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Unc119, a novel activator of Lck/Fyn, is essential for T cell activation.

Gorska MM, Stafford SJ, Cen O, Sur S, Alam R - J. Exp. Med. (2004)

Bottom Line: Reconstitution of cells with Unc119 reverses the signaling and functional outcome.Thus, Unc119 is a receptor-associated activator of Src-type kinases.It provides a novel mechanism of signal generation in the TCR complex.

View Article: PubMed Central - PubMed

Affiliation: Division of Allergy and Immunology, Department of Internal Medicine, University of Texas Medical Branch, Galveston 77555, USA.

ABSTRACT
The first step in T cell receptor for antigen (TCR) signaling is the activation of the receptor-bound Src kinases, Lck and Fyn. The exact mechanism of this process is unknown. Here, we report that the novel Src homology (SH) 3/SH2 ligand-Uncoordinated 119 (Unc119) associates with CD3 and CD4, and activates Lck and Fyn. Unc119 overexpression increases Lck/Fyn activity in T cells. In Unc119-deficient T cells, Lck/Fyn activity is dramatically reduced with concomitant decrease in interleukin 2 production and cellular proliferation. Reconstitution of cells with Unc119 reverses the signaling and functional outcome. Thus, Unc119 is a receptor-associated activator of Src-type kinases. It provides a novel mechanism of signal generation in the TCR complex.

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Unc119 interacts with the TCR complex. (A) Unc119 is expressed in human primary T cells and the Jurkat T cell line. Cell lysates were Western blotted with anti-Unc119 (left) or anti-Unc119 antibody–depleted (right) serum. Recombinant Unc119 (rUnc119) was electrophoresed to mark its position. NS, nonspecific band. (B) Unc119 specifically coprecipitates with CD3 in T cells. Human primary T cells were incubated with (+) or without (−) anti-CD3 and lysed. As a control, the anti-CD3 antibody was added to unstimulated sample after lysis. Lysates were immunoprecipitated (IP) with anti-Unc119 and Western blotted with antibodies against indicated molecules. The membranes were reprobed with anti-Unc119 to demonstrate equal protein loading (bottom). (Pellet) Immunoprecipitated proteins. (Supernatant) Cell lysate after removal of immunoprecipitated proteins. (C) Unc119 coprecipitates with CD4 in T cells. T cells, prepared as in B, were immunoprecipitated (IP) with indicated antibodies. Pellets and/or supernatants (B) were Western blotted with anti-Unc119. The membrane (left) was reprobed with anti-CD4. (D) GST-Unc119 interacts with CD3 and CD4. T cells, prepared as in B, were subjected to GST or GST-Unc119 pulldown. Pellets and/or supernatants (B) were Western blotted with antibodies against indicated molecules. Membranes from all experiments were reprobed with an anti-GST antibody. A representative blot is shown (bottom left).
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fig1: Unc119 interacts with the TCR complex. (A) Unc119 is expressed in human primary T cells and the Jurkat T cell line. Cell lysates were Western blotted with anti-Unc119 (left) or anti-Unc119 antibody–depleted (right) serum. Recombinant Unc119 (rUnc119) was electrophoresed to mark its position. NS, nonspecific band. (B) Unc119 specifically coprecipitates with CD3 in T cells. Human primary T cells were incubated with (+) or without (−) anti-CD3 and lysed. As a control, the anti-CD3 antibody was added to unstimulated sample after lysis. Lysates were immunoprecipitated (IP) with anti-Unc119 and Western blotted with antibodies against indicated molecules. The membranes were reprobed with anti-Unc119 to demonstrate equal protein loading (bottom). (Pellet) Immunoprecipitated proteins. (Supernatant) Cell lysate after removal of immunoprecipitated proteins. (C) Unc119 coprecipitates with CD4 in T cells. T cells, prepared as in B, were immunoprecipitated (IP) with indicated antibodies. Pellets and/or supernatants (B) were Western blotted with anti-Unc119. The membrane (left) was reprobed with anti-CD4. (D) GST-Unc119 interacts with CD3 and CD4. T cells, prepared as in B, were subjected to GST or GST-Unc119 pulldown. Pellets and/or supernatants (B) were Western blotted with antibodies against indicated molecules. Membranes from all experiments were reprobed with an anti-GST antibody. A representative blot is shown (bottom left).

Mentions: We have shown previously that Unc119 activates Lyn kinase in eosinophils (11). To elucidate its function in other hematopoietic cells, we investigated the presence of Unc119 in human blood T cells and Jurkat T cells through Western blotting (Fig. 1 A). Sequence analysis predicts that Unc119 has a mass of ∼37–39 kD. We observed the appearance of three bands (one prominent and two faint, in this molecular weight range). The prominent middle band migrated in the gel in the same position as the recombinant Unc119. Furthermore, the anti-Unc119–depleted serum failed to detect the middle band and rUnc119 on the blot, whereas other bands were still present, suggesting that the middle band was indeed Unc119. Our results suggest that Unc119 is present in T cells.


Unc119, a novel activator of Lck/Fyn, is essential for T cell activation.

Gorska MM, Stafford SJ, Cen O, Sur S, Alam R - J. Exp. Med. (2004)

Unc119 interacts with the TCR complex. (A) Unc119 is expressed in human primary T cells and the Jurkat T cell line. Cell lysates were Western blotted with anti-Unc119 (left) or anti-Unc119 antibody–depleted (right) serum. Recombinant Unc119 (rUnc119) was electrophoresed to mark its position. NS, nonspecific band. (B) Unc119 specifically coprecipitates with CD3 in T cells. Human primary T cells were incubated with (+) or without (−) anti-CD3 and lysed. As a control, the anti-CD3 antibody was added to unstimulated sample after lysis. Lysates were immunoprecipitated (IP) with anti-Unc119 and Western blotted with antibodies against indicated molecules. The membranes were reprobed with anti-Unc119 to demonstrate equal protein loading (bottom). (Pellet) Immunoprecipitated proteins. (Supernatant) Cell lysate after removal of immunoprecipitated proteins. (C) Unc119 coprecipitates with CD4 in T cells. T cells, prepared as in B, were immunoprecipitated (IP) with indicated antibodies. Pellets and/or supernatants (B) were Western blotted with anti-Unc119. The membrane (left) was reprobed with anti-CD4. (D) GST-Unc119 interacts with CD3 and CD4. T cells, prepared as in B, were subjected to GST or GST-Unc119 pulldown. Pellets and/or supernatants (B) were Western blotted with antibodies against indicated molecules. Membranes from all experiments were reprobed with an anti-GST antibody. A representative blot is shown (bottom left).
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fig1: Unc119 interacts with the TCR complex. (A) Unc119 is expressed in human primary T cells and the Jurkat T cell line. Cell lysates were Western blotted with anti-Unc119 (left) or anti-Unc119 antibody–depleted (right) serum. Recombinant Unc119 (rUnc119) was electrophoresed to mark its position. NS, nonspecific band. (B) Unc119 specifically coprecipitates with CD3 in T cells. Human primary T cells were incubated with (+) or without (−) anti-CD3 and lysed. As a control, the anti-CD3 antibody was added to unstimulated sample after lysis. Lysates were immunoprecipitated (IP) with anti-Unc119 and Western blotted with antibodies against indicated molecules. The membranes were reprobed with anti-Unc119 to demonstrate equal protein loading (bottom). (Pellet) Immunoprecipitated proteins. (Supernatant) Cell lysate after removal of immunoprecipitated proteins. (C) Unc119 coprecipitates with CD4 in T cells. T cells, prepared as in B, were immunoprecipitated (IP) with indicated antibodies. Pellets and/or supernatants (B) were Western blotted with anti-Unc119. The membrane (left) was reprobed with anti-CD4. (D) GST-Unc119 interacts with CD3 and CD4. T cells, prepared as in B, were subjected to GST or GST-Unc119 pulldown. Pellets and/or supernatants (B) were Western blotted with antibodies against indicated molecules. Membranes from all experiments were reprobed with an anti-GST antibody. A representative blot is shown (bottom left).
Mentions: We have shown previously that Unc119 activates Lyn kinase in eosinophils (11). To elucidate its function in other hematopoietic cells, we investigated the presence of Unc119 in human blood T cells and Jurkat T cells through Western blotting (Fig. 1 A). Sequence analysis predicts that Unc119 has a mass of ∼37–39 kD. We observed the appearance of three bands (one prominent and two faint, in this molecular weight range). The prominent middle band migrated in the gel in the same position as the recombinant Unc119. Furthermore, the anti-Unc119–depleted serum failed to detect the middle band and rUnc119 on the blot, whereas other bands were still present, suggesting that the middle band was indeed Unc119. Our results suggest that Unc119 is present in T cells.

Bottom Line: Reconstitution of cells with Unc119 reverses the signaling and functional outcome.Thus, Unc119 is a receptor-associated activator of Src-type kinases.It provides a novel mechanism of signal generation in the TCR complex.

View Article: PubMed Central - PubMed

Affiliation: Division of Allergy and Immunology, Department of Internal Medicine, University of Texas Medical Branch, Galveston 77555, USA.

ABSTRACT
The first step in T cell receptor for antigen (TCR) signaling is the activation of the receptor-bound Src kinases, Lck and Fyn. The exact mechanism of this process is unknown. Here, we report that the novel Src homology (SH) 3/SH2 ligand-Uncoordinated 119 (Unc119) associates with CD3 and CD4, and activates Lck and Fyn. Unc119 overexpression increases Lck/Fyn activity in T cells. In Unc119-deficient T cells, Lck/Fyn activity is dramatically reduced with concomitant decrease in interleukin 2 production and cellular proliferation. Reconstitution of cells with Unc119 reverses the signaling and functional outcome. Thus, Unc119 is a receptor-associated activator of Src-type kinases. It provides a novel mechanism of signal generation in the TCR complex.

Show MeSH
Related in: MedlinePlus