Limits...
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.

Show MeSH

Related in: MedlinePlus

Unc119 activates Lck and Fyn. (A) Unc119-Lck association correlates with kinase activity. T cells were stimulated for indicated time and lysed. Lysates were immunoprecipitated with indicated antibodies. The anti-Unc119 and anti-CD3ζ precipitates were Western blotted with anti-Lck (I) and antiphosphotyrosine (V, pTyr), respectively, and reprobed with anti-Unc119 (II) and anti-CD3ζ (VI). Anti-Lck precipitates were used in the kinase assay with enolase as a substrate in the presence of [32P]γ-ATP and autoradiographed (III). The kinase assay membrane was blotted with monoclonal anti-Lck (IV). (B–D) Recombinant Unc119 activates Lck and Fyn. Lck (B and D) or Fyn (C) were immunoprecipitated (IP) with rabbit antikinase antibodies, subjected to the kinase assay in the presence of recombinant purified Unc119 (B and C) or a CD2-derived peptide (D), and autoradiographed. Membranes were Western blotted (bottom) with monoclonal anti-Lck (B and D) or anti-Fyn (C). (E) Generation of Unc119-overexpressing Jurkat clones. Jurkat cells were transfected with pMSCVneo (empty vector) or pMSCVneo-Unc119 (Unc119 vector). Lysates of select clones were Western blotted with anti-Unc119 and reprobed with antitubulin. (F) Recruitment of Unc119 to Lck is enhanced in overexpressing cells. Unc119-overexpressing (Unc119) and empty vector (empty) clones were incubated with (+) or without (−) anti-CD3 and lysed. Lysates were immunoprecipitated with anti-Lck, Western blotted with anti-Unc119 (top), and reprobed with anti-Lck (bottom). (G) Overexpressed Unc119 activates Lck and Fyn. Lck and Fyn was immunoprecipitated (IP) from Unc119-overexpressing and empty vector Jurkat lysates (F), assayed for kinase activity as in A, and autoradiographed. Membranes were Western blotted with rabbit anti-Lck or anti-Fyn antibodies (bottom). IgH, immunoglobulin heavy chain.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211793&req=5

fig4: Unc119 activates Lck and Fyn. (A) Unc119-Lck association correlates with kinase activity. T cells were stimulated for indicated time and lysed. Lysates were immunoprecipitated with indicated antibodies. The anti-Unc119 and anti-CD3ζ precipitates were Western blotted with anti-Lck (I) and antiphosphotyrosine (V, pTyr), respectively, and reprobed with anti-Unc119 (II) and anti-CD3ζ (VI). Anti-Lck precipitates were used in the kinase assay with enolase as a substrate in the presence of [32P]γ-ATP and autoradiographed (III). The kinase assay membrane was blotted with monoclonal anti-Lck (IV). (B–D) Recombinant Unc119 activates Lck and Fyn. Lck (B and D) or Fyn (C) were immunoprecipitated (IP) with rabbit antikinase antibodies, subjected to the kinase assay in the presence of recombinant purified Unc119 (B and C) or a CD2-derived peptide (D), and autoradiographed. Membranes were Western blotted (bottom) with monoclonal anti-Lck (B and D) or anti-Fyn (C). (E) Generation of Unc119-overexpressing Jurkat clones. Jurkat cells were transfected with pMSCVneo (empty vector) or pMSCVneo-Unc119 (Unc119 vector). Lysates of select clones were Western blotted with anti-Unc119 and reprobed with antitubulin. (F) Recruitment of Unc119 to Lck is enhanced in overexpressing cells. Unc119-overexpressing (Unc119) and empty vector (empty) clones were incubated with (+) or without (−) anti-CD3 and lysed. Lysates were immunoprecipitated with anti-Lck, Western blotted with anti-Unc119 (top), and reprobed with anti-Lck (bottom). (G) Overexpressed Unc119 activates Lck and Fyn. Lck and Fyn was immunoprecipitated (IP) from Unc119-overexpressing and empty vector Jurkat lysates (F), assayed for kinase activity as in A, and autoradiographed. Membranes were Western blotted with rabbit anti-Lck or anti-Fyn antibodies (bottom). IgH, immunoglobulin heavy chain.

Mentions: Next, we asked if the association of Unc119 with Lck is correlated with an increase in the kinase activity and induction of downstream signaling events. Toward this goal, we stimulated peripheral blood T cells for increasing periods of time with anti-CD3 antibody. The lysates of these cells were used for Lck/Unc119 coprecipitation experiment, Lck kinase assay, and for the measurement of CD3ζ phosphorylation. Fig. 4 A shows that Unc119 associated with Lck from 30 s to 5 min of stimulation with the highest binding at the 2-min time point (Fig. 4 A, I). Interestingly, Lck activity slightly increased at 30 s as compared with nonstimulated kinase, reached maximum at 2 min, dramatically decreased at 10 min, and went back to basal level after the latter time point (Fig. 4 A, III). Therefore, the binding of Unc119 to Lck correlated well with the augmented kinase activity. Conversely, the dissociation of Unc119 from Lck correlated with the decrease in the kinase activity. We also examined the kinetics of CD3ζ phosphorylation in these cells. CD3ζ was phosphorylated to some extent at 30 s and maximally at 2 min, and became undetectable at 60 min (Fig. 4 A, V). Overall, the association of Unc119 with Lck was always concomitant with enhanced signaling through TCR. Both the Lck activity and CD3ζ phosphorylation reached the peak at the time when the Unc119–Lck interaction was maximum.


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 activates Lck and Fyn. (A) Unc119-Lck association correlates with kinase activity. T cells were stimulated for indicated time and lysed. Lysates were immunoprecipitated with indicated antibodies. The anti-Unc119 and anti-CD3ζ precipitates were Western blotted with anti-Lck (I) and antiphosphotyrosine (V, pTyr), respectively, and reprobed with anti-Unc119 (II) and anti-CD3ζ (VI). Anti-Lck precipitates were used in the kinase assay with enolase as a substrate in the presence of [32P]γ-ATP and autoradiographed (III). The kinase assay membrane was blotted with monoclonal anti-Lck (IV). (B–D) Recombinant Unc119 activates Lck and Fyn. Lck (B and D) or Fyn (C) were immunoprecipitated (IP) with rabbit antikinase antibodies, subjected to the kinase assay in the presence of recombinant purified Unc119 (B and C) or a CD2-derived peptide (D), and autoradiographed. Membranes were Western blotted (bottom) with monoclonal anti-Lck (B and D) or anti-Fyn (C). (E) Generation of Unc119-overexpressing Jurkat clones. Jurkat cells were transfected with pMSCVneo (empty vector) or pMSCVneo-Unc119 (Unc119 vector). Lysates of select clones were Western blotted with anti-Unc119 and reprobed with antitubulin. (F) Recruitment of Unc119 to Lck is enhanced in overexpressing cells. Unc119-overexpressing (Unc119) and empty vector (empty) clones were incubated with (+) or without (−) anti-CD3 and lysed. Lysates were immunoprecipitated with anti-Lck, Western blotted with anti-Unc119 (top), and reprobed with anti-Lck (bottom). (G) Overexpressed Unc119 activates Lck and Fyn. Lck and Fyn was immunoprecipitated (IP) from Unc119-overexpressing and empty vector Jurkat lysates (F), assayed for kinase activity as in A, and autoradiographed. Membranes were Western blotted with rabbit anti-Lck or anti-Fyn antibodies (bottom). IgH, immunoglobulin heavy chain.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211793&req=5

fig4: Unc119 activates Lck and Fyn. (A) Unc119-Lck association correlates with kinase activity. T cells were stimulated for indicated time and lysed. Lysates were immunoprecipitated with indicated antibodies. The anti-Unc119 and anti-CD3ζ precipitates were Western blotted with anti-Lck (I) and antiphosphotyrosine (V, pTyr), respectively, and reprobed with anti-Unc119 (II) and anti-CD3ζ (VI). Anti-Lck precipitates were used in the kinase assay with enolase as a substrate in the presence of [32P]γ-ATP and autoradiographed (III). The kinase assay membrane was blotted with monoclonal anti-Lck (IV). (B–D) Recombinant Unc119 activates Lck and Fyn. Lck (B and D) or Fyn (C) were immunoprecipitated (IP) with rabbit antikinase antibodies, subjected to the kinase assay in the presence of recombinant purified Unc119 (B and C) or a CD2-derived peptide (D), and autoradiographed. Membranes were Western blotted (bottom) with monoclonal anti-Lck (B and D) or anti-Fyn (C). (E) Generation of Unc119-overexpressing Jurkat clones. Jurkat cells were transfected with pMSCVneo (empty vector) or pMSCVneo-Unc119 (Unc119 vector). Lysates of select clones were Western blotted with anti-Unc119 and reprobed with antitubulin. (F) Recruitment of Unc119 to Lck is enhanced in overexpressing cells. Unc119-overexpressing (Unc119) and empty vector (empty) clones were incubated with (+) or without (−) anti-CD3 and lysed. Lysates were immunoprecipitated with anti-Lck, Western blotted with anti-Unc119 (top), and reprobed with anti-Lck (bottom). (G) Overexpressed Unc119 activates Lck and Fyn. Lck and Fyn was immunoprecipitated (IP) from Unc119-overexpressing and empty vector Jurkat lysates (F), assayed for kinase activity as in A, and autoradiographed. Membranes were Western blotted with rabbit anti-Lck or anti-Fyn antibodies (bottom). IgH, immunoglobulin heavy chain.
Mentions: Next, we asked if the association of Unc119 with Lck is correlated with an increase in the kinase activity and induction of downstream signaling events. Toward this goal, we stimulated peripheral blood T cells for increasing periods of time with anti-CD3 antibody. The lysates of these cells were used for Lck/Unc119 coprecipitation experiment, Lck kinase assay, and for the measurement of CD3ζ phosphorylation. Fig. 4 A shows that Unc119 associated with Lck from 30 s to 5 min of stimulation with the highest binding at the 2-min time point (Fig. 4 A, I). Interestingly, Lck activity slightly increased at 30 s as compared with nonstimulated kinase, reached maximum at 2 min, dramatically decreased at 10 min, and went back to basal level after the latter time point (Fig. 4 A, III). Therefore, the binding of Unc119 to Lck correlated well with the augmented kinase activity. Conversely, the dissociation of Unc119 from Lck correlated with the decrease in the kinase activity. We also examined the kinetics of CD3ζ phosphorylation in these cells. CD3ζ was phosphorylated to some extent at 30 s and maximally at 2 min, and became undetectable at 60 min (Fig. 4 A, V). Overall, the association of Unc119 with Lck was always concomitant with enhanced signaling through TCR. Both the Lck activity and CD3ζ phosphorylation reached the peak at the time when the Unc119–Lck interaction was maximum.

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