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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.

Type

Journal article

Journal

Biochem J

Publication Date

15/11/1992

Volume

288 ( Pt 1)

Pages

69 - 77

Keywords

Adenosine Triphosphate, Amino Acid Sequence, Antibodies, Monoclonal, Antigens, CD2, Antigens, CD3, Antigens, Differentiation, T-Lymphocyte, Bacterial Proteins, Binding Sites, Cell Membrane Permeability, Cross-Linking Reagents, Cytosol, Humans, Immunosorbent Techniques, Lymphocyte Activation, Molecular Sequence Data, Phorbol 12,13-Dibutyrate, Phosphoric Monoester Hydrolases, Phosphorylation, Protein Kinase C, Receptors, Immunologic, Streptolysins, T-Lymphocytes