In vitro characterization of major ligands for Src homology 2 domains derived from protein tyrosine kinases, from the adaptor protein SHC and from GTPase-activating protein in Ramos B cells

Treatment of B lymphocytes with antibodies to membrane immunoglobulin (Ig) stimulates protein tyrosine phosphorylation. We have examined the phosphorylation in vitro of proteins associated with membrane Ig. The Src family protein tyrosine kinases p53/56lyn, p59fyn, and p56lck are associated with membrane Ig in spleen B cells and B-cell lines and undergo phosphorylation in vitro. The pattern of expression of Src family protein tyrosine kinases in B cells varied. Our studies suggest that multiple kinases can potentially interact with membrane Ig and that within any one B-cell type, all of the Src family kinases expressed can be found in association with membrane Ig. We also observed that the Ig-associated Ig alpha protein, multiple forms of Ig beta, and proteins of 100 and 25 kDa were tyrosine phosphorylated in vitro. The 100- and 25-kDa proteins remain unidentified.

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A Dual Role for Src Homology 2 Domain–Containing Inositol-5-Phosphatase (Ship) in Immunity

Journal of Experimental Medicine ◽

10.1084/jem.191.5.781 ◽

2000 ◽

Vol 191 (5) ◽

pp. 781-794 ◽

Cited By ~ 115

Author(s):

Cheryl D. Helgason ◽

Christian P. Kalberer ◽

Jacqueline E. Damen ◽

Suzanne M. Chappel ◽

Nicolas Pineault ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Lymphoid Development ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain ◽

B Lymphoid

In this report, we demonstrate that the Src homology 2 domain–containing inositol-5-phosphatase (SHIP) plays a critical role in regulating both B cell development and responsiveness to antigen stimulation. SHIP−/− mice exhibit a transplantable alteration in B lymphoid development that results in reduced numbers of precursor B (fraction C) and immature B cells in the bone marrow. In vitro, purified SHIP−/− B cells exhibit enhanced proliferation in response to B cell receptor stimulation in both the presence and absence of Fcγ receptor IIB coligation. This enhancement is associated with increased phosphorylation of both mitogen-activated protein kinase and Akt, as well as with increased survival and cell cycling. SHIP−/− mice manifest elevated serum immunoglobulin (Ig) levels and an exaggerated IgG response to the T cell–independent type 2 antigen trinitrophenyl Ficoll. However, only altered B cell development was apparent upon transplantation into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice. The in vitro hyperresponsiveness, together with the in vivo findings, suggests that SHIP regulates B lymphoid development and antigen responsiveness by both intrinsic and extrinsic mechanisms.

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Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases.

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2315 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2315-2321 ◽

Cited By ~ 113

Author(s):

M A Campbell ◽

B M Sefton

Keyword(s):

B Cells ◽

B Cell ◽

Tyrosine Kinases ◽

B Lymphocyte ◽

Protein Tyrosine Kinases ◽

Cell Type ◽

Protein Tyrosine ◽

Membrane Immunoglobulin ◽

Family Protein

Treatment of B lymphocytes with antibodies to membrane immunoglobulin (Ig) stimulates protein tyrosine phosphorylation. We have examined the phosphorylation in vitro of proteins associated with membrane Ig. The Src family protein tyrosine kinases p53/56lyn, p59fyn, and p56lck are associated with membrane Ig in spleen B cells and B-cell lines and undergo phosphorylation in vitro. The pattern of expression of Src family protein tyrosine kinases in B cells varied. Our studies suggest that multiple kinases can potentially interact with membrane Ig and that within any one B-cell type, all of the Src family kinases expressed can be found in association with membrane Ig. We also observed that the Ig-associated Ig alpha protein, multiple forms of Ig beta, and proteins of 100 and 25 kDa were tyrosine phosphorylated in vitro. The 100- and 25-kDa proteins remain unidentified.

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OC.13.1 IN VITRO CHARACTERIZATION OF CDH1 VARIANTS FIRST BY IMMORTALIZATION OF PERIPHERAL B-CELLS OF PATIENTS

Digestive and Liver Disease ◽

10.1016/s1590-8658(14)60082-7 ◽

2014 ◽

Vol 46 ◽

pp. S30

Author(s):

L. Caggiari ◽

M. De Zorzi ◽

P. De Paoli ◽

V. Canzonieri ◽

S. Maiero ◽

...

Keyword(s):

B Cells ◽

In Vitro Characterization ◽

Peripheral B Cells

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p56lck interacts via its src homology 2 domain with the ZAP-70 kinase.

Journal of Experimental Medicine ◽

10.1084/jem.179.4.1163 ◽

1994 ◽

Vol 179 (4) ◽

pp. 1163-1172 ◽

Cited By ~ 105

Author(s):

P Duplay ◽

M Thome ◽

F Hervé ◽

O Acuto

Keyword(s):

Tyrosine Kinases ◽

Intracellular Signaling ◽

Specific Binding ◽

Sh2 Domain ◽

Jurkat Cells ◽

Vitro Assay ◽

Phosphorylated Proteins ◽

Src Homology 2 ◽

Src Homology

p56lck, a member of the src family of protein tyrosine kinases, is an essential component in T cell receptor (TCR) signal transduction. p56lck contains a src homology 2 (SH2) domain found in a number of proteins involved in intracellular signaling. SH2 domains have been implicated in protein-protein interactions by binding to sequences in target proteins containing phosphorylated tyrosine. Using an in vitro assay, we have studied specific binding of tyrosine-phosphorylated proteins to a recombinant p56lck SH2 domain. In nonactivated Jurkat cells, two tyrosine-phosphorylated proteins were detected. Stimulation with anti-CD3 monoclonal antibodies induced the binding of seven additional tyrosine-phosphorylated proteins to the SH2 domain of p56lck. We have identified the zeta-associated tyrosine kinase, ZAP-70, as one of these proteins. Evidence suggests that binding of ZAP-70 to p56lck SH2 is direct and not mediated by zeta. The significance of this interaction was further investigated in vivo. p56lck could be coprecipitated with the zeta/ZAP-70 complex and conversely, ZAP-70 was detected in p56lck immunoprecipitates of activated Jurkat cells. The physical association of p56lck and ZAP-70 during activation supports the recently proposed functional cooperation of these two tyrosine kinases in TCR signaling.

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Cloning and Characterization of Human Lnk, an Adaptor Protein with Pleckstrin Homology and Src Homology 2 Domains that Can Inhibit T Cell Activation

The Journal of Immunology ◽

10.4049/jimmunol.164.10.5199 ◽

2000 ◽

Vol 164 (10) ◽

pp. 5199-5206 ◽

Cited By ~ 75

Author(s):

Yijin Li ◽

Xiaoqing He ◽

Josephine Schembri-King ◽

Scott Jakes ◽

Jun Hayashi

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Adaptor Protein ◽

Pleckstrin Homology ◽

Src Homology 2 ◽

Src Homology

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In Vitro Characterization of Human CD24hiCD38hi Regulatory B Cells Shows CD9 Is Not a Stable Breg Cell Marker

International Journal of Molecular Sciences ◽

10.3390/ijms22094583 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4583

Author(s):

Fatin N. Mohd Jaya ◽

Sergio G. Garcia ◽

Francesc E. Borras ◽

Dolores Guerrero ◽

Godfrey C. F. Chan ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Activation ◽

B Cell Activation ◽

Functional Studies ◽

In Vitro Characterization ◽

Reliable Marker ◽

The Stability

Regulatory B (Breg) cells are endowed with immune suppressive functions. Various human and murine Breg subtypes have been reported. While interleukin (IL)-10 intracellular staining remains the most reliable way to identify Breg cells, this technique hinders further essential functional studies. Recent findings suggest that CD9 is an effective surface marker of murine IL-10 competent Breg cells. However, the stability of CD9 and its relevance as a unique marker for human Breg cells, which have been widely characterized as CD24hiCD38hi, have not been investigated. Here, we demonstrate that CD9 expression is sensitive to in vitro B cell stimulations. CD9 expression could either be re-expressed or downregulated in purified CD9-negative B cells and CD9-positive B cells, respectively. We found no significant differences in the Breg differentiation capacity of the CD9-negative and CD9-positive B cells. Furthermore, CD9-positive B cells co-express CD40 and CD86, suggesting their nature as B cell activation or co-stimulatory molecules, rather than regulatory ones. Therefore, we report the relatively unstable CD9 as a distinct surface molecule, indicating the need for further research for a more reliable marker to purify human Breg cells.

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Rapid and efficient purification of Src homology 2 domain-containing proteins: Fyn, Csk and phosphatidylinositol 3-kinase p85

Biochemical Journal ◽

10.1042/bj3020737 ◽

1994 ◽

Vol 302 (3) ◽

pp. 737-744 ◽

Cited By ~ 24

Author(s):

M Koegl ◽

R M Kypta ◽

M Bergman ◽

K Alitalo ◽

S A Courtneidge

Keyword(s):

Tyrosine Kinases ◽

Intramolecular Interaction ◽

Affinity Purification ◽

Sh2 Domain ◽

Exchange Chromatography ◽

Phosphatidylinositol 3 Kinase ◽

Src Homology 2 ◽

Src Homology ◽

Phosphatidylinositol 3

To analyse the regulation of Src family tyrosine kinases in vitro, we have purified Fyn and Csk, a kinase capable of regulating Fyn activity by phosphorylation, from baculovirus-infected insect cells. The proteins were purified by affinity purification over a phosphotyrosine column. Highly purified proteins were eluted from the resin by a salt gradient and further purified by ion-exchange chromatography. This purification scheme was successfully applied to a third, unrelated protein that also contains the Src homology 2 (SH2) domain, namely the 85 kDa subunit of phosphatidylinositol 3-kinase, indicating that this method is versatile and should prove applicable to any protein with an accessible SH2 domain. The binding of Csk to different phosphopeptides was tested, and specificity for the autophosphorylation site of Fyn was demonstrated. Pure Csk was used to phosphorylate Fyn and down-regulate its kinase activity, and the kinetic parameters of both the active and the repressed forms of Fyn were determined. Repression of Fyn activity by Csk reduced binding of Fyn to phosphopeptides to undetectable levels, supporting the model that predicts an intramolecular interaction of the Fyn SH2 domain with a C-terminal phosphotyrosine residue.

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