The Phosphorylation State of CD3γ Influences T Cell Responsiveness and Controls T Cell Receptor Cycling

doi:10.1074/jbc.273.37.24232

The Phosphorylation State of CD3γ Influences T Cell Responsiveness and Controls T Cell Receptor Cycling*

From the ^‡Institute of Medical Microbiology and Immunology and ^**Institute of Medical Anatomy, University of Copenhagen, The Panum Institute, Building 18.3, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark, the ^¶Malaghan Institute of Medical Research, Wellington, New Zealand, and the ^‡Basel Institute for Immunology, CH-4005 Basel, Switzerland

Abstract

The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3γ. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the mechanisms involved in the sorting events following PKC-induced internalization are not known. In this study, we demonstrated that following PKC-induced internalization, the TCR is recycled back to the cell surface in a functional state. TCR recycling was dependent on dephosphorylation of CD3γ, probably mediated by the serine/threonine protein phosphatase-2A, but independent on microtubules or actin polymerization. Furthermore, in contrast to ligand-mediated TCR sorting, recycling of the TCR was independent of the tyrosine phosphatase CD45 and the Src tyrosine kinases p56^Lckand p59^Fyn. Studies of mutated TCR and chimeric CD4-CD3γ molecules demonstrated that CD3γ did not contain a recycling signal in itself. In contrast, the only sorting information in CD3γ was the Leu-based motif that mediated lysosomal sorting of chimeric CD4-CD3γ molecules. Finally, we found a correlation between the phosphorylation state of CD3γ and T cell responsiveness. Based on these observations a physiological role of CD3γ and TCR cycling is proposed.

Footnotes

↵* This work was supported in part by The Danish Cancer Society, The Novo Nordisk Foundation, The Danish Medical Research Council, The Danish Natural Science Research Council, Director Ib Henriksens Foundation, Gerda and Aage Haensch’s Foundation, and Director Leo Nielsen and wife Karen Magrethe Nielsen Foundation for Medical Basic Research. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche LTD, Basel, Switzerland.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Recipient of a post-doctoral fellowship from the Danish Medical Research Counsel. To whom correspondence should be addressed. Tel.: 45 3532 7886; Fax: 45 3532 7881; E-mail: vader{at}biobase.dk.
↵‖ Recipient of a Ph.D. scholarship from the University of Copenhagen.
↵§§ Member of The Biotechnology Center for Cellular Communication.
Abbreviations:

PKC

protein kinase C

TCR

T cell receptor

Tyr-based

tyrosine-based

PE

phycoerythrin

PDBu

phorbol 12,13-dibutyrate

MFI

mean fluorescence intensity

ECL

enhanced chemiluminescence

HRP

horseradish peroxidase

PBS

phosphate-buffered saline

FCS

fetal calf serum

FACS

fluorescence-activated cell sorter

[Ca²⁺]_i

intracellular calcium

mAb

monoclonal antibody.
- Received March 9, 1998.
- Revision received May 27, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.