Unc119 interacts with the TCR complex. (A) Unc119 is expressed in human primary T cells and the Jurkat T cell line. Cell lysates were Western blotted with anti-Unc119 (left) or anti-Unc119 antibody–depleted (right) serum. Recombinant Unc119 (rUnc119) was electrophoresed to mark its position. NS, nonspecific band. (B) Unc119 specifically coprecipitates with CD3 in T cells. Human primary T cells were incubated with (+) or without (−) anti-CD3 and lysed. As a control, the anti-CD3 antibody was added to unstimulated sample after lysis. Lysates were immunoprecipitated (IP) with anti-Unc119 and Western blotted with antibodies against indicated molecules. The membranes were reprobed with anti-Unc119 to demonstrate equal protein loading (bottom). (Pellet) Immunoprecipitated proteins. (Supernatant) Cell lysate after removal of immunoprecipitated proteins. (C) Unc119 coprecipitates with CD4 in T cells. T cells, prepared as in B, were immunoprecipitated (IP) with indicated antibodies. Pellets and/or supernatants (B) were Western blotted with anti-Unc119. The membrane (left) was reprobed with anti-CD4. (D) GST-Unc119 interacts with CD3 and CD4. T cells, prepared as in B, were subjected to GST or GST-Unc119 pulldown. Pellets and/or supernatants (B) were Western blotted with antibodies against indicated molecules. Membranes from all experiments were reprobed with an anti-GST antibody. A representative blot is shown (bottom left).

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.

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.

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.

Lck/Fyn activity in Unc119-deficient cells. (A) Unc119 level in antisense Unc119 cDNA-expressing T cells. Cells were transfected retrovirally with empty vectors (GFP-RV for primary T cells and pMSCV for Jurkat cells) or same vectors containing Unc119 antisense cDNA (antisense). GFP⁺ primary T cells were sorted by flow cytometry and infected Jurkat cells were selected and cloned on G418. The presence of Unc119 (Unc), CD3ζ, and tubulin was analyzed by Western blotting (left and middle). NS, nonspecific band. CD3 surface expression on Unc119-deficient cells was measured by flow cytometry (right graph, dashed line, isotype control; thin line, empty vector; and thick line, antisense vector). (B) Lck and Fyn activity in Unc119-deficient cells. GFP⁺ Unc119-deficient primary T cells were immunoprecipitated with rabbit anti-Lck and anti-Fyn, analyzed for kinase activity as in Fig. 4 A, and autoradiographed. Membranes were Western blotted with monoclonal anti-Lck or anti-Fyn antibodies (bottom).

Downstream signaling and phenotype of Unc119-deficient cells. (A) Protein tyrosine phosphorylation in Unc119-deficient cells. Antisense Jurkat cells were incubated with (+) or without (−) anti-CD3 antibody, lysed, Western blotted with antiphosphotyrosine antibody, and reprobed with the antitubulin. (B) ZAP70 and Itk activity in Unc119-deficient cells. Antisense Jurkat cell lysates, prepared as in A, were immunoprecipitated with anti-ZAP70 or anti-Itk antibodies, analyzed for kinase activity as in Fig. 4 A, and autoradiographed. Membranes were Western blotted with anti-ZAP70 and anti-Itk antibodies (bottom). (C) LAT phosphorylation in Unc119-deficient cells. Antisense Jurkat cell lysates, prepared as in A, were immunoprecipitated with anti-LAT, Western blotted with antiphosphotyrosine, and reprobed with the anti-LAT antibody. (D) ERK phosphorylation in Unc119-deficient cells. Antisense Jurkat cell lysates, prepared as in A, were Western blotted with anti-phosphoERK and reprobed with anti-ERK2. (E) IL-2 production by Unc119-deficient cells. GFP⁺ primary T cells or Jurkat clones were cultured without (−) or with anti-CD3 (CD3) or Concanavalin A (left, ConA) or Phorbol myristate acetate + Ionomycin (right, PMA + Io) for 36 h. The IL-2 concentration (ELISA) in the supernatant was adjusted to the number of live cells (Trypan blue) at the conclusion of the culture. E, empty vector. A, antisense vector. (F) Proliferation of Unc119-deficient cells. GFP⁺ primary T cells were cultured for 72 h on anti-CD3 coated plates (+) or left unstimulated (−). [³H]Thymidine incorporation was measured. E, empty vector. A, anti-sense vector.

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).

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).