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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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Lck/Fyn activity in Unc119-deficient cells. (A) Unc119 level in antisense Unc119 cDNA-expressing T cells. Cells were transfected retrovirally with empty vectors (GFP-RV for primary T cells and pMSCV for Jurkat cells) or same vectors containing Unc119 antisense cDNA (antisense). GFP+ primary T cells were sorted by flow cytometry and infected Jurkat cells were selected and cloned on G418. The presence of Unc119 (Unc), CD3ζ, and tubulin was analyzed by Western blotting (left and middle). NS, nonspecific band. CD3 surface expression on Unc119-deficient cells was measured by flow cytometry (right graph, dashed line, isotype control; thin line, empty vector; and thick line, antisense vector). (B) Lck and Fyn activity in Unc119-deficient cells. GFP+ Unc119-deficient primary T cells were immunoprecipitated with rabbit anti-Lck and anti-Fyn, analyzed for kinase activity as in Fig. 4 A, and autoradiographed. Membranes were Western blotted with monoclonal anti-Lck or anti-Fyn antibodies (bottom).
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fig5: Lck/Fyn activity in Unc119-deficient cells. (A) Unc119 level in antisense Unc119 cDNA-expressing T cells. Cells were transfected retrovirally with empty vectors (GFP-RV for primary T cells and pMSCV for Jurkat cells) or same vectors containing Unc119 antisense cDNA (antisense). GFP+ primary T cells were sorted by flow cytometry and infected Jurkat cells were selected and cloned on G418. The presence of Unc119 (Unc), CD3ζ, and tubulin was analyzed by Western blotting (left and middle). NS, nonspecific band. CD3 surface expression on Unc119-deficient cells was measured by flow cytometry (right graph, dashed line, isotype control; thin line, empty vector; and thick line, antisense vector). (B) Lck and Fyn activity in Unc119-deficient cells. GFP+ Unc119-deficient primary T cells were immunoprecipitated with rabbit anti-Lck and anti-Fyn, analyzed for kinase activity as in Fig. 4 A, and autoradiographed. Membranes were Western blotted with monoclonal anti-Lck or anti-Fyn antibodies (bottom).

Mentions: To develop Unc119-deficient T cells, we stably transfected human primary T cells and Jurkat cells with the bicistronic GFP-RV and pMSCV retroviral constructs, respectively, containing the Unc119 antisense cDNA. The antisense construct was created by ligating the Unc119 cDNA into vectors in reverse orientation. After transfection, GFP+ primary T cells were sorted by flow cytometry. Transfected Jurkat cells were selected on G418 and cloned. We have isolated five Unc119-deficient clones. The expression of Unc119 in sorted T cells (Fig. 5 A, left) and Jurkat clones (Fig. 5 A, right, and Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20030589/DC1) was substantially reduced. Reprobing the membrane with antitubulin ruled out the possibility of unequal protein loading (Fig. 5 A and Fig. S1 A, bottom). Unc119-deficient primary T cells and Jurkat clones demonstrated a dramatic reduction in anti-CD3–stimulated Fyn and/or Lck activation (Fig. 5 B, top, and Fig. S1 B, top, respectively). Because the introduction of the antisense vector did not alter total or surface (only slight elevation) CD3 (Fig. 5 A, bottom left and flow cytometry graph), CD4 (not depicted), and Lck and Fyn expression (Fig. 5 B, bottom), the inability of the anti-CD3 antibody to stimulate these kinases is likely due to the decrease in Unc119 concentration. The results strongly suggest that Unc119 is physiologically relevant for Lck and Fyn activation 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)

Lck/Fyn activity in Unc119-deficient cells. (A) Unc119 level in antisense Unc119 cDNA-expressing T cells. Cells were transfected retrovirally with empty vectors (GFP-RV for primary T cells and pMSCV for Jurkat cells) or same vectors containing Unc119 antisense cDNA (antisense). GFP+ primary T cells were sorted by flow cytometry and infected Jurkat cells were selected and cloned on G418. The presence of Unc119 (Unc), CD3ζ, and tubulin was analyzed by Western blotting (left and middle). NS, nonspecific band. CD3 surface expression on Unc119-deficient cells was measured by flow cytometry (right graph, dashed line, isotype control; thin line, empty vector; and thick line, antisense vector). (B) Lck and Fyn activity in Unc119-deficient cells. GFP+ Unc119-deficient primary T cells were immunoprecipitated with rabbit anti-Lck and anti-Fyn, analyzed for kinase activity as in Fig. 4 A, and autoradiographed. Membranes were Western blotted with monoclonal anti-Lck or anti-Fyn antibodies (bottom).
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Related In: Results  -  Collection

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fig5: Lck/Fyn activity in Unc119-deficient cells. (A) Unc119 level in antisense Unc119 cDNA-expressing T cells. Cells were transfected retrovirally with empty vectors (GFP-RV for primary T cells and pMSCV for Jurkat cells) or same vectors containing Unc119 antisense cDNA (antisense). GFP+ primary T cells were sorted by flow cytometry and infected Jurkat cells were selected and cloned on G418. The presence of Unc119 (Unc), CD3ζ, and tubulin was analyzed by Western blotting (left and middle). NS, nonspecific band. CD3 surface expression on Unc119-deficient cells was measured by flow cytometry (right graph, dashed line, isotype control; thin line, empty vector; and thick line, antisense vector). (B) Lck and Fyn activity in Unc119-deficient cells. GFP+ Unc119-deficient primary T cells were immunoprecipitated with rabbit anti-Lck and anti-Fyn, analyzed for kinase activity as in Fig. 4 A, and autoradiographed. Membranes were Western blotted with monoclonal anti-Lck or anti-Fyn antibodies (bottom).
Mentions: To develop Unc119-deficient T cells, we stably transfected human primary T cells and Jurkat cells with the bicistronic GFP-RV and pMSCV retroviral constructs, respectively, containing the Unc119 antisense cDNA. The antisense construct was created by ligating the Unc119 cDNA into vectors in reverse orientation. After transfection, GFP+ primary T cells were sorted by flow cytometry. Transfected Jurkat cells were selected on G418 and cloned. We have isolated five Unc119-deficient clones. The expression of Unc119 in sorted T cells (Fig. 5 A, left) and Jurkat clones (Fig. 5 A, right, and Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20030589/DC1) was substantially reduced. Reprobing the membrane with antitubulin ruled out the possibility of unequal protein loading (Fig. 5 A and Fig. S1 A, bottom). Unc119-deficient primary T cells and Jurkat clones demonstrated a dramatic reduction in anti-CD3–stimulated Fyn and/or Lck activation (Fig. 5 B, top, and Fig. S1 B, top, respectively). Because the introduction of the antisense vector did not alter total or surface (only slight elevation) CD3 (Fig. 5 A, bottom left and flow cytometry graph), CD4 (not depicted), and Lck and Fyn expression (Fig. 5 B, bottom), the inability of the anti-CD3 antibody to stimulate these kinases is likely due to the decrease in Unc119 concentration. The results strongly suggest that Unc119 is physiologically relevant for Lck and Fyn activation 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