about 30 proteins that was predominantly based on dynamic interactions. However, a stimulation-independent and constant association of SLP65 with the Cbl-interacting protein of 85 kDa (CIN85) was requisite for SLP65 phosphorylation and its inducible plasma membrane translocation. In the absence of a steady SLP65/CIN85 complex, BCR-induced Ca(2+) and NF-κB responses were abrogated. Finally, live cell imaging and co-immunoprecipitation experiments further confirmed that both SLP65 and CIN85 are key components of the BCR-associated primary transducer module required for the onset and progression phases of BCR signal transduction.

The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85.

Recruitment of the growth factor receptor-bound protein 2 (Grb2) by the plasma membrane-associated adapter protein downstream of kinase 3 (Dok-3) attenuates signals transduced by the B cell antigen receptor (BCR). Here we describe molecular details of Dok-3/Grb2 signal integration and function, showing that the Lyn-dependent activation of the BCR transducer kinase Syk is attenuated by Dok-3/Grb2 in a site-specific manner. This process is associated with the SH3 domain-dependent translocation of Dok-3/Grb2 complexes into BCR microsignalosomes and augmented phosphorylation of the inhibitory Lyn target SH2 domain-containing inositol 5' phosphatase. Hence, our findings imply that Dok-3/Grb2 modulates the balance between activatory and inhibitory Lyn functions with the aim to adjust BCR signaling efficiency.

The Dok-3/Grb2 protein signal module attenuates Lyn kinase-dependent activation of Syk kinase in B cell antigen receptor microclusters.

Mutations in the chromodomain helicase DNA binding protein 7 gene (CHD7) lead to CHARGE syndrome, an autosomal dominant multiple malformation disorder. Proteins involved in chromatin remodeling typically act in multiprotein complexes. We previously demonstrated that a part of human CHD7 interacts with a part of human CHD8, another chromodomain helicase DNA binding protein presumably being involved in the pathogenesis of neurodevelopmental (NDD) and autism spectrum disorders (ASD). Because identification of novel CHD7 and CHD8 interacting partners will provide further insights into the pathogenesis of CHARGE syndrome and ASD/NDD, we searched for additional associated polypeptides using the method of stable isotope labeling by amino acids in cell culture (SILAC) in combination with mass spectrometry.

Identification and characterization of FAM124B as a novel component of a CHD7 and CHD8 containing complex.

Spleen tyrosine kinase (Syk) induces cell survival and proliferation in a high proportion of acute myeloid leukemia (AML) blasts, but the underlying molecular events of Syk signaling have not been investigated. Proteomic techniques have allowed us to identify the multiprotein complex that is nucleated by constitutively active Syk in AML cells. This complex differs from the B-lymphoid Syk interactome with respect to several proteins, especially the integrin receptor Mac-1, the Fc-γ receptor I (FcγRI), and the transcription factors STAT3 and STAT5. We show in several AML cell line models that tonic signals derived from the Fc-γ chain lead to Syk-dependent activation of STAT3 and STAT5, which in turn induces cell survival and proliferation. Moreover, stimulation of Mac-1 or FcγRI intensifies the constitutive Syk-mediated STAT3/5 activation in AML cells, a scenario likely to take place in the bone marrow niche. In accordance with these findings, we observed that β2 integrins, including Mac-1, trigger proliferation of AML cells in an AML cell/stroma coculture model. Taken together, we identified an oncogenic integrin/Syk/STAT3/5 signaling axis that might serve as a therapeutic target of AML in the future.

β2 integrin-derived signals induce cell survival and proliferation of AML blasts by activating a Syk/STAT signaling axis.

The adaptor Nck links receptor signaling to cytoskeleton regulation. Here we found that Nck also controlled the phosphatidylinositol-3-OH kinase (PI(3)K)-kinase Akt pathway by recruiting the adaptor BCAP after activation of B cells. Nck bound directly to the B cell antigen receptor (BCR) via the non-immunoreceptor tyrosine-based activation motif (ITAM) phosphorylated tyrosine residue at position 204 in the tail of the immunoglobulin-α component. Genetic ablation of Nck resulted in defective BCR signaling, which led to hampered survival and proliferation of B cells in vivo. Indeed, antibody responses in Nck-deficient mice were also considerably impaired. Thus, we demonstrate a previously unknown adaptor function for Nck in recruiting BCAP to sites of BCR signaling and thereby modulating the PI(3)K-Akt pathway in B cells.

Nck-mediated recruitment of BCAP to the BCR regulates the PI(3)K-Akt pathway in B cells.

Ag-mediated B cell stimulation relies on phospholipase Cγ2 (PLCγ2) for Ca(2+) mobilization. Enzymatic activity of PLCγ2 is triggered upon Src homology 2 domain-mediated binding to the tyrosine-phosphorylated adaptor SLP65. However, SLP65 phosphorylation outlasts the elevation of cytosolic Ca(2+) concentration suggesting additional levels of PLCγ2 regulation. We show in this article that the functionality of the PLCγ2/SLP65 complex is controlled by the weakly characterized C2 domain of PLCγ2. Usually C2 domains bind membrane lipids, but that of PLCγ2 docks in a Ca(2+)-regulated manner to a distinct phosphotyrosine of SLP65. Hence, early Ca(2+) fluxing provides feed-forward signal amplification by promoting anchoring of the PLCγ2 C2 domain to phospho-SLP65. As the cellular Ca(2+) resources become exhausted, the concomitant decline of Ca(2+) dampens the C2-phosphotyrosine interaction so that PLCγ2 activation terminates despite sustained SLP65 phosphorylation.

Cutting edge: feed-forward activation of phospholipase Cγ2 via C2 domain-mediated binding to SLP65.

The histone methyltransferase EZH2 is frequently mutated in germinal center-derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2(Y641F) is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2(Y641F) by crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2(Y641F). The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2(Y641F) can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.

A transgenic mouse model demonstrating the oncogenic role of mutations in the polycomb-group gene EZH2 in lymphomagenesis.

The transcription factor Tal1 is a critical activator or repressor of gene expression in hematopoiesis and leukaemia. The mechanism by which Tal1 differentially influences transcription of distinct genes is not fully understood. Here we show that Tal1 interacts with the peptidylarginine deiminase IV (PADI4). We demonstrate that PADI4 can act as an epigenetic coactivator through influencing H3R2me2a. At the Tal1/PADI4 target gene IL6ST the repressive H3R2me2a mark triggered by PRMT6 is counteracted by PADI4, which augments the active H3K4me3 mark and thus increases IL6ST expression. In contrast, at the CTCF promoter PADI4 acts as a repressor. We propose that the influence of PADI4 on IL6ST transcription plays a role in the control of IL6ST expression during lineage differentiation of hematopoietic stem/progenitor cells. These results open the possibility to pharmacologically influence Tal1 in leukaemia.

PADI4 acts as a coactivator of Tal1 by counteracting repressive histone arginine methylation.

T cell antigen receptor (TCR) signaling depends on three interacting adaptor proteins: SLP-76, Gads, and LAT. Their mechanisms of signaling have been extensively explored, with the aid of fortuitously isolated LAT- and SLP-76-deficient T cell lines, but no such tools were available for Gads, a Grb2-family adaptor that bridges the TCR-inducible interaction between SLP-76 and LAT. TALEN-directed genome editing was applied to disrupt the first coding exon of human Gads in the Jurkat T cell line. Gads was dispensable for TCR-induced phosphorylation of SLP-76, but was a dose-dependent amplifier of TCR-induced CD69 expression. Gads conferred responsiveness to weak TCR stimuli, leading to PLC-γ1 phosphorylation and calcium flux. TALEN-derived, Gads-deficient T cell lines provide a uniquely tractable genetic platform for exploring its regulatory features, such as Gads phosphorylation at T262, which we observed by mass spectrometry. Upon mutation of this site, TCR responsiveness and sensitivity to weak TCR stimuli were increased. This study demonstrates the feasibility of TALEN-based reverse genetics in Jurkat T cells, while enriching our understanding of Gads as a regulated modulator of TCR sensitivity.

Modulation of TCR responsiveness by the Grb2-family adaptor, Gads.

Acute myeloid leukemia (AML) is driven by niche-derived and cell-autonomous stimuli. While many cell-autonomous disease drivers are known, niche-dependent signaling in the context of the genetic disease heterogeneity has been difficult to investigate. Here, we analyzed the role of Bruton's tyrosine kinase (BTK) in AML. BTK was frequently expressed and its inhibition strongly impaired the proliferation and survival of AML cells - also in the presence of bone marrow stroma. By interactome analysis, (phospho)proteomics and transcriptome sequencing, we characterized BTK signaling networks. We show that BTK-dependent signaling is highly context-dependent. In FLT3-ITD-positive AML, BTK mediates FLT3-ITD-dependent Myc- and STAT5 activation and combined targeting of FLT3-ITD and BTK showed additive effects. In FLT3-ITD-negative AML, BTK couples Toll-like receptor 9 (TLR9) activation to NFκB and STAT5. Both BTK-dependent transcriptional programs were relevant for cell cycle progression and apoptosis regulation. Thus, we identify context-dependent oncogenic driver events that may guide subtype-specific treatment strategies and point for the first time to a role of TLR9 in AML. Clinical evaluation of BTK inhibitors in AML seems warranted.

Article	Year
SLP-65 phosphorylation dynamics reveals a functional basis for signal integration by receptor-proximal adaptor proteins. Molecular & cellular proteomics : MCP\| PubMed ID: 19372136	2009
The B-lymphoid Grb2 interaction code. Immunological reviews\| PubMed ID: 19909361	2009
Fc gamma receptor IIb modulates the molecular Grb2 interaction network in activated B cells. Cellular signalling\| PubMed ID: 21262349	2011
Complex phosphorylation dynamics control the composition of the Syk interactome in B cells. European journal of immunology\| PubMed ID: 21469132	2011
B cell receptor-mediated antigen gathering requires ubiquitin ligase Cbl and adaptors Grb2 and Dok-3 to recruit dynein to the signaling microcluster. Immunity\| PubMed ID: 21703542	2011
Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells. The EMBO journal\| PubMed ID: 21725281	2011
The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85. The EMBO journal\| PubMed ID: 21822214	2011
The Dok-3/Grb2 protein signal module attenuates Lyn kinase-dependent activation of Syk kinase in B cell antigen receptor microclusters. The Journal of biological chemistry\| PubMed ID: 23223229	2013
Identification and characterization of FAM124B as a novel component of a CHD7 and CHD8 containing complex. PloS one\| PubMed ID: 23285124	2012
β2 integrin-derived signals induce cell survival and proliferation of AML blasts by activating a Syk/STAT signaling axis. Blood\| PubMed ID: 23509157	2013
Nck-mediated recruitment of BCAP to the BCR regulates the PI(3)K-Akt pathway in B cells. Nature immunology\| PubMed ID: 23913047	2013
Cutting edge: feed-forward activation of phospholipase Cγ2 via C2 domain-mediated binding to SLP65. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 24166973	2013
A transgenic mouse model demonstrating the oncogenic role of mutations in the polycomb-group gene EZH2 in lymphomagenesis. Blood\| PubMed ID: 24802772	2014
PADI4 acts as a coactivator of Tal1 by counteracting repressive histone arginine methylation. Nature communications\| PubMed ID: 24874575	2014
Modulation of TCR responsiveness by the Grb2-family adaptor, Gads. Cellular signalling\| PubMed ID: 25452106	2015
FLT3-ITD and TLR9 employ Bruton's tyrosine kinase to activate distinct transcriptional programs mediating AML cell survival and proliferation. Blood\| PubMed ID: 25605370	2015

Thomas Oellerich

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