(A) Lck and Fyn regulate TCR stimulation–induced tyrosine phosphorylation of shared and distinct substrates. Thymocytes isolated from wild-type (Wt), lck^−/−, or fyn^−/− mice were either left unstimulated (−) or stimulated (+) with the anti-CD3 mAb 145-2C11 for 3 min. Cells were lysed, and tyrosine phosphorylation of whole cell lysates was analyzed by immunoblotting with the anti-phosphotyrosine (anti–P-Tyr) mAb 4G10. The migration of tyrosine-phosphorylated TCR-ζ chain is indicated. Molecular mass markers are also indicated. (B) Tyrosine phosphorylation of ZAP-70 requires Lck but not Fyn. Thymocytes isolated from wild-type (Wt), lck^−/−, or fyn^−/− mice were either left unstimulated or stimulated with the anti-CD3 mAb for 3 min. Cells were lysed, ZAP70 was immunoprecipitated (IP) with an anti–ZAP-70 mAb (1E7.2), and analyzed by anti–P-Tyr immunoblotting using mAb 4G10. The same blot was stripped and reprobed with anti–ZAP-70 (mAb 1E7.2).

Fyn, but not Lck, is required for TCR-stimulated Pyk2 tyrosine phosphorylation. (A) Pyk2 phosphorylation in Jurkat T cells. Left, Jurkat cells were stimulated with anti-TCR (mAb C305) for the indicated period of times. Right, Jurkat cells were stimulated with antiTCR or ionomycin (IONO) for 2 min. Cells were lysed, Pyk2 was immunoprecipitated with anti-Pyk2 (antibody no. 3), and analyzed by immunoblotting using anti–P-Tyr mAb RC20. The level of Pyk2 in each sample was verified either by immunoblotting an aliquot of anti-Pyk2 immunoprecipitates with antiPyk2 (antibody no. 3) (left) or by stripping and reprobing the same blot with anti-Pyk2 (right). (B) Thymocytes isolated from wildtype (Wt), lck^−/−, or fyn^−/− mice were either left unstimulated or stimulated with the anti-CD3 mAb for 3 min. Cells were lysed, Pyk2 was immunoprecipitated with anti-Pyk2 (antibody no. 600), and analyzed by immunoblotting using anti-P-Tyr mAb 4G10. The same blot was stripped and reprobed with antiPyk2 (antibody no. 3).

Pyk2 activation is selectively induced by Fyn, but not Lck. (A) Tyrosine phosphorylation of Pyk2. Pyk2 or the kinase-inactive ZAP-70 mutant, ZAP-70 (K369A), was transiently cotransfected with vector, Lck, or Fyn (T) into COS-7 cells. Tyrosine phosphorylation of Pyk2 or ZAP-70 (K369A) was determined by immunoblotting using anti–P-Tyr mAb 4G10 (right), after immunoprecipitation with anti-Pyk2 (antibody no. 1) or anti– ZAP-70 (antibody no. 1598). The expression of Pyk2, ZAP-70 (K369A), Fyn (T), or Lck in each transfected sample was determined by immunoblotting whole cell lysates with anti-Pyk2 (antibody no. 1), anti–ZAP-70 (mAb 2F3), a rabbit anti-Fyn antibody, or anti-Lck (mAb 1F6) (left). (B) Phosphorylation of an exogenous substrate by Pyk2. Pyk2 was transiently transfected into COS-7 cells with vector, Lck, or Fyn (T). Cells were lysed, and Pyk2 was immunoprecipitated with anti-Pyk2 (antibody no. 600). The immunoprecipitates were extensively washed with RIPA buffer, and subjected to kinase reactions in vitro in the presence of γ-[³²P]ATP and the exogenous substrate poly(Glu/Tyr) (4:1). The phosphorylated products were resolved by SDS-PAGE, analyzed by autoradiography (top), and quantitated by a phosphorimager and Image Quant software (Molecular Dynamics). The kinase activities relative to vector cotransfected cells are indicated (middle). The expression of Pyk2 in each sample was determined by immunoblotting whole cell lysates with antiPyk2 (antibody no. 1) (bottom). (C) The kinase activity of Pyk2 is required for the ability of Pyk2 to phosphorylate an exogenous substrate but not Pyk2 tyrosine phosphorylation induced by Fyn (T). Pyk2 or the kinase inactive mutant Pyk2 with a point mutation at the ATP-binding site (PKM) was cotransfected with Fyn (T) into COS-7 cells. Cells were lysed, and Pyk2 or PKM was immunoprecipitated with anti-Pyk2 (antibody no. 600). Phosphorylation of poly(Glu/Tyr) (4:1) by the immunoprecipitates in in vitro kinase reactions was determined (left). The relative kinase activities to Pyk2 and vector cotransfected cells (data not shown) are also indicated. Tyrosine phosphorylation of Pyk2 and PKM was analyzed by immunoblotting using anti– P-Tyr mAb 4G10 (right). The expression of Pyk2, PKM, or Fyn (T) in each transfected sample was determined by immunoblotting whole cell lysates with the anti-Pyk2 (antibody no. 1) or an anti-Fyn mAb. (D) Phosphorylation of PKM by purified Lck or Fyn. COS-7 cells transfected with PKM were lysed, and lysates were immunoprecipitated with either normal rabbit serum (NRS) or an affinity-purified anti-Pyk2 (antibody D1). The immunoprecipitates were extensively washed with RIPA buffer and used in in vitro kinase reactions in the absence (−) or presence (+) of purified Lck or Fyn (3 U/reaction).

Physical association of Fyn with Pyk2 in T cells. (A) Thymocytes isolated from C57BL/6 mice were either left unstimulated or stimulated with the anti-CD3 mAb for 3 min. Cells were lysed, lysates were immunoprecipitated with NRS, a rabbit anti-Fyn antibody, or a rabbit anti-Lck antibody, and analyzed by immunoblotting with an affinitypurified anti-Pyk2 (antibody D1), an anti-Fyn mAb, and anti-Lck (mAb 1F6). (B) HA-tagged Pyk2 was transiently transfected into TAg Jurkat T cells. 40 h later, transfected cells were either left unstimulated or stimulated with anti-TCR (mAb C305) for 2 min. Cells were lysed, lysates were immunoprecipitated with either a control (Ctrl) antibody (OKT8) or antiHA (mAb 12CA5), and analyzed by immunoblotting with an anti-HA mAb (12CA5) and an anti-Fyn mAb. The positions of HA–Pyk2 and Fyn are indicated by arrows.