CD3 and CD2 antigen-mediated CD3 gamma-chain phosphorylation in permeabilized human T cells. Regulation by cytosolic phosphatases.
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.
Alexander
D R
DR
Department of Immunology, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, U.K.
Brown
M H
MH
Tutt
A L
AL
Crumpton
M J
MJ
Shivnan
E
E
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D018801:CD2 Antigens; D017252:CD3 Complex / Q000378:metabolism*; D002463:Cell Membrane Permeability; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007163:Immunosorbent Techniques; D008213:Lymphocyte Activation / Q000187:drug effects; D008969:Molecular Sequence Data; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000097:blood*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism*; D013301:Streptolysins; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D001426:Bacterial Proteins; D018801:CD2 Antigens; D017252:CD3 Complex; D003432:Cross-Linking Reagents; D011971:Receptors, Immunologic; D013301:Streptolysins; C025964:streptolysin O; D015240:Phorbol 12,13-Dibutyrate; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases