Advertisement

Article Figures & Data

Figures

  • Figure 1.

    Defective basal and CD3-induced TCR-ζ chain phosphorylation in primary Syk+ T cells from a ZAP-70–deficient patient are restored following introduction of ZAP-70. CD4+ T cells from a healthy individual (ZAP+/Syk), a ZAP-70–deficient patient (ZAP/Syk+), and following introduction of ectopic ZAP-70 (ZAPE/Syk+)27,45 were either left unstimulated (–) or stimulated for 3′ with a cross-linked αCD3 mAb (+). (A) Phosphorylation of the TCR-ζ chain was monitored in whole cell lysates by the presence of the p21 and p23 TCR-ζ isoforms. Nonphosphorylated TCR-ζ migrates with a molecular weight of 16 kDa (p16). ZAP-70 and Syk levels in these whole cell lysates were assessed with ZAP-70– and Syk-specific Abs, and the relative amount of protein in each lane was determined by blotting for Erk2. Note that the ectopic ZAP-70 bears a terminal vesicular stomatitis virus-protein G epitope tag allowing endogenous and ectopic ZAP-70 proteins to be distinguished on the basis of their molecular weights. (B) TCR-ζ was immunoprecipitated from unstimulated (–) or αCD3/αCD4-stimulated (3′, +) control (ZAP+/Syk), and patient (ZAP/Syk+) CD4+ T cells. The levels of ζ-associated ZAP-70 and Syk molecules were determined by immunoblotting with αZAP-70 and αSyk Abs, and tyrosine phosphorylation of these proteins was monitored by immunoblotting with an α-phospho-tyrosine (PY) mAb. The amount of immunoprecipitated p16-ζ in each lane was monitored with an α-ζ mAb.

  • Figure 2.

    Generation of T-cell lines expressing equivalent levels of ZAP-70 and Syk. (A) p116 Jurkat cells, expressing neither ZAP-70 nor Syk (ZAP/Syk), were transduced with a ZAP-70/EGFP or Syk/EGFP retroviral vector, and cells expressing similar levels of EGFP were sorted on a FACS Vantage cytometer. The EGFP fluorescence of the sorted p116 cell lines expressing ectopic ZAP-70 (ZAPE/Syk) and Syk (ZAP/SykE) are shown. Background fluorescence of p116 cells not expressing EGFP is shown in a filled histogram. (B) ZAP-70 and Syk expression in these cells was quantified by comparison with known concentrations of human recombinant ZAP-70 and GST-Syk proteins (2-50 ng). Amido-black staining of these recombinant proteins and the corresponding immunoblots with Syk and ZAP-70 antibodies are shown. The quantification of ectopic ZAP-70 and Syk expressed in the transduced cell lines (A) was determined by comparison of the respective signals in several cell concentrations (15-60 × 103 cells) with that obtained for the recombinant protein (using the NIHimage software program). (C) ZAP-70 and Syk expression in the ZAPE/Syk and ZAP/SykE cells as well as in other Jurkat cell lines (Table 1) were monitored by immunoblotting of total cellular lysates with αZAP-70 and αSyk Abs. The blot was then stripped and reprobed with an αErk2 mAb to control for protein loading.

  • Figure 3.

    Ectopic ZAP-70 and Syk expression are associated with distinct TCR-ζ phosphorylation profiles in p116 Jurkat cells. p116 Jurkat cells (ZAP/Syk) expressing equivalent levels of ectopic ZAP-70 (ZAPE/Syk) or Syk (ZAP/SykE) (Figure 2) were stimulated via CD3 cross-linking (3′) or pervanadate (VO4)(5′). Immunoprecipitations were performed with an αTCR-ζ Ab, and the levels of the p21 and p23 phosphorylated TCR-ζ isoforms as well as the nonphosphorylated p16 isoform following stimulation are shown. The presence of ζ-associated ZAP-70 and Syk molecules as well as their relative phosphorylation status were determined by immunoblotting the ζ immunoprecipitates with αZAP-70, αSyk, and αPY Abs, respectively.

  • Figure 4.

    Relative binding of Syk to fully phosphorylated TCR-ζ is significantly lower than that of ZAP-70. (A) Induction of the phosphorylated p21 and p23 TCR-ζ isoforms was assessed in Jurkat cells expressing endogenous ZAP-70 and Syk (ZAP+/Syk+) following a 3′ CD3 ligation. Phosphorylation was monitored using an αPY mAb, and total levels of p16-ζ were assessed with an α-ζ mAb. (B) TCR-ζ was immunoprecipitated from lysates of 10 × 106 of these ZAP+/Syk+ Jurkat cells following CD3 cross-linking or pervanadate treatment, and the presence of associated ZAP-70 and Syk was detected by immunoblotting. To determine the relative levels of association of these 2 kinases with TCR-ζ, the total cellular levels of ZAP-70 and Syk present in 5 × 106, 1 × 106, and 0.5 × 106 of these Jurkat cells was concomitantly monitored on the same blot. (C) Western blots were quantified using scanning densitometry, and the relative levels of TCR-ζ–associated ZAP-70 and Syk in unstimulated and stimulated cells from the experiment shown in panel B are shown.

  • Figure 5.

    Role of Tyr319 and ZAP-70 kinase activity in TCR-ζ phosphorylation. (A) TCR-ζ was immunoprecipitated from p116 Jurkat cells (ZAP/Syk) expressing similar levels of ectopic ZAP-70 (ZAPE/Syk) or Syk (ZAP/SykE) following CD3 cross-linking (+), and the level of associated Lck was determined by immunoblotting with an Lck pAb. The positions of the p56 and p59 Lck isoforms are indicated, and the level of immunoprecipitated TCR-ζ is shown. (B) TCR-ζ was immunoprecipitated following CD3 cross-linking or pervanadate treatment. The levels of TCR-ζ–associated ZAP-70 and Syk phosphorylated on Tyr319 and Tyr352, respectively, were monitored using a pAb recognizing these homologous phosphorylated tyrosine residues. The levels of total TCR-ζ–associated ZAP-70 and Syk were revealed with ZAP-70– and Syk-specific antibodies, respectively. (C) TCR-ζ phosphorylation was monitored in whole cell lysates of unstimulated (–) or CD3-stimulated (+) p116 cells stably expressing WT ZAP-70, Y319F ZAP-70, or a kinase-dead (KD) ZAP-70. The relative levels of ZAP-70 phosphorylated on Tyr319, total ZAP-70, phosphorylated Erk1/Erk2 (P-MAPK), and total Erk2 were assessed by immunoblotting with the appropriate antibodies.

  • Figure 6.

    CD3-induced phosphorylation of Syk is augmented in the presence of ZAP-70. The relative phosphorylation levels of ZAP-70 and Syk in Jurkat cells engineered to express either ZAP-70 or Syk alone (ZAPE/Syk and ZAP/SykE cells, respectively) or together (ZAP+/SykE) were monitored following their joint immunoprecipitation. Immunoprecipitates were immunoblotted with an αPY mAb, and blots were then reprobed with αZAP-70 and αSyk Abs.

  • Figure 7.

    Downstream signaling is altered in Syk-expressing p116 cells. (A) Erk phosphorylation in the parental p116 cells and the derived ZAPE/Syk and ZAP/SykE cell lines was monitored 3, 5, and 10 minutes after stimulation with an αCD3 mAb at 37° C. Cell lysates (1 × 106 cell equivalents) were immunoblotted with a polyclonal Ab that recognizes the doubly phosphorylated forms of Erk1 and Erk2. The blot was then stripped and reprobed with an αErk2 mAb. (B) CD69 expression was assessed following overnight culture in serum-free media in the absence or presence of immobilized αCD3 mAb (1 μg/mL). Constitutive (filled histograms) and CD3-induced (open histograms) CD69 expression in the parental p116 cells and the derived ZAPE/Syk and ZAP/SykE cell lines were detected using a phycoerythrin (PE)–conjugated anti-CD69 mAb and analyzed on a FACScan. The mean fluorescence intensity of CD69 expression in the absence or presence of αCD3 is indicated in each histogram.

Tables

  • Table 1.

    Nomenclature of parental and transduced Jurkat cell lines

    Parental cellsIntroduced geneNomenclatureMean CD3 expression ± standard deviation*
    P116 ZAP-/Syk- 440 ± 79
    P116 ZAP ZAPE/Syk- 401 ± 84
    P116 Syk ZAP-/SykE 392 ± 54
    E6.1 Syk ZAP+/SykE 462 ± 91
    77-6.8 ZAP+/Syk+ 234 ± 64
    • + indicates endogenous expression; E, ectopic expression; and —, no expression.

    • * Mean fluorescence intensity of CD3 expression as monitored by flow cytometry using an αCD3 mAb, mean ± standard deviation in 5 independent experiments.