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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 specifically interacts with Lck and Fyn. (A) Unc119 specifically interacts with Lck in T cells. T cells were prepared as in Fig. 1 B, immunoprecipitated (IP) with indicated antibodies. Pellets and/or supernatants (see Fig. 1 B) were Western blotted with anti-Unc119 antibody and reprobed with anti-Lck. NS, nonspecific band. (B) Unc119 interacts with Fyn in T cells. T cells were prepared as in Fig. 1 B and immunoprecipitated (IP) with anti-Unc119. Pellets and/or supernatants (Fig. 1 B) were Western blotted with antibodies against indicated proteins and reprobed with anti-Unc119 (bottom). (C) GST-Unc119 interacts with Lck and Fyn. Cells lysates, prepared as aforementioned, were precipitated with GST or GST-Unc119. Pellets and/or supernatants were Western blotted with antibodies against indicated proteins.
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fig3: Unc119 specifically interacts with Lck and Fyn. (A) Unc119 specifically interacts with Lck in T cells. T cells were prepared as in Fig. 1 B, immunoprecipitated (IP) with indicated antibodies. Pellets and/or supernatants (see Fig. 1 B) were Western blotted with anti-Unc119 antibody and reprobed with anti-Lck. NS, nonspecific band. (B) Unc119 interacts with Fyn in T cells. T cells were prepared as in Fig. 1 B and immunoprecipitated (IP) with anti-Unc119. Pellets and/or supernatants (Fig. 1 B) were Western blotted with antibodies against indicated proteins and reprobed with anti-Unc119 (bottom). (C) GST-Unc119 interacts with Lck and Fyn. Cells lysates, prepared as aforementioned, were precipitated with GST or GST-Unc119. Pellets and/or supernatants were Western blotted with antibodies against indicated proteins.

Mentions: CD3 and CD4 are known to interact with Fyn and Lck tyrosine kinases, respectively. We examined the association of Unc119 with Lck and Fyn. Coprecipitation (Fig. 3, A and B, left) and GST-Unc119 pulldown (Fig. 3 C, left) experiments with primary T cells showed the recruitment of Unc119 to Lck and Fyn upon anti-CD3 stimulation. We observed a low level of interaction between GST-Unc119 and the kinases in pulldown experiments with the unstimulated cell lysate. We believe that it is due to the excess of the recombinant protein used. Like other members of the Src-type kinases, Lck and Fyn have SH2 and SH3 domains in their sequence. We have shown previously that Unc119 interacts with the SH2 and SH3 domains of Lyn and Fyn (11). To determine specificity of this interaction, we examined the association of Unc119 with other SH2- and/or SH3-containing kinases as follows: p85 of phosphatidyl inositol-3 kinase (Fig. 3 A, right) and ZAP70 and Itk (Fig. 3 B, right) in coprecipitation experiments. ZAP70 as well as Abl, FAK, p85 subunit of phosphatidyl inositol-3 kinase, and the serine threonine kinases Raf-1, ERK2, and p38 MAPK were studied in GST-Unc119 pulldown experiments (Fig. 3 C, right). Unc119 and GST-Unc119 did not precipitate the foregoing kinases (pellet). However, our antibodies detected the kinases in the supernatant fraction. Previously, we reported that Unc119 did not interact with the SH2/SH3 adaptor proteins Grb2 and Shc (11). The results suggest that the interaction of Unc119 is relatively specific for the SH3/SH2 domains of Src family kinases.


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 specifically interacts with Lck and Fyn. (A) Unc119 specifically interacts with Lck in T cells. T cells were prepared as in Fig. 1 B, immunoprecipitated (IP) with indicated antibodies. Pellets and/or supernatants (see Fig. 1 B) were Western blotted with anti-Unc119 antibody and reprobed with anti-Lck. NS, nonspecific band. (B) Unc119 interacts with Fyn in T cells. T cells were prepared as in Fig. 1 B and immunoprecipitated (IP) with anti-Unc119. Pellets and/or supernatants (Fig. 1 B) were Western blotted with antibodies against indicated proteins and reprobed with anti-Unc119 (bottom). (C) GST-Unc119 interacts with Lck and Fyn. Cells lysates, prepared as aforementioned, were precipitated with GST or GST-Unc119. Pellets and/or supernatants were Western blotted with antibodies against indicated proteins.
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Related In: Results  -  Collection

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fig3: Unc119 specifically interacts with Lck and Fyn. (A) Unc119 specifically interacts with Lck in T cells. T cells were prepared as in Fig. 1 B, immunoprecipitated (IP) with indicated antibodies. Pellets and/or supernatants (see Fig. 1 B) were Western blotted with anti-Unc119 antibody and reprobed with anti-Lck. NS, nonspecific band. (B) Unc119 interacts with Fyn in T cells. T cells were prepared as in Fig. 1 B and immunoprecipitated (IP) with anti-Unc119. Pellets and/or supernatants (Fig. 1 B) were Western blotted with antibodies against indicated proteins and reprobed with anti-Unc119 (bottom). (C) GST-Unc119 interacts with Lck and Fyn. Cells lysates, prepared as aforementioned, were precipitated with GST or GST-Unc119. Pellets and/or supernatants were Western blotted with antibodies against indicated proteins.
Mentions: CD3 and CD4 are known to interact with Fyn and Lck tyrosine kinases, respectively. We examined the association of Unc119 with Lck and Fyn. Coprecipitation (Fig. 3, A and B, left) and GST-Unc119 pulldown (Fig. 3 C, left) experiments with primary T cells showed the recruitment of Unc119 to Lck and Fyn upon anti-CD3 stimulation. We observed a low level of interaction between GST-Unc119 and the kinases in pulldown experiments with the unstimulated cell lysate. We believe that it is due to the excess of the recombinant protein used. Like other members of the Src-type kinases, Lck and Fyn have SH2 and SH3 domains in their sequence. We have shown previously that Unc119 interacts with the SH2 and SH3 domains of Lyn and Fyn (11). To determine specificity of this interaction, we examined the association of Unc119 with other SH2- and/or SH3-containing kinases as follows: p85 of phosphatidyl inositol-3 kinase (Fig. 3 A, right) and ZAP70 and Itk (Fig. 3 B, right) in coprecipitation experiments. ZAP70 as well as Abl, FAK, p85 subunit of phosphatidyl inositol-3 kinase, and the serine threonine kinases Raf-1, ERK2, and p38 MAPK were studied in GST-Unc119 pulldown experiments (Fig. 3 C, right). Unc119 and GST-Unc119 did not precipitate the foregoing kinases (pellet). However, our antibodies detected the kinases in the supernatant fraction. Previously, we reported that Unc119 did not interact with the SH2/SH3 adaptor proteins Grb2 and Shc (11). The results suggest that the interaction of Unc119 is relatively specific for the SH3/SH2 domains of Src family kinases.

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