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Cytotoxic T lymphocyte antigen 4 (CTLA-4) interferes with extracellular signal-regulated kinase (ERK) and Jun NH2-terminal kinase (JNK) activation, but does not affect phosphorylation of T cell receptor zeta and ZAP70.

Calvo CR, Amsen D, Kruisbeek AM - J. Exp. Med. (1997)

Bottom Line: We here report that CTLA-4 engagement strikingly selectively shuts off activation of downstream T cell receptor (TCR)/CD28 signaling events, i.e., activation of the microtubule-associated protein kinase (MAPKs) ERK and JNK.In sharp contrast, proximal TCR signaling events such as ZAP70 and TCR-zeta chain phosphorylation are not affected by CTLA-4 engagement on activated T cells.Since activation of the ERK and JNK kinases is required for stimulation of interleukin (IL)-2 transcription, these data provide a molecular explanation for the block in IL-2 production imposed by CTLA-4.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute, Amsterdam.

ABSTRACT
Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an important regulator of T cell homeostasis. Ligation of this receptor leads to prominent downregulation of T cell proliferation, mainly as a consequence of interference with IL-2 production. We here report that CTLA-4 engagement strikingly selectively shuts off activation of downstream T cell receptor (TCR)/CD28 signaling events, i.e., activation of the microtubule-associated protein kinase (MAPKs) ERK and JNK. In sharp contrast, proximal TCR signaling events such as ZAP70 and TCR-zeta chain phosphorylation are not affected by CTLA-4 engagement on activated T cells. Since activation of the ERK and JNK kinases is required for stimulation of interleukin (IL)-2 transcription, these data provide a molecular explanation for the block in IL-2 production imposed by CTLA-4.

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CTLA-4 reduces  JNK activity induced by CD3  and CD28 triggering. Preactivated T cells were coated with  different combinations of antibodies and lysed 10 min after addition of warm cross-linking antibody. Lysates were tested for  JNK activity by precipitation  with GST–c-jun precoupled to  glutathione beads followed by in  vitro kinase reactions as described in Materials and Methods. Phosphorylated GST–c-jun is  indicated by the arrow (top). The  same lysates were tested for JNK  protein abundance on immunoblot (middle). JNK activities were  quantified using a phosphorimager and represented as relative  values compared to JNK activity  from unstimulated cells (unstimulated = 1; bottom). Data for the  10-min time point are shown  because no JNK activity could  be measured at earlier times.
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Figure 3: CTLA-4 reduces JNK activity induced by CD3 and CD28 triggering. Preactivated T cells were coated with different combinations of antibodies and lysed 10 min after addition of warm cross-linking antibody. Lysates were tested for JNK activity by precipitation with GST–c-jun precoupled to glutathione beads followed by in vitro kinase reactions as described in Materials and Methods. Phosphorylated GST–c-jun is indicated by the arrow (top). The same lysates were tested for JNK protein abundance on immunoblot (middle). JNK activities were quantified using a phosphorimager and represented as relative values compared to JNK activity from unstimulated cells (unstimulated = 1; bottom). Data for the 10-min time point are shown because no JNK activity could be measured at earlier times.

Mentions: Like the ERKs, the JNK family of MAP kinases have been implicated in induction of IL-2 gene transcription, as these enzymes phosphorylate the NH2 terminus of c-Jun and thereby enhance its transcriptional activity (20, 21, 25, 28, 29). We therefore tested whether the activity of JNK might also be regulated by CTLA-4. JNKs bind with high affinity to c-Jun, such that these kinases can be precipitated from cell lysates with immobilized GST–c-Jun coupled to beads (25), after which in vitro kinase reactions can be performed. It has been shown in both Jurkat T cells and in T cell clones that activation of JNKs by TCR triggering is dependent on costimulation (20, 27). Indeed, we find that also in these preactivated primary T cells, JNK activity is only induced if CD3 triggering is accompanied by CD28 engagement (Fig. 3). Cocross-linking with anti–CTLA-4 results in a clear reduction in JNK activity, demonstrating that CTLA-4 also interferes with the activation or activity of these kinases involved in induction of IL-2 transcription. Whether this reflects a direct effect of CTLA-4 on JNK pathway components or is a consequence of CTLA-4– induced abrogation of TCR signaling remains to be investigated.


Cytotoxic T lymphocyte antigen 4 (CTLA-4) interferes with extracellular signal-regulated kinase (ERK) and Jun NH2-terminal kinase (JNK) activation, but does not affect phosphorylation of T cell receptor zeta and ZAP70.

Calvo CR, Amsen D, Kruisbeek AM - J. Exp. Med. (1997)

CTLA-4 reduces  JNK activity induced by CD3  and CD28 triggering. Preactivated T cells were coated with  different combinations of antibodies and lysed 10 min after addition of warm cross-linking antibody. Lysates were tested for  JNK activity by precipitation  with GST–c-jun precoupled to  glutathione beads followed by in  vitro kinase reactions as described in Materials and Methods. Phosphorylated GST–c-jun is  indicated by the arrow (top). The  same lysates were tested for JNK  protein abundance on immunoblot (middle). JNK activities were  quantified using a phosphorimager and represented as relative  values compared to JNK activity  from unstimulated cells (unstimulated = 1; bottom). Data for the  10-min time point are shown  because no JNK activity could  be measured at earlier times.
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Related In: Results  -  Collection

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Figure 3: CTLA-4 reduces JNK activity induced by CD3 and CD28 triggering. Preactivated T cells were coated with different combinations of antibodies and lysed 10 min after addition of warm cross-linking antibody. Lysates were tested for JNK activity by precipitation with GST–c-jun precoupled to glutathione beads followed by in vitro kinase reactions as described in Materials and Methods. Phosphorylated GST–c-jun is indicated by the arrow (top). The same lysates were tested for JNK protein abundance on immunoblot (middle). JNK activities were quantified using a phosphorimager and represented as relative values compared to JNK activity from unstimulated cells (unstimulated = 1; bottom). Data for the 10-min time point are shown because no JNK activity could be measured at earlier times.
Mentions: Like the ERKs, the JNK family of MAP kinases have been implicated in induction of IL-2 gene transcription, as these enzymes phosphorylate the NH2 terminus of c-Jun and thereby enhance its transcriptional activity (20, 21, 25, 28, 29). We therefore tested whether the activity of JNK might also be regulated by CTLA-4. JNKs bind with high affinity to c-Jun, such that these kinases can be precipitated from cell lysates with immobilized GST–c-Jun coupled to beads (25), after which in vitro kinase reactions can be performed. It has been shown in both Jurkat T cells and in T cell clones that activation of JNKs by TCR triggering is dependent on costimulation (20, 27). Indeed, we find that also in these preactivated primary T cells, JNK activity is only induced if CD3 triggering is accompanied by CD28 engagement (Fig. 3). Cocross-linking with anti–CTLA-4 results in a clear reduction in JNK activity, demonstrating that CTLA-4 also interferes with the activation or activity of these kinases involved in induction of IL-2 transcription. Whether this reflects a direct effect of CTLA-4 on JNK pathway components or is a consequence of CTLA-4– induced abrogation of TCR signaling remains to be investigated.

Bottom Line: We here report that CTLA-4 engagement strikingly selectively shuts off activation of downstream T cell receptor (TCR)/CD28 signaling events, i.e., activation of the microtubule-associated protein kinase (MAPKs) ERK and JNK.In sharp contrast, proximal TCR signaling events such as ZAP70 and TCR-zeta chain phosphorylation are not affected by CTLA-4 engagement on activated T cells.Since activation of the ERK and JNK kinases is required for stimulation of interleukin (IL)-2 transcription, these data provide a molecular explanation for the block in IL-2 production imposed by CTLA-4.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute, Amsterdam.

ABSTRACT
Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an important regulator of T cell homeostasis. Ligation of this receptor leads to prominent downregulation of T cell proliferation, mainly as a consequence of interference with IL-2 production. We here report that CTLA-4 engagement strikingly selectively shuts off activation of downstream T cell receptor (TCR)/CD28 signaling events, i.e., activation of the microtubule-associated protein kinase (MAPKs) ERK and JNK. In sharp contrast, proximal TCR signaling events such as ZAP70 and TCR-zeta chain phosphorylation are not affected by CTLA-4 engagement on activated T cells. Since activation of the ERK and JNK kinases is required for stimulation of interleukin (IL)-2 transcription, these data provide a molecular explanation for the block in IL-2 production imposed by CTLA-4.

Show MeSH