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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 translocates to the TCR complex upon anti-CD3 stimulation. (A) Expression of GFP-Unc119 fusion protein. Jurkat cells were stably transfected with pLEGFP-N1 containing cDNA for GFP alone (1) or GFP-Unc119 fusion protein (2) and examined by fluorescence microscopy. (B) Unc119 translocates to CD3 upon stimulation. GFP-Unc119 (1, 2, and 3) or GFP (4) Jurkat cells were incubated with anti-CD3 (1 and 4) or anti-CD28 (2) or isotype-control antibody (3)–coated protein A/G agarose beads, fixed, and analyzed by fluorescence and light (inset) microscopy. (C) Unc119 translocates to CD4 upon stimulation. Receptors of GFP-IRIP (1–6) or GFP (7–9) Jurkat cells were capped with anti-CD4 (1–3, 7–9), anti-CD28 (4–6), or isotype controls (not depicted) and examined by fluorescence or light (inset) microscopy. (left) GFP/GFP-IRIP fluorescence. (middle) Rhodamine (CD4 or CD28) fluorescence. (right) Color overlay.
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fig2: Unc119 translocates to the TCR complex upon anti-CD3 stimulation. (A) Expression of GFP-Unc119 fusion protein. Jurkat cells were stably transfected with pLEGFP-N1 containing cDNA for GFP alone (1) or GFP-Unc119 fusion protein (2) and examined by fluorescence microscopy. (B) Unc119 translocates to CD3 upon stimulation. GFP-Unc119 (1, 2, and 3) or GFP (4) Jurkat cells were incubated with anti-CD3 (1 and 4) or anti-CD28 (2) or isotype-control antibody (3)–coated protein A/G agarose beads, fixed, and analyzed by fluorescence and light (inset) microscopy. (C) Unc119 translocates to CD4 upon stimulation. Receptors of GFP-IRIP (1–6) or GFP (7–9) Jurkat cells were capped with anti-CD4 (1–3, 7–9), anti-CD28 (4–6), or isotype controls (not depicted) and examined by fluorescence or light (inset) microscopy. (left) GFP/GFP-IRIP fluorescence. (middle) Rhodamine (CD4 or CD28) fluorescence. (right) Color overlay.

Mentions: In translocation experiments, GFP or GFP-Unc119-expressing Jurkat cells, prepared as aforementioned, were mixed with anti-CD3, anti-CD28, or isotype-control antibody-coated protein A/G agarose beads and incubated at 37°C for 1–5 min. The bead–cell conjugates were fixed in 1% paraformaldehyde and examined under the microscope. Capping experiments were performed as described previously (17). For each antibody (bead or capping test), three independent experiments were performed, and 50–70 cells (or bead–cell conjugates) were examined per experiment. More than 85% of cells demonstrated interaction with bead or capping and >75% of bead-bound or capped cells showed GFP/GFP-IRIP localization similar to that shown in Fig. 2 (B and C), respectively.


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 translocates to the TCR complex upon anti-CD3 stimulation. (A) Expression of GFP-Unc119 fusion protein. Jurkat cells were stably transfected with pLEGFP-N1 containing cDNA for GFP alone (1) or GFP-Unc119 fusion protein (2) and examined by fluorescence microscopy. (B) Unc119 translocates to CD3 upon stimulation. GFP-Unc119 (1, 2, and 3) or GFP (4) Jurkat cells were incubated with anti-CD3 (1 and 4) or anti-CD28 (2) or isotype-control antibody (3)–coated protein A/G agarose beads, fixed, and analyzed by fluorescence and light (inset) microscopy. (C) Unc119 translocates to CD4 upon stimulation. Receptors of GFP-IRIP (1–6) or GFP (7–9) Jurkat cells were capped with anti-CD4 (1–3, 7–9), anti-CD28 (4–6), or isotype controls (not depicted) and examined by fluorescence or light (inset) microscopy. (left) GFP/GFP-IRIP fluorescence. (middle) Rhodamine (CD4 or CD28) fluorescence. (right) Color overlay.
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Related In: Results  -  Collection

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fig2: Unc119 translocates to the TCR complex upon anti-CD3 stimulation. (A) Expression of GFP-Unc119 fusion protein. Jurkat cells were stably transfected with pLEGFP-N1 containing cDNA for GFP alone (1) or GFP-Unc119 fusion protein (2) and examined by fluorescence microscopy. (B) Unc119 translocates to CD3 upon stimulation. GFP-Unc119 (1, 2, and 3) or GFP (4) Jurkat cells were incubated with anti-CD3 (1 and 4) or anti-CD28 (2) or isotype-control antibody (3)–coated protein A/G agarose beads, fixed, and analyzed by fluorescence and light (inset) microscopy. (C) Unc119 translocates to CD4 upon stimulation. Receptors of GFP-IRIP (1–6) or GFP (7–9) Jurkat cells were capped with anti-CD4 (1–3, 7–9), anti-CD28 (4–6), or isotype controls (not depicted) and examined by fluorescence or light (inset) microscopy. (left) GFP/GFP-IRIP fluorescence. (middle) Rhodamine (CD4 or CD28) fluorescence. (right) Color overlay.
Mentions: In translocation experiments, GFP or GFP-Unc119-expressing Jurkat cells, prepared as aforementioned, were mixed with anti-CD3, anti-CD28, or isotype-control antibody-coated protein A/G agarose beads and incubated at 37°C for 1–5 min. The bead–cell conjugates were fixed in 1% paraformaldehyde and examined under the microscope. Capping experiments were performed as described previously (17). For each antibody (bead or capping test), three independent experiments were performed, and 50–70 cells (or bead–cell conjugates) were examined per experiment. More than 85% of cells demonstrated interaction with bead or capping and >75% of bead-bound or capped cells showed GFP/GFP-IRIP localization similar to that shown in Fig. 2 (B and C), respectively.

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