The Vav binding site (Y315) in ZAP-70 is critical for antigen receptor-mediated signal transduction

Abstract

Stimulation of antigen receptors in T and B cells leads to the activation of the Src and Syk families of protein tyrosine kinases (PTK). These PTKs subsequently phosphorylate numerous intracellular substrates, including the 95-kD protooncogene product Vav. Vav is essential for both T and B cell development and T and B cell antigen receptor-mediated signal transduction. After receptor ligation, Vav associates with phosphorylated Syk and ZAP-70 PTKs, an interaction that depends upon its SH2 domain. Here we demonstrate that a point mutation of tyrosine 315 (Y315F) in ZAP-70, a putative Vav SH2 domain binding site, eliminated the Vav- ZAP-70 interaction. Moreover, the Y315 mutation impaired the function of ZAP-70 in antigen receptor signaling. Surprisingly, this mutation also resulted in marked reduction in the tyrosine phosphorylation of ZAP-70, Vav, SLP-76, and Shc. These data demonstrate that the Vav binding site in ZAP-70 plays a critical role in antigen receptor-mediated signal transduction.

Figure 1

Mutation of Y315 in ZAP-70 impairs its function in BCRmediated signal transduction. Syk-deficient DT-40 cells were transiently co-transfected with 20 μg of NFAT-Luc along with 30 μg of either an empty vector, wild-type ZAP-70, or ZAP-70(Y315F). After transfection (24–40 h), cells were either left unstimulated or stimulated with either anti-BCR (M4, 2 mg/ml) or PMA (50 ng/ml) plus ionomycin (1 μM) for 6–8 h, and subsequently assayed for luciferase activity. The results are shown as the fold induction of luciferase activity as compared with the activity in unstimulated cells transfected with the empty vector, which is ∼200 arbitrary units. Luciferase activity was determined in triplicate in each experimental condition. The data are representative of at least three independent experiments. The lower panel represents anti–ZAP-70 blot (mAb 2F3.2) of equivalent amount of lysates from different transfectants in the luciferase assay described above.

Figure 2

Y315 in ZAP-70 is required for its binding to the Vav SH2 domain. (A) Syk-deficient DT-40 cells were transiently transfected with either an empty vector, wild-type ZAP-70, or ZAP-70(Y315F). Cells were then either left unstimulated or stimulated with either anti-BCR or pervanadate, and lysed. The lysates were first precleared with GST alone and then mixed with a GST fusion protein containing the Vav SH2 domain. The protein complexes were blotted with anti–ZAP-70 mAb (2F3.2). The lysate lanes represent approximately one tenth volume of the total cell lysates used for GST precipitations. (B) Mutation of Y315 in ZAP-70 did not affect its ability to interact with the Lck SH2 domain. Syk-deficient cells were transfected as described in A and were either left unstimulated or stimulated with anti-BCR or pervanadate. The lysates were first precleared with GST alone and then precipitated with GST fusion protein containing the Lck SH2 domain. The protein complexes were then blotted with anti–ZAP-70 mAb (2F3.2).

Figure 2

Y315 in ZAP-70 is required for its binding to the Vav SH2 domain. (A) Syk-deficient DT-40 cells were transiently transfected with either an empty vector, wild-type ZAP-70, or ZAP-70(Y315F). Cells were then either left unstimulated or stimulated with either anti-BCR or pervanadate, and lysed. The lysates were first precleared with GST alone and then mixed with a GST fusion protein containing the Vav SH2 domain. The protein complexes were blotted with anti–ZAP-70 mAb (2F3.2). The lysate lanes represent approximately one tenth volume of the total cell lysates used for GST precipitations. (B) Mutation of Y315 in ZAP-70 did not affect its ability to interact with the Lck SH2 domain. Syk-deficient cells were transfected as described in A and were either left unstimulated or stimulated with anti-BCR or pervanadate. The lysates were first precleared with GST alone and then precipitated with GST fusion protein containing the Lck SH2 domain. The protein complexes were then blotted with anti–ZAP-70 mAb (2F3.2).

Figure 3

(A) Vav is not phosphorylated in cells transfected with ZAP-70(Y315F). Lyn/Syk double-deficient DT-40 cells were transiently co-transfected with human Vav (pCI115) along with either an empty vector, wild-type ZAP-70, ZAP-70(Y315F), or Syk. After transfection (20–40 h), cells were either left unstimulated or stimulated with anti-BCR (M4, 2 μg/ml) for 2 min, and then lysed. The lysates were immunoprecipitated with anti-Vav polyclonal Ab and the immune complexes were blotted with antiphosphotyrosine Ab (4G10; top). The blot was then stripped and reblotted with antiVav polyclonal Ab (middle). Equivalent amount of lysates were taken from each experimental condition and resolved on a SDS-PAGE and then blotted with anti–ZAP-70 mAb (2F3.2; bottom). (B) Mutation of Y315 in ZAP-70 reduces ZAP-70–mediated SLP-76 tyrosine phosphorylation. Syk-deficient DT-40 cells were transiently transfected with FLAG epitope–tagged human SLP-76 (FLAG-SLP-76) along with either an empty vector, wild-type ZAP70, or ZAP-70(Y315F). Cells were stimulated and lysed as described in A. The lysates were immunoprecipitated with anti-FLAG epitope antibody (M2) and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with anti-FLAG antibody (bottom). Anti–ZAP-70 Western blot revealed equivalent expression between wild-type ZAP-70 and ZAP-70 (Y315F) (data not shown). (C) Mutation of Y315 in ZAP-70 also reduces ZAP-70–mediated Shc tyrosine phosphorylation. Lyn/Syk double-deficient DT-40 cells were transiently transfected with human Shc cDNA along with either an empty vector, wild-type ZAP-70, or ZAP-70 (Y315F). Cells were stimulated and lysed as described in A. The lysates were immunoprecipitated with anti-Shc mAb and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with polyclonal anti-Shc antibody (bottom). Anti–ZAP-70 Western blot showed equivalent expression between wild-type ZAP-70 and ZAP-70(Y315F) (data not shown). (D) Mutation of Y315 reduces BCR-mediated ZAP-70 tyrosine phosphorylation. Syk-deficient DT-40 cells were transiently transfected with either a vector, a myc epitope–tagged wild type or ZAP-70(Y315F). Cells were either left unstimulated or stimulated with anti-BCR or pervanadate for 2 min, and then lysed. The lysates were then immunoprecipitated with anti-myc antibody (9E10) and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with anti–ZAP-70 mAb (2F3.2) (bottom).

Figure 3

(A) Vav is not phosphorylated in cells transfected with ZAP-70(Y315F). Lyn/Syk double-deficient DT-40 cells were transiently co-transfected with human Vav (pCI115) along with either an empty vector, wild-type ZAP-70, ZAP-70(Y315F), or Syk. After transfection (20–40 h), cells were either left unstimulated or stimulated with anti-BCR (M4, 2 μg/ml) for 2 min, and then lysed. The lysates were immunoprecipitated with anti-Vav polyclonal Ab and the immune complexes were blotted with antiphosphotyrosine Ab (4G10; top). The blot was then stripped and reblotted with antiVav polyclonal Ab (middle). Equivalent amount of lysates were taken from each experimental condition and resolved on a SDS-PAGE and then blotted with anti–ZAP-70 mAb (2F3.2; bottom). (B) Mutation of Y315 in ZAP-70 reduces ZAP-70–mediated SLP-76 tyrosine phosphorylation. Syk-deficient DT-40 cells were transiently transfected with FLAG epitope–tagged human SLP-76 (FLAG-SLP-76) along with either an empty vector, wild-type ZAP70, or ZAP-70(Y315F). Cells were stimulated and lysed as described in A. The lysates were immunoprecipitated with anti-FLAG epitope antibody (M2) and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with anti-FLAG antibody (bottom). Anti–ZAP-70 Western blot revealed equivalent expression between wild-type ZAP-70 and ZAP-70 (Y315F) (data not shown). (C) Mutation of Y315 in ZAP-70 also reduces ZAP-70–mediated Shc tyrosine phosphorylation. Lyn/Syk double-deficient DT-40 cells were transiently transfected with human Shc cDNA along with either an empty vector, wild-type ZAP-70, or ZAP-70 (Y315F). Cells were stimulated and lysed as described in A. The lysates were immunoprecipitated with anti-Shc mAb and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with polyclonal anti-Shc antibody (bottom). Anti–ZAP-70 Western blot showed equivalent expression between wild-type ZAP-70 and ZAP-70(Y315F) (data not shown). (D) Mutation of Y315 reduces BCR-mediated ZAP-70 tyrosine phosphorylation. Syk-deficient DT-40 cells were transiently transfected with either a vector, a myc epitope–tagged wild type or ZAP-70(Y315F). Cells were either left unstimulated or stimulated with anti-BCR or pervanadate for 2 min, and then lysed. The lysates were then immunoprecipitated with anti-myc antibody (9E10) and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with anti–ZAP-70 mAb (2F3.2) (bottom).

Figure 3

(A) Vav is not phosphorylated in cells transfected with ZAP-70(Y315F). Lyn/Syk double-deficient DT-40 cells were transiently co-transfected with human Vav (pCI115) along with either an empty vector, wild-type ZAP-70, ZAP-70(Y315F), or Syk. After transfection (20–40 h), cells were either left unstimulated or stimulated with anti-BCR (M4, 2 μg/ml) for 2 min, and then lysed. The lysates were immunoprecipitated with anti-Vav polyclonal Ab and the immune complexes were blotted with antiphosphotyrosine Ab (4G10; top). The blot was then stripped and reblotted with antiVav polyclonal Ab (middle). Equivalent amount of lysates were taken from each experimental condition and resolved on a SDS-PAGE and then blotted with anti–ZAP-70 mAb (2F3.2; bottom). (B) Mutation of Y315 in ZAP-70 reduces ZAP-70–mediated SLP-76 tyrosine phosphorylation. Syk-deficient DT-40 cells were transiently transfected with FLAG epitope–tagged human SLP-76 (FLAG-SLP-76) along with either an empty vector, wild-type ZAP70, or ZAP-70(Y315F). Cells were stimulated and lysed as described in A. The lysates were immunoprecipitated with anti-FLAG epitope antibody (M2) and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with anti-FLAG antibody (bottom). Anti–ZAP-70 Western blot revealed equivalent expression between wild-type ZAP-70 and ZAP-70 (Y315F) (data not shown). (C) Mutation of Y315 in ZAP-70 also reduces ZAP-70–mediated Shc tyrosine phosphorylation. Lyn/Syk double-deficient DT-40 cells were transiently transfected with human Shc cDNA along with either an empty vector, wild-type ZAP-70, or ZAP-70 (Y315F). Cells were stimulated and lysed as described in A. The lysates were immunoprecipitated with anti-Shc mAb and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with polyclonal anti-Shc antibody (bottom). Anti–ZAP-70 Western blot showed equivalent expression between wild-type ZAP-70 and ZAP-70(Y315F) (data not shown). (D) Mutation of Y315 reduces BCR-mediated ZAP-70 tyrosine phosphorylation. Syk-deficient DT-40 cells were transiently transfected with either a vector, a myc epitope–tagged wild type or ZAP-70(Y315F). Cells were either left unstimulated or stimulated with anti-BCR or pervanadate for 2 min, and then lysed. The lysates were then immunoprecipitated with anti-myc antibody (9E10) and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with anti–ZAP-70 mAb (2F3.2) (bottom).

Figure 3

(A) Vav is not phosphorylated in cells transfected with ZAP-70(Y315F). Lyn/Syk double-deficient DT-40 cells were transiently co-transfected with human Vav (pCI115) along with either an empty vector, wild-type ZAP-70, ZAP-70(Y315F), or Syk. After transfection (20–40 h), cells were either left unstimulated or stimulated with anti-BCR (M4, 2 μg/ml) for 2 min, and then lysed. The lysates were immunoprecipitated with anti-Vav polyclonal Ab and the immune complexes were blotted with antiphosphotyrosine Ab (4G10; top). The blot was then stripped and reblotted with antiVav polyclonal Ab (middle). Equivalent amount of lysates were taken from each experimental condition and resolved on a SDS-PAGE and then blotted with anti–ZAP-70 mAb (2F3.2; bottom). (B) Mutation of Y315 in ZAP-70 reduces ZAP-70–mediated SLP-76 tyrosine phosphorylation. Syk-deficient DT-40 cells were transiently transfected with FLAG epitope–tagged human SLP-76 (FLAG-SLP-76) along with either an empty vector, wild-type ZAP70, or ZAP-70(Y315F). Cells were stimulated and lysed as described in A. The lysates were immunoprecipitated with anti-FLAG epitope antibody (M2) and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with anti-FLAG antibody (bottom). Anti–ZAP-70 Western blot revealed equivalent expression between wild-type ZAP-70 and ZAP-70 (Y315F) (data not shown). (C) Mutation of Y315 in ZAP-70 also reduces ZAP-70–mediated Shc tyrosine phosphorylation. Lyn/Syk double-deficient DT-40 cells were transiently transfected with human Shc cDNA along with either an empty vector, wild-type ZAP-70, or ZAP-70 (Y315F). Cells were stimulated and lysed as described in A. The lysates were immunoprecipitated with anti-Shc mAb and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with polyclonal anti-Shc antibody (bottom). Anti–ZAP-70 Western blot showed equivalent expression between wild-type ZAP-70 and ZAP-70(Y315F) (data not shown). (D) Mutation of Y315 reduces BCR-mediated ZAP-70 tyrosine phosphorylation. Syk-deficient DT-40 cells were transiently transfected with either a vector, a myc epitope–tagged wild type or ZAP-70(Y315F). Cells were either left unstimulated or stimulated with anti-BCR or pervanadate for 2 min, and then lysed. The lysates were then immunoprecipitated with anti-myc antibody (9E10) and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with anti–ZAP-70 mAb (2F3.2) (bottom).

Figure 4

(A) Mutation of Y315 in ZAP-70 does not affect its intrinsic tyrosine kinase activity. Lyn/Syk double-deficient DT-40 cells were transient transfected with either an empty vector, a myc epitope–tagged wildtype ZAP-70, or ZAP-70(Y315F). After transfection (24–40 h), the lysates were immunoprecipitated with anti-myc epitope (9E10) and subjected to an in vitro kinase assay. The products were separated on a SDS-PAGE gel and transferred to polyvinylidene difluoride membrane. The membrane was subjected to KOH treatment and then in vitro phosphorylated proteins were detected by autoradiography (top). Expression of ZAP-70 was detected by immunoblotting the same membrane with anti–ZAP-70 mAb (2F3.2) (bottom). (B) Mutation of Y315 in ZAP-70 does not affect its binding to receptor ITAMs. Syk-deficient cells were transfected with either an empty vector, ZAP-70, or ZAP-70(Y315F). After transfection (24–40 h), the lysates were mixed with 1 μg of doubly phosphorylated peptide encompassing the second ITAM of TCR ζ chain, followed by the addition of avidin beads to collect complexes. The complexes were then blotted with anti–ZAP-70 mAb (2F3.2).

Figure 4

(A) Mutation of Y315 in ZAP-70 does not affect its intrinsic tyrosine kinase activity. Lyn/Syk double-deficient DT-40 cells were transient transfected with either an empty vector, a myc epitope–tagged wildtype ZAP-70, or ZAP-70(Y315F). After transfection (24–40 h), the lysates were immunoprecipitated with anti-myc epitope (9E10) and subjected to an in vitro kinase assay. The products were separated on a SDS-PAGE gel and transferred to polyvinylidene difluoride membrane. The membrane was subjected to KOH treatment and then in vitro phosphorylated proteins were detected by autoradiography (top). Expression of ZAP-70 was detected by immunoblotting the same membrane with anti–ZAP-70 mAb (2F3.2) (bottom). (B) Mutation of Y315 in ZAP-70 does not affect its binding to receptor ITAMs. Syk-deficient cells were transfected with either an empty vector, ZAP-70, or ZAP-70(Y315F). After transfection (24–40 h), the lysates were mixed with 1 μg of doubly phosphorylated peptide encompassing the second ITAM of TCR ζ chain, followed by the addition of avidin beads to collect complexes. The complexes were then blotted with anti–ZAP-70 mAb (2F3.2).