Fine-tuning of proximal TCR signaling by ZAP-70 tyrosine residues in jurkat cells

M. Szabo, T. Czömpöly, K. Kvell, G. Talaber, D. Bartis, P. Németh, T. Berki, F. Boldizsár

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Zeta-chain-associated protein kinase of 70kDa (ZAP-70) kinase is a key regulator in the early steps of TCR signaling but some aspects of its fine regulation are still unclear. From its 31 tyrosine (Y) residues, 11 phosphorylation sites have been identified, some with activator (Y315 and Y493) or inhibitory (Y292 and Y492) and others with unknown function (Y069, Y126 and Y178). In our present work, we aimed to elucidate the role of different Y residues of ZAP-70, especially those with unknown function, in calcium signaling and the autoregulation of the kinase. ZAP-70-deficient Jurkat cells (P116) were stably reconstituted with point-mutated ZAP-70 constructs where tyrosine residues 069, 126, 178, 238, 292, 315, 492 or 493 were replaced with phenylalanine (F). The anti-CD3-elicited calcium signal increased in F069-, F292- and F492-ZAP-70-expressing cell lines but decreased in the F126-, F315- and F493-ZAP-70-expressing cell lines. ZAP-70 point mutations led to phosphorylation changes predominantly in SH2 domain containing leukocyte protein of 76kDa (SLP-76) but not linker of activated T cells (LAT) during CD3-activation; moreover, we detected basal hyperphosphorylation of SLP-76 Y128 in the F126-, F178- and F492-ZAP-70-expressing cell lines. In summary, Y069, Y178, Y292 and Y492 have inhibitory, while Y126, Y315 and Y493 activator role in anti-CD3-induced T-cell activation. Phosphorylation changes in LAT and SLP-76 suggest that fine regulation of ZAP-70 on calcium signaling is rather transmitted through SLP-76 not LAT. Additionally, negative or positive autoregulatory function of Y292 and Y493 or Y315, respectively, was revealed in ZAP-70. These data indicate that previously not characterized Y069, Y126 and Y178 in ZAP-70 participate in the fine regulation of TCR signaling.

Original languageEnglish
Pages (from-to)79-87
Number of pages9
JournalInternational Immunology
Volume24
Issue number2
DOIs
Publication statusPublished - 2012

Fingerprint

Jurkat Cells
Tyrosine
ZAP-70 Protein-Tyrosine Kinase
T-Lymphocytes
Calcium Signaling
Phosphorylation
Cell Line
src Homology Domains
Phenylalanine
Point Mutation
Leukocytes
Homeostasis
Phosphotransferases
Calcium
Proteins

Keywords

  • Lentiviral transfection
  • Site-directed mutagenesis
  • TCR signaling
  • ZAP-70

ASJC Scopus subject areas

  • Immunology

Cite this

Fine-tuning of proximal TCR signaling by ZAP-70 tyrosine residues in jurkat cells. / Szabo, M.; Czömpöly, T.; Kvell, K.; Talaber, G.; Bartis, D.; Németh, P.; Berki, T.; Boldizsár, F.

In: International Immunology, Vol. 24, No. 2, 2012, p. 79-87.

Research output: Contribution to journalArticle

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abstract = "Zeta-chain-associated protein kinase of 70kDa (ZAP-70) kinase is a key regulator in the early steps of TCR signaling but some aspects of its fine regulation are still unclear. From its 31 tyrosine (Y) residues, 11 phosphorylation sites have been identified, some with activator (Y315 and Y493) or inhibitory (Y292 and Y492) and others with unknown function (Y069, Y126 and Y178). In our present work, we aimed to elucidate the role of different Y residues of ZAP-70, especially those with unknown function, in calcium signaling and the autoregulation of the kinase. ZAP-70-deficient Jurkat cells (P116) were stably reconstituted with point-mutated ZAP-70 constructs where tyrosine residues 069, 126, 178, 238, 292, 315, 492 or 493 were replaced with phenylalanine (F). The anti-CD3-elicited calcium signal increased in F069-, F292- and F492-ZAP-70-expressing cell lines but decreased in the F126-, F315- and F493-ZAP-70-expressing cell lines. ZAP-70 point mutations led to phosphorylation changes predominantly in SH2 domain containing leukocyte protein of 76kDa (SLP-76) but not linker of activated T cells (LAT) during CD3-activation; moreover, we detected basal hyperphosphorylation of SLP-76 Y128 in the F126-, F178- and F492-ZAP-70-expressing cell lines. In summary, Y069, Y178, Y292 and Y492 have inhibitory, while Y126, Y315 and Y493 activator role in anti-CD3-induced T-cell activation. Phosphorylation changes in LAT and SLP-76 suggest that fine regulation of ZAP-70 on calcium signaling is rather transmitted through SLP-76 not LAT. Additionally, negative or positive autoregulatory function of Y292 and Y493 or Y315, respectively, was revealed in ZAP-70. These data indicate that previously not characterized Y069, Y126 and Y178 in ZAP-70 participate in the fine regulation of TCR signaling.",
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T1 - Fine-tuning of proximal TCR signaling by ZAP-70 tyrosine residues in jurkat cells

AU - Szabo, M.

AU - Czömpöly, T.

AU - Kvell, K.

AU - Talaber, G.

AU - Bartis, D.

AU - Németh, P.

AU - Berki, T.

AU - Boldizsár, F.

PY - 2012

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AB - Zeta-chain-associated protein kinase of 70kDa (ZAP-70) kinase is a key regulator in the early steps of TCR signaling but some aspects of its fine regulation are still unclear. From its 31 tyrosine (Y) residues, 11 phosphorylation sites have been identified, some with activator (Y315 and Y493) or inhibitory (Y292 and Y492) and others with unknown function (Y069, Y126 and Y178). In our present work, we aimed to elucidate the role of different Y residues of ZAP-70, especially those with unknown function, in calcium signaling and the autoregulation of the kinase. ZAP-70-deficient Jurkat cells (P116) were stably reconstituted with point-mutated ZAP-70 constructs where tyrosine residues 069, 126, 178, 238, 292, 315, 492 or 493 were replaced with phenylalanine (F). The anti-CD3-elicited calcium signal increased in F069-, F292- and F492-ZAP-70-expressing cell lines but decreased in the F126-, F315- and F493-ZAP-70-expressing cell lines. ZAP-70 point mutations led to phosphorylation changes predominantly in SH2 domain containing leukocyte protein of 76kDa (SLP-76) but not linker of activated T cells (LAT) during CD3-activation; moreover, we detected basal hyperphosphorylation of SLP-76 Y128 in the F126-, F178- and F492-ZAP-70-expressing cell lines. In summary, Y069, Y178, Y292 and Y492 have inhibitory, while Y126, Y315 and Y493 activator role in anti-CD3-induced T-cell activation. Phosphorylation changes in LAT and SLP-76 suggest that fine regulation of ZAP-70 on calcium signaling is rather transmitted through SLP-76 not LAT. Additionally, negative or positive autoregulatory function of Y292 and Y493 or Y315, respectively, was revealed in ZAP-70. These data indicate that previously not characterized Y069, Y126 and Y178 in ZAP-70 participate in the fine regulation of TCR signaling.

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