Subcellular localization of Grb2 by the adaptor protein Dok-3 restricts the intensity of Ca2+ signaling in B cells

Stork, Björn; Neumann, Konstantin; Goldbeck, Ingo; Alers, Sebastian; Kähne, Thilo; Naumann, Michael; Engelke, Michael; Wienands, Jürgen

doi:10.1038/sj.emboj.7601557

Cited by 64 publications

(155 citation statements)

References 50 publications

Supporting

Mentioning

147

Contrasting

Order By: Relevance

“…Despite a number of known interactors for the Grb2 SH2 domain, the mandatory Grb2-recruiting protein in BCR-stimulated cells appears to be Dok-3 because BCR-induced Grb2 relocalization hardly occurs in Dok-3-deficient DT40 B cells [21]. Our data show that BCR/FcγRIIb co-stimulation evokes a more pronounced tyrosine phosphorylation of Dok-3 than BCR stimulation alone.…”

Section: Discussionmentioning

confidence: 60%

“…Previous findings of our laboratory emphasize Dok-3 to be the main BCR-proximal Grb2-interacting protein under positive signaling conditions [21]. Moreover, Dok-3 and Grb2 bind to the FcγRIIb effector SHIP [27][28][29] implying that Dok-3/Grb2 might play a role under negative signaling conditions, too.…”

Section: Co-engagement Of the Bcr And Fcγriib Leads To Increased Formmentioning

confidence: 78%

“…BCR stimulation was performed for indicated times using 10 μg/ml F(ab) 2 fragments of goat anti-mouse IgM or 15 μg/ml complete goat anti-mouse IgM (Jackson Immuno Research Laboratories). Cells were lysed as described in [21]. For immunoprecipitation rabbit antiDok-3 S-20 (Santa Cruz Biotechnology) was used.…”

Section: Cells Abs and Reagentsmentioning

confidence: 99%

“…Cells were washed and prepared for measurements as described earlier [21]. Changes in the ratio of fluorescence intensities at 405 nm and 510 nm were monitored on a LSRII flow cytometer (Becton Dickinson) and analyzed with FlowJo (TriStar).…”

Section: Calcium Measurementsmentioning

confidence: 99%

“…In complex with the adapter protein downstream of kinase-3 (Dok-3) Grb2 attenuates PLC-γ2-dependent production of inositol-1,4,5-trisphosphate [21]. Dok-3-deficient mice have increased serum IgM titers and enhanced humoral immune responses to T cell-independent antigens [22].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Neumann

Oellerich

Heine

et al. 2011

Cellular Signalling

Self Cite

View full text Add to dashboard Cite

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 domaincontaining 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.

show abstract

Section: Discussionmentioning

confidence: 60%

Section: Co-engagement Of the Bcr And Fcγriib Leads To Increased Formmentioning

confidence: 78%

Section: Cells Abs and Reagentsmentioning

confidence: 99%

Section: Calcium Measurementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Neumann

Oellerich

Heine

et al. 2011

Cellular Signalling

Self Cite

View full text Add to dashboard Cite

show abstract

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

et al. 2011

Self Cite

View full text Add to dashboard Cite

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 14-3-3c 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.Key words: B-cell activation . Interactome . Spleen tyrosine kinase . 14-3-3 Supporting Information available online IntroductionThe 72 kDa spleen tyrosine kinase Syk provides catalytic activity to hematopoietic cell surface receptors encompassing ITAMs in their signaling subunits [1]. Following ligand-induced receptor aggregation, doubly phosphorylated ITAMs recruit Syk by virtue of its N-terminal Src homology 2 (SH2) domains. Interdomain A of Syk links the two SH2 domains, which are connected to the C-terminal kinase domain by interdomain B. Two Syk isoforms can be generated by alternative splicing, which leads to the presence or absence of 23 amino acids, called the linker insert region, in interdomain B [2,3]. Several mechanisms operate in concert to control Syk activity. The phospho-ITAM/(SH2) 2 interaction leads to allosteric activation most likely by changing the conformation of Syk from a closed inactive form to an open active structure [4,5]. Moreover, phospho-ITAMs act as inducible membrane anchors for cytosolic Syk and the accompanied subcellular relocalization provides Syk with access to key substrates [6]. Phosphorylation of tyrosine residues within the kinase domain or interdomain B boosts the catalytic activity of Syk or generates docking sites for SH2 domain-containing effector proteins, respectively [7]. Termination of Syk activity can be achieved by dephosphorylation through protein tyrosine phosphatases such as SHP1 or proteasomal degradation induced Ã These authors contributed equally to this work. 1550Frontline by binding of the E3 ubiquitin ligase Cbl to a distinct phosphotyrosine residue in interdomain B [8,9].Syk activation and triggering of downstream effector cascades have been extensively studied in B lymphocytes. In fact, Syk was initially identified as a B-lymphoid tyrosine kinase associated with BCR [10,11]. BCRs comprise membrane-bound Igs of different cla...

show abstract

The Dok‐3/Grb2 adaptor module promotes inducible association of the lipid phosphatase SHIP with the BCR in a coreceptor‐independent manner

et al. 2016

Self Cite

View full text Add to dashboard Cite

The SH2 domain-containing inositol 5'-phosphatase (SHIP) plays a key role in preventing autoimmune phenomena by limiting antigen-mediated B cell activation. SHIP function is thought to require the dual engagement of the BCR and negative regulatory coreceptors as only the latter appear capable of recruiting SHIP from the cytosol to the plasma membrane by the virtue of phosphorylated immunoreceptor tyrosine-based inhibitory motifs. Here, we demonstrate a coreceptor-independent membrane recruitment and function of SHIP in B cells. In the absence of coreceptor ligation, SHIP translocates to sites of BCR activation through a concerted action of the protein adaptor unit Dok-3/Grb2 and phosphorylated BCR signaling components. Our data reveal auto-inhibitory SHIP activation by the activated BCR and suggest an unexpected negative-regulatory capacity of immunoreceptor tyrosine-based activation motifs in Igα and Igβ.

show abstract

Subcellular localization of Grb2 by the adaptor protein Dok-3 restricts the intensity of Ca2+ signaling in B cells

Cited by 64 publications

References 50 publications

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

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

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

The Dok‐3/Grb2 adaptor module promotes inducible association of the lipid phosphatase SHIP with the BCR in a coreceptor‐independent manner

Contact Info

Product

Resources

About