CD3 and CD2 antigen-mediated CD3 γ-chain phosphorylation in permeabilized human T cells : regulation by cytosolic phosphatases

• Published in: Biochemical journal 1992-11, Vol.288 (1), p.69-77
• Main Authors: ALEXANDER, D. R, BROWN, M. H, TUTT, A. L, CRUMPTON, M. J, SHIVNAN, E
• Format: Article
• Language: English
• Subjects: activity; Adenosine Triphosphate - pharmacology; Amino Acid Sequence; Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte - metabolism; Bacterial Proteins; Binding Sites; Biological and medical sciences; CD2 Antigens; CD3 antigen; CD3 Complex - metabolism; Cell Membrane Permeability; Cross-Linking Reagents; Cytosol - enzymology; Fundamental and applied biological sciences. Psychology; Fundamental immunology; gamma -chain; Humans; Immunobiology; Immunosorbent Techniques; Lymphocyte Activation - drug effects; lymphocytes T; Lymphoid cells: ontogeny, maturation, markers, receptors, circulation and recirculation; man; Molecular Sequence Data; Phorbol 12,13-Dibutyrate - pharmacology; phosphoprotein phosphatase; Phosphoric Monoester Hydrolases - antagonists & inhibitors; Phosphoric Monoester Hydrolases - blood; Phosphorylation; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - metabolism; Receptors, Immunologic - metabolism; Streptolysins; T-Lymphocytes - enzymology; T-Lymphocytes - immunology
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2880069

The role of cytosolic and membrane-associated phosphatases in regulating dephosphorylation of the CD3 antigen gamma-chain has been investigated using streptolysin-O-permeabilized T lymphoblasts and Jurkat T leukaemia cells. Permeabilization of T cells caused a rapid extrusion of cytosolic type 2A phosphatases, but a membrane-associated phosphorylase phosphatase activity remained inside the cells. This activity had the properties characteristic of type 2A phosphatases, being resistant to inhibition by type 1 phosphatase inhibitors, though it was inhibited in a time-dependent manner by ATP or by non-hydrolysable ATP analogues, but not by GTP, CTP, ITP or PPi. The membrane-associated type 2A phosphatase in permeabilized cells did not dephosphorylate the CD3 antigen gamma-chain, suggesting that cytosolic phosphatases dephosphorylate the gamma-chain in situ. Cross-linking the CD2 and CD3 antigens with a bivalent monoclonal antibody in the absence of cytosolic phosphatases induced marked phosphorylation of the CD3 gamma-chain, immunoprecipitated using a novel gamma-chain peptide analogue directed antiserum (TG1). Phosphorylation was inhibited by a protein kinase C (PKC) pseudosubstrate inhibitor, indicating that CD2/CD3-induced gamma-chain phosphorylation is a PKC-mediated event. Activation of T cells either with phorbol 12,13-dibutyrate or by CD2-CD3 cross-linking caused [32P]Pi incorporation into the same gamma-chain Ser residues. The site-mapping data suggested that PKC in situ may incorporate phosphate at the CD3 gamma-chain Ser-123 and Ser-126 residues, but that phosphate is rapidly lost from Ser-123 by cytosolic phosphatase action. Our findings underline the importance of the dual actions of kinases and phosphatases as potential regulators of T cell antigen-receptor complex function.