A protein kinase C pseudosubstrate peptide inhibits phosphorylation of the CD3 antigen in streptolysin-O-permeabilized human T lymphocytes.
Activation of human T lymphocytes leads to the phosphorylation of the CD3-antigen gamma polypeptide. We have investigated a possible role for protein kinase C (PKC) in mediating this phosphorylation event by using T cells permeabilized with streptolysin-O in the presence of 120 mM-K+ buffers containing Ca2+-EGTA. The gamma-chain was phosphorylated by [gamma-32P]ATP in permeabilized T lymphoblasts in the presence of phorbol 12,13-dibutyrate (Pdbu) or phytohaemagglutinin (PHA). Ca2+ alone in the range 0.5-1.0 microM also induced gamma-chain phosphorylation in some T-lymphoblast preparations; that in Jurkat-6 cells occurred at lower concentrations (50-500 nM). Two experimental approaches were used to investigate the possible involvement of PKC. Firstly, when permeabilization was carried out in buffer lacking free Ca2+, PKC was lost from the cells, and gamma-chain phosphorylation could then no longer be induced on subsequent addition of Pdbu or PHA in 400 nM-Ca2+, or 800 nM-Ca2+ alone, to permeabilized cells. However, when permeabilization was carried out in the presence of these three agents, PKC was translocated to intracellular membranes, and subsequent addition of [gamma-32P]ATP to these cells then resulted in gamma-chain phosphorylation. In the second approach, induction of gamma-chain phosphorylation by Pdbu, 1-oleoyl-2-acetylglycerol, 1,2-diolein, PHA or Ca2+ alone was effectively blocked by permeabilizing T cells in the presence of a PKC pseudosubstrate peptide (50 microM). Pseudosubstrate concentrations in the range 7-20 microM inhibited gamma-chain phosphorylation by 50%. In contrast, addition of four other 'irrelevant' basic peptides (50 microM) did not result in detectable inhibition, and 50 microM-pseudosubstrate did not inhibit the phosphorylation of 17 other polypeptides isolated from permeabilized T cells. These data suggest that Pdbu-, 1,2-diacylglycerol-, PHA- and Ca2+-induced phosphorylation of the CD3-antigen gamma chain in permeabilized T cells is mediated by PKC.
Alexander
D R
DR
Imperial Cancer Research Fund, London, U.K.
Hexham
J M
JM
Lucas
S C
SC
Graves
J D
JD
Cantrell
D A
DA
Crumpton
M J
MJ
D000945:Antigens, Differentiation, T-Lymphocyte; D001426:Bacterial Proteins; D017252:CD3 Complex; D002352:Carrier Proteins / Q000494:pharmacology; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D011948:Receptors, Antigen, T-Cell; D013301:Streptolysins; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*
D000945:Antigens, Differentiation, T-Lymphocyte; D001426:Bacterial Proteins; D017252:CD3 Complex; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D013301:Streptolysins; C015482:protein kinase modulator; C025964:streptolysin O; D011493:Protein Kinase C