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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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Reconstitution of Unc119-deficient Jurkat cells with Unc119. (A) Unc119 level in reconstituted cells. Unc119-deficient Jurkat clones were incubated with purified recombinant Unc119 (U) or human serum albumin (A) in the presence of the protein-transducing reagent Chariot (left area of top and bottom). The right area of the panels shows reconstitution by retroviral infection. Antisense clone was infected with the empty virus (pMSCV) or Unc119 virus (pMSCV-Unc119). An empty vector Jurkat clone was infected with empty virus. Reconstituted cell lysates were Western blotted with anti-Unc119 antibody (top) and reprobed with antitubulin (bottom). (B) Lck activity in Unc119 reconstituted cells. Unc119-deficient cells were reconstituted with Unc119 (U) or albumin (A) in the Chariot experiment and incubated with (+) or without (−) anti-CD3. Samples were analyzed for kinase activity as in Fig. 4 A, autoradiographed, and Western blotted with a monoclonal anti-Lck antibody. (C) Apoptosis in Unc119-deficient cells and the effect of Unc119 reconstitution. Empty vector and antisense Jurkat cells were reconstituted with albumin (left and middle) or Unc119 (right) as in A and B, cultured for 72 h, stained with annexin V–FITC/propidium iodide, and analyzed by flow cytometry. Anti-CD3 stimulation did not influence apoptosis in Unc119-deficient cells (not depicted). (D) Effect of Unc119 reconstitution on IL-2 production of antisense Jurkat cells. Antisense cells were infected with the empty virus (E→A Jurkat) or Unc119 virus (U→A Jurkat) with 30% infection efficiency as in A. The empty vector Jurkat clone was infected with empty virus (E→E Jurkat). Transiently infected cells were incubated on anti-CD3–coated plates (CD3) or left unstimulated (−). IL-2 production was measured as in Fig. 6 E. E→E Jurkat cells produced 30 ± 10 and 240 ± 20 pg/ml, without and with anti-CD3 stimulation, respectively (similar to noninfected cells).
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fig7: Reconstitution of Unc119-deficient Jurkat cells with Unc119. (A) Unc119 level in reconstituted cells. Unc119-deficient Jurkat clones were incubated with purified recombinant Unc119 (U) or human serum albumin (A) in the presence of the protein-transducing reagent Chariot (left area of top and bottom). The right area of the panels shows reconstitution by retroviral infection. Antisense clone was infected with the empty virus (pMSCV) or Unc119 virus (pMSCV-Unc119). An empty vector Jurkat clone was infected with empty virus. Reconstituted cell lysates were Western blotted with anti-Unc119 antibody (top) and reprobed with antitubulin (bottom). (B) Lck activity in Unc119 reconstituted cells. Unc119-deficient cells were reconstituted with Unc119 (U) or albumin (A) in the Chariot experiment and incubated with (+) or without (−) anti-CD3. Samples were analyzed for kinase activity as in Fig. 4 A, autoradiographed, and Western blotted with a monoclonal anti-Lck antibody. (C) Apoptosis in Unc119-deficient cells and the effect of Unc119 reconstitution. Empty vector and antisense Jurkat cells were reconstituted with albumin (left and middle) or Unc119 (right) as in A and B, cultured for 72 h, stained with annexin V–FITC/propidium iodide, and analyzed by flow cytometry. Anti-CD3 stimulation did not influence apoptosis in Unc119-deficient cells (not depicted). (D) Effect of Unc119 reconstitution on IL-2 production of antisense Jurkat cells. Antisense cells were infected with the empty virus (E→A Jurkat) or Unc119 virus (U→A Jurkat) with 30% infection efficiency as in A. The empty vector Jurkat clone was infected with empty virus (E→E Jurkat). Transiently infected cells were incubated on anti-CD3–coated plates (CD3) or left unstimulated (−). IL-2 production was measured as in Fig. 6 E. E→E Jurkat cells produced 30 ± 10 and 240 ± 20 pg/ml, without and with anti-CD3 stimulation, respectively (similar to noninfected cells).

Mentions: Next, we examined the phenotypic outcome of Unc119 deficiency in T cells. Lck and Fyn regulate IL-2 production, T cell proliferation, and survival (1–3, 18). For this reason, we examined the effect of Unc119 deficiency on the foregoing T cell functions. Unc119-deficient primary T cells and Jurkat clones produced negligible amounts of IL-2 after anti-CD3 or Concanavalin A stimulation (Fig. 6 E, left). This blockade of IL-2 production was significantly reversed upon stimulation with ionomycin and phorbol ester, which activate signaling molecules downstream of the Src kinases (Fig. 6 E, right). The decrease in IL-2 production and its incomplete reversal after ionomycin/PMA treatment can be partially attributed to an increase in basal apoptosis of Jurkat cells (see Fig. 7 C). In contrast to Jurkat cells, Unc119-deficient primary T cells did not show elevated apoptosis (unpublished data). We observed a fourfold reduction in [3H]thymidine incorporation in antisense primary T cells, as compared with empty vector cells (Fig. 6 F). Overall, the phenotype of Unc119-deficient cells largely resembled the phenotype of T cell clones transfected with Lck antisense cDNA (18).


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)

Reconstitution of Unc119-deficient Jurkat cells with Unc119. (A) Unc119 level in reconstituted cells. Unc119-deficient Jurkat clones were incubated with purified recombinant Unc119 (U) or human serum albumin (A) in the presence of the protein-transducing reagent Chariot (left area of top and bottom). The right area of the panels shows reconstitution by retroviral infection. Antisense clone was infected with the empty virus (pMSCV) or Unc119 virus (pMSCV-Unc119). An empty vector Jurkat clone was infected with empty virus. Reconstituted cell lysates were Western blotted with anti-Unc119 antibody (top) and reprobed with antitubulin (bottom). (B) Lck activity in Unc119 reconstituted cells. Unc119-deficient cells were reconstituted with Unc119 (U) or albumin (A) in the Chariot experiment and incubated with (+) or without (−) anti-CD3. Samples were analyzed for kinase activity as in Fig. 4 A, autoradiographed, and Western blotted with a monoclonal anti-Lck antibody. (C) Apoptosis in Unc119-deficient cells and the effect of Unc119 reconstitution. Empty vector and antisense Jurkat cells were reconstituted with albumin (left and middle) or Unc119 (right) as in A and B, cultured for 72 h, stained with annexin V–FITC/propidium iodide, and analyzed by flow cytometry. Anti-CD3 stimulation did not influence apoptosis in Unc119-deficient cells (not depicted). (D) Effect of Unc119 reconstitution on IL-2 production of antisense Jurkat cells. Antisense cells were infected with the empty virus (E→A Jurkat) or Unc119 virus (U→A Jurkat) with 30% infection efficiency as in A. The empty vector Jurkat clone was infected with empty virus (E→E Jurkat). Transiently infected cells were incubated on anti-CD3–coated plates (CD3) or left unstimulated (−). IL-2 production was measured as in Fig. 6 E. E→E Jurkat cells produced 30 ± 10 and 240 ± 20 pg/ml, without and with anti-CD3 stimulation, respectively (similar to noninfected cells).
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fig7: Reconstitution of Unc119-deficient Jurkat cells with Unc119. (A) Unc119 level in reconstituted cells. Unc119-deficient Jurkat clones were incubated with purified recombinant Unc119 (U) or human serum albumin (A) in the presence of the protein-transducing reagent Chariot (left area of top and bottom). The right area of the panels shows reconstitution by retroviral infection. Antisense clone was infected with the empty virus (pMSCV) or Unc119 virus (pMSCV-Unc119). An empty vector Jurkat clone was infected with empty virus. Reconstituted cell lysates were Western blotted with anti-Unc119 antibody (top) and reprobed with antitubulin (bottom). (B) Lck activity in Unc119 reconstituted cells. Unc119-deficient cells were reconstituted with Unc119 (U) or albumin (A) in the Chariot experiment and incubated with (+) or without (−) anti-CD3. Samples were analyzed for kinase activity as in Fig. 4 A, autoradiographed, and Western blotted with a monoclonal anti-Lck antibody. (C) Apoptosis in Unc119-deficient cells and the effect of Unc119 reconstitution. Empty vector and antisense Jurkat cells were reconstituted with albumin (left and middle) or Unc119 (right) as in A and B, cultured for 72 h, stained with annexin V–FITC/propidium iodide, and analyzed by flow cytometry. Anti-CD3 stimulation did not influence apoptosis in Unc119-deficient cells (not depicted). (D) Effect of Unc119 reconstitution on IL-2 production of antisense Jurkat cells. Antisense cells were infected with the empty virus (E→A Jurkat) or Unc119 virus (U→A Jurkat) with 30% infection efficiency as in A. The empty vector Jurkat clone was infected with empty virus (E→E Jurkat). Transiently infected cells were incubated on anti-CD3–coated plates (CD3) or left unstimulated (−). IL-2 production was measured as in Fig. 6 E. E→E Jurkat cells produced 30 ± 10 and 240 ± 20 pg/ml, without and with anti-CD3 stimulation, respectively (similar to noninfected cells).
Mentions: Next, we examined the phenotypic outcome of Unc119 deficiency in T cells. Lck and Fyn regulate IL-2 production, T cell proliferation, and survival (1–3, 18). For this reason, we examined the effect of Unc119 deficiency on the foregoing T cell functions. Unc119-deficient primary T cells and Jurkat clones produced negligible amounts of IL-2 after anti-CD3 or Concanavalin A stimulation (Fig. 6 E, left). This blockade of IL-2 production was significantly reversed upon stimulation with ionomycin and phorbol ester, which activate signaling molecules downstream of the Src kinases (Fig. 6 E, right). The decrease in IL-2 production and its incomplete reversal after ionomycin/PMA treatment can be partially attributed to an increase in basal apoptosis of Jurkat cells (see Fig. 7 C). In contrast to Jurkat cells, Unc119-deficient primary T cells did not show elevated apoptosis (unpublished data). We observed a fourfold reduction in [3H]thymidine incorporation in antisense primary T cells, as compared with empty vector cells (Fig. 6 F). Overall, the phenotype of Unc119-deficient cells largely resembled the phenotype of T cell clones transfected with Lck antisense cDNA (18).

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