Phosphorylation of Extracellular Domains of T-Lymphocyte Surface Proteins

doi:10.1074/jbc.271.41.25677

Phosphorylation of Extracellular Domains of T-Lymphocyte Surface Proteins

CONSTITUTIVE SERINE AND THREONINE PHOSPHORYLATION OF THE T CELL ANTIGEN RECEPTOR ECTODOMAINS*

From the Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892

^‡ To whom correspondence and reprint requests should be addressed.

Abstract

The extracellular accumulation of ATP after activation of T-lymphocytes, as well as the presence of ecto-protein kinases in these cells, led us to propose that T cell surface receptors could be regulated through the reversible phosphorylation of their extracellular domains (ectodomains). Here, in a model system, we used T cell transfectants which express T cell antigen receptor chains lacking intracellular and transmembrane protein domains and ³²P_i metabolic labeling of cells to definitively demonstrate phosphorylation of ectodomains of T cell surface proteins. We show that αβTCR ectodomains were phosphorylated intracellularly and constitutively on serine and threonine residues and were then expressed on the T cell surface in phosphorylated form. TCR ectodomains also could be phosphorylated at the cell surface when extracellular [γ-³²P]ATP or [γ-³²P]GTP were used as phosphate donors with the same cells. Consensus phosphorylation sites for serine and threonine protein kinases were found to be strongly evolutionary conserved in both α and β TCR chains constant regions. These results are consistent with the hypothesis, where T cell surface proteins which are phosphorylated intracellularly on their ectodomains, could subsequently be expressed at the cell surface and then be reversibly modified by ectoprotein phosphatase(s) and by ectokinase(s). Such modifications may change T cells cognate interactions by, e.g. affecting TCR-multimolecular complex formation and antigen binding affinity. It is suggested that αβTCR ectodomain phosphorylation could serve as a potential mechanism for regulation of αβTCR-mediated T-lymphocytes response.

Footnotes

↵* 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.
↵¹ The abbreviations used are:

MHC

major histocompatibility complex

αβTCR

αβ T cell receptor

CKII

casein II kinase

GPI

glycophosphatidylinositol

mAb

monoclonal antibody

PLC

phospholipase C

³²P_i

inorganic phosphate

PAGE

polyacrylamide gel electrophoresis.
↵² S. G. Apasov and M. V. Sitkovsky, unpublished observations.
↵³ S. Apasov, unpublished observations.
↵⁴ S. A. Redegeld, P. Smith, and M. Sitkovsky, unpublished observations.
↵⁵ F. Redegeld, unpublished observations.
↵⁶ P. Smith, S. Apasov, and M. Sitkovsky, unpublished results.
- Received June 5, 1996.
- Revision received July 26, 1996.