Correction: Binding of phosphatidylinositol-3-OH kinase to CD28 is required for T-cell signalling

Abstract

not shown). Consistent with previous results I I ,Jak-3 is expressed in breast-tis sue-derived ceIl lines (K.S.L. and E.T.L., unpublished observations). To assess Jak-3 in sign aIling, cytokine-induced tyrosine phosphorylation was examined. Phosphorylation was not seen with erythropoietin, IL-3, granulocyte macrophage colony-stimulating factor (GM-CSF), G-CSF, interferon-a (IFN-a), IFN-y or IL-6 (data not shown), but was seen in IL-2-or IL-4-stimulated cells. In CTLL ceIls, IL-2 and IL-4 induced tyrosine phosphorylation of several proteins (Fig. 4), including proteins of 120K and 130K, as recently shown 16. IL-2 and IL-4 induced tyrosine phosphorylation of Jak-3 (aJak-3), which co migrated with the major l20K substrate, and Jak-l (aJak-l), which comigrated with the 130K substrate. No Jak-2 or Tyk-2 phosphorylation was detected (data not shown). Last, the Jak-3/Jak-l-crossreactive Tyk-2 antiserum immunoprecipitated proteins that comigrated with Jak-l and Jak-3 but not Tyk-2. Phosphorylation was detectable within 1 min, peaked at 20-30 min and subsequently declined (data not shown). Cytokine-induced tyrosine phosphorylation of Jaks activates their in vitro kinase activityl.2,4,7,9. Therefore, we examined the effects of IL-2 or IL-4 on kinase activity. No kinase activity was detected in immunoprecipitates of Jak-l or immunoprecipitates of Jak-3 obtained with the Jak-3-specific antiserum (data not shown). However, the Jak-3 antiserum is against a peptide containing the autophosphorylation site (KDYY; single-letter amino-acid code) and could interfere with activity. Therefore immunoprecipitates obtained with the Jak-l/Jak-3 crossreactive antiserum were examined. With these, IL-2-and IL-4-induced in vitro kinase activity was readily detected (Fig. 4b) and the single phosphorylated protein comigrated with Jak-3. No detectable Jak-l phoshorylation was seen. Amino-acid analysis of phosphorylated Jak-3 detected exclusively phosphotyrosine (data not shown). To assess the specificity of Jak-3 activation, we examined CTLL cells expressing the erythropoietin receptor, which shares homology in the cytoplasmic domain with the IL-2 receptor [3-chain17. Transfected CTLL ceIls express levels of high-affinity erythropoietin receptors comparable to transfected myeloid ceIls (O.M. and J.N.J, unpublished data). Erythropoietin, but neither IL-2 nor IL-4, induced tyrosine phosphorylation of Jak-2 (Fig. 4c). Conversely, IL-2, but not erythropoietin, induced phosphorylation of Jak-3. The IL-2 receptor consists of a-, [3-and y-chains IR • The ychain is also used in the IL-4 receptor l9 ,20 and is essential for function. The [3-chain is also essential and contains two functional domains. An acidic domain is required for association and activation of p56 lck , p59" y n or p53/561yn18 but is dispensable for mitogenesis, In contrast, the membrane-proximal, serine-rich domain is required for mitogenesis. To assess their role in Jak-3 activation, the wild-type and receptors lacking the acidic (Amutant) or serine-rich (S-mutant) domains were intro...

Malignant plasma cell lines express a functional CD28 molecule

“…Human CD28 transfectant (DWT6.11) was obtained by transfecting full-length human CD28 cDNA in pH␤-pr-neo vector into murine T cell hybridoma. 6 These cell lines were cultured in culture medium. All cell lines were free of mycoplasma contamination as assayed using an ELISA kit from Boehringer (Mannheim, Germany).…”

“…This was not surprising since phosphorylation of CD28 T cell molecules was rarely found 3 except for CD28 transfectants that expressed a very large density of CD28 molecules. 6 …”

“…1 CD28 activation has been shown to be critical in inducing IL-2 secretion in activated T cells. 1,2 More recently, activation of CD28 molecules was shown to induce activation of phosphatidyl-inositol 3-kinase (PI-3) in T cells [3][4][5][6][7] and to promote survival by upregulating bcl-X L expression. 8 In an early report, CD28 molecules were found to be present on human malignant plasma cells.…”

The Role of Glycogen Synthase Kinase 3-β in Immunity and Cell Cycle: Implications in Esophageal Cancer

“…Other studies demonstrated that GSK3 was an important effector molecule involved in the B7-CD28 co-stimulatory signaling pathway. CD28 co-stimulation of T cells was shown to increase PI3K activity and mediate the inactivation of GSK3 (Pages et al 1994;Wood et al 2006). In addition, studies using the Leishmania major infection model have shown that GSK3 inhibition suppressed Th1 responses (Ohtani et al 2008) on a systematic level.…”

Rôle de CTLA-4 dans la cosignalisation négative du système immunitaire

“…La partie intracytoplasmique (IC) de CD28 (41 acides aminés chez l'homme) ne comporte aucune activité enzymatique démontrée, mais l'analyse de la séquence peptidique a permis d'y identifier des motifs qui lui permettent de s'associer à des enzymes et des protéines adaptatrices impliquées dans la transmission du signal intracellulaire. Parmi les motifs les plus importants, on peut citer YMNM qui rend compte, après la phosphorylation de la tyrosine, de la fixation des domaines SH2 de la sous-unité p85 de la phosphoinositide 3-kinase (PI3K) et de Grb2 et GADS (Grb2-related adaptor downstream of Shc) [10,11]. Deux motifs riches en proline, PRRP et PYAP, recrutent les domaines SH3 des PTK (protéine tyrosine kinase) Tec et Itk (PRRP) et Lck et Grb2 (PYAP) ainsi que la filamine A (FLNA) [11].…”