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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 binds and activates Lck through the SH3 motif. (A) An SH3 motif peptide from Unc119 activates Lck. Lck immunoprecipitates (IP) were assayed for kinase activity as in Fig. 4 A in the presence of the SH3 motif (left) or mutated peptide (right) from Unc119 (11). Membranes were Western blotted (bottom) with a monoclonal anti-Lck antibody. (B) Expression of a SH3 motif mutant in Jurkat cells. Jurkat cells (left, single transfection experiment) or the Unc119-deficient Jurkat clone (right, reconstitution experiment) were infected with the GFP-RV empty vector (empty) or the SH3 motif mutant of Unc119-FLAG-GFP-RV vector (mut) or the Unc119-FLAG-GFP-RV vector (wt). As an additional control, the empty vector clone was infected with GFP-RV. GFP+ cells (left and right) were sorted, Western blotted (top) with anti-FLAG antibody, and reprobed with antitubulin (bottom). (C) The SH3 motif mutant of Unc119 does not coprecipitate with Lck. GFP+ cells (B, single transfection) were incubated with (+) or without (−) anti-CD3, lysed, and immunoprecipitated with anti-Lck. Pellets and supernatants (Fig. 1 B) were Western blotted with anti-FLAG (top) and reprobed with anti-Lck (bottom). (D) The SH3 motif mutant inhibits Lck activation. GFP+ cells (B, single transfection) were treated as in C, analyzed for kinase activity as in Fig. 4 A, autoradiographed (top), and Western blotted with anti-Lck antibody (bottom). (E) The SH3 motif mutant is unable to rescue Lck activity in Unc119-deficient cells. GFP+ cells (B, reconstitution) were incubated with (+) or without (−) anti-CD3 antibody, analyzed for kinase activity as in Fig. 4 A, autoradiographed (top), and Western blotted with anti-Lck antibody (bottom).
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fig8: Unc119 binds and activates Lck through the SH3 motif. (A) An SH3 motif peptide from Unc119 activates Lck. Lck immunoprecipitates (IP) were assayed for kinase activity as in Fig. 4 A in the presence of the SH3 motif (left) or mutated peptide (right) from Unc119 (11). Membranes were Western blotted (bottom) with a monoclonal anti-Lck antibody. (B) Expression of a SH3 motif mutant in Jurkat cells. Jurkat cells (left, single transfection experiment) or the Unc119-deficient Jurkat clone (right, reconstitution experiment) were infected with the GFP-RV empty vector (empty) or the SH3 motif mutant of Unc119-FLAG-GFP-RV vector (mut) or the Unc119-FLAG-GFP-RV vector (wt). As an additional control, the empty vector clone was infected with GFP-RV. GFP+ cells (left and right) were sorted, Western blotted (top) with anti-FLAG antibody, and reprobed with antitubulin (bottom). (C) The SH3 motif mutant of Unc119 does not coprecipitate with Lck. GFP+ cells (B, single transfection) were incubated with (+) or without (−) anti-CD3, lysed, and immunoprecipitated with anti-Lck. Pellets and supernatants (Fig. 1 B) were Western blotted with anti-FLAG (top) and reprobed with anti-Lck (bottom). (D) The SH3 motif mutant inhibits Lck activation. GFP+ cells (B, single transfection) were treated as in C, analyzed for kinase activity as in Fig. 4 A, autoradiographed (top), and Western blotted with anti-Lck antibody (bottom). (E) The SH3 motif mutant is unable to rescue Lck activity in Unc119-deficient cells. GFP+ cells (B, reconstitution) were incubated with (+) or without (−) anti-CD3 antibody, analyzed for kinase activity as in Fig. 4 A, autoradiographed (top), and Western blotted with anti-Lck antibody (bottom).

Mentions: Previously, we reported activation of Lyn kinase by a SH3 motif peptide and to a lesser extent SH2 and phospho-SH2 peptides from Unc119 (11). We believe that Unc119 activation of Src kinases is mainly mediated through its interaction with the SH3 domain. In this paper, we examined the activation of Lck by the SH3 motif peptide from Unc119 (Fig. 8 A, left). To study the importance of motif-related conserved residues, we designed another peptide in which critical proline and arginine residues of the motif peptide were substituted with alanine (Fig. 8 A, right). The SH3 peptide, but not mutated peptide, activated Lck at a concentration as low as 0.01 μM. We obtained similar results for Fyn kinase (unpublished data).


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 binds and activates Lck through the SH3 motif. (A) An SH3 motif peptide from Unc119 activates Lck. Lck immunoprecipitates (IP) were assayed for kinase activity as in Fig. 4 A in the presence of the SH3 motif (left) or mutated peptide (right) from Unc119 (11). Membranes were Western blotted (bottom) with a monoclonal anti-Lck antibody. (B) Expression of a SH3 motif mutant in Jurkat cells. Jurkat cells (left, single transfection experiment) or the Unc119-deficient Jurkat clone (right, reconstitution experiment) were infected with the GFP-RV empty vector (empty) or the SH3 motif mutant of Unc119-FLAG-GFP-RV vector (mut) or the Unc119-FLAG-GFP-RV vector (wt). As an additional control, the empty vector clone was infected with GFP-RV. GFP+ cells (left and right) were sorted, Western blotted (top) with anti-FLAG antibody, and reprobed with antitubulin (bottom). (C) The SH3 motif mutant of Unc119 does not coprecipitate with Lck. GFP+ cells (B, single transfection) were incubated with (+) or without (−) anti-CD3, lysed, and immunoprecipitated with anti-Lck. Pellets and supernatants (Fig. 1 B) were Western blotted with anti-FLAG (top) and reprobed with anti-Lck (bottom). (D) The SH3 motif mutant inhibits Lck activation. GFP+ cells (B, single transfection) were treated as in C, analyzed for kinase activity as in Fig. 4 A, autoradiographed (top), and Western blotted with anti-Lck antibody (bottom). (E) The SH3 motif mutant is unable to rescue Lck activity in Unc119-deficient cells. GFP+ cells (B, reconstitution) were incubated with (+) or without (−) anti-CD3 antibody, analyzed for kinase activity as in Fig. 4 A, autoradiographed (top), and Western blotted with anti-Lck antibody (bottom).
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fig8: Unc119 binds and activates Lck through the SH3 motif. (A) An SH3 motif peptide from Unc119 activates Lck. Lck immunoprecipitates (IP) were assayed for kinase activity as in Fig. 4 A in the presence of the SH3 motif (left) or mutated peptide (right) from Unc119 (11). Membranes were Western blotted (bottom) with a monoclonal anti-Lck antibody. (B) Expression of a SH3 motif mutant in Jurkat cells. Jurkat cells (left, single transfection experiment) or the Unc119-deficient Jurkat clone (right, reconstitution experiment) were infected with the GFP-RV empty vector (empty) or the SH3 motif mutant of Unc119-FLAG-GFP-RV vector (mut) or the Unc119-FLAG-GFP-RV vector (wt). As an additional control, the empty vector clone was infected with GFP-RV. GFP+ cells (left and right) were sorted, Western blotted (top) with anti-FLAG antibody, and reprobed with antitubulin (bottom). (C) The SH3 motif mutant of Unc119 does not coprecipitate with Lck. GFP+ cells (B, single transfection) were incubated with (+) or without (−) anti-CD3, lysed, and immunoprecipitated with anti-Lck. Pellets and supernatants (Fig. 1 B) were Western blotted with anti-FLAG (top) and reprobed with anti-Lck (bottom). (D) The SH3 motif mutant inhibits Lck activation. GFP+ cells (B, single transfection) were treated as in C, analyzed for kinase activity as in Fig. 4 A, autoradiographed (top), and Western blotted with anti-Lck antibody (bottom). (E) The SH3 motif mutant is unable to rescue Lck activity in Unc119-deficient cells. GFP+ cells (B, reconstitution) were incubated with (+) or without (−) anti-CD3 antibody, analyzed for kinase activity as in Fig. 4 A, autoradiographed (top), and Western blotted with anti-Lck antibody (bottom).
Mentions: Previously, we reported activation of Lyn kinase by a SH3 motif peptide and to a lesser extent SH2 and phospho-SH2 peptides from Unc119 (11). We believe that Unc119 activation of Src kinases is mainly mediated through its interaction with the SH3 domain. In this paper, we examined the activation of Lck by the SH3 motif peptide from Unc119 (Fig. 8 A, left). To study the importance of motif-related conserved residues, we designed another peptide in which critical proline and arginine residues of the motif peptide were substituted with alanine (Fig. 8 A, right). The SH3 peptide, but not mutated peptide, activated Lck at a concentration as low as 0.01 μM. We obtained similar results for Fyn kinase (unpublished data).

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