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PMID: 2527499 Published · ppublish English Journal Article

A protein kinase C pseudosubstrate peptide inhibits phosphorylation of the CD3 antigen in streptolysin-O-permeabilized human T lymphocytes.

The Biochemical journal ·Vol. 260 ·No. 3 ·1989-06-15 ·Pages 893-901

Alexander DR, Hexham JM, Lucas SC, Graves JD, Cantrell DA, Crumpton MJ

Abstract

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.

MeSH Terms
Antigens, Differentiation, T-Lymphocyte Bacterial Proteins CD3 Complex Carrier Proteins/pharmacology Cell Membrane Permeability Cells, Cultured Humans Intracellular Signaling Peptides and Proteins Membrane Glycoproteins/metabolism Peptides/pharmacology Phosphorylation Protein Kinase C/antagonists & inhibitors Receptors, Antigen, T-Cell Streptolysins T-Lymphocytes/drug effects,metabolism
Chemicals
Antigens, Differentiation, T-Lymphocyte Bacterial Proteins CD3 Complex Carrier Proteins Intracellular Signaling Peptides and Proteins Membrane Glycoproteins Peptides Receptors, Antigen, T-Cell Streptolysins protein kinase modulator streptolysin O Protein Kinase C
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Alexander D R
Imperial Cancer Research Fund, London, U.K.
Hexham J M
Lucas S C
Graves J D
Cantrell D A
Crumpton M J
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2600893
Published
1989-06-15
Pages
893-901
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1138760
Subset
IM
External Links
PubMed source DOI: 10.1042/bj2600893 PMC Full Text View journal details
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