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

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.

SUBMITTER: Alexander DR

PROVIDER: S-EPMC1138760 | biostudies-other | 1989 Jun

REPOSITORIES: biostudies-other

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Project description:Human platelets containing dense granules labelled with 5-hydroxy[14C]tryptamine ([14C]5-HT) were permeabilized by exposure to streptolysin O (SLO) in the presence of 4 mM [gamma-32P]ATP. Addition of either 100 nM phorbol 12-myristate 13-acetate (PMA) or of Ca2+ (pCa 5) at the same time as SLO induced secretion of dense-granule [14C]5-HT and the phosphorylation of pleckstrin by protein kinase C (PKC). Ca2+ also induced phosphorylation of myosin P-light chains. Guanosine 5'-[gamma-thio]triphosphate (GTP[S], 100 microM) did not stimulate secretion from SLO-permeabilized platelets in the absence of Ca2+ (pCa>9), but greatly potentiated secretion in the presence of low PMA (10 nM) or low Ca2+ (pCa 6). However, GTP[S] did stimulate myosin P-light-chain phosphorylation in the absence of Ca2+, an effect that was associated with morphological changes, including granule centralization. Inhibition of PKC and of pleckstrin phosphorylation by Ro 31-8220 blocked secretion induced by PMA or by GTP[S] and PMA in the absence of Ca2+, but did not prevent the GTP[S]-induced phosphorylation of myosin P-light chains or secretion induced by Ca2+ at pCa 5. When the time period between exposure of platelets to SLO and challenge at pCa>9 with PMA or with GTP[S] and PMA was increased, there were rapid and parallel decreases in the secretion and pleckstrin phosphorylation responses, which were lost after 3-5 min. In contrast, the responsiveness of secretion to Ca2+ (pCa 5) or to GTP[S] and Ca2+ (pCa 6) persisted for at least 10 min after exposure of platelets to SLO, although the ability of pleckstrin to undergo phosphorylation was still lost after 3-5 min. Both PKC and pleckstrin were undetectable within platelets after 5 min exposure to SLO. The results suggest that the loss of responsiveness to PMA or to GTP[S] and PMA is attributable to the leakage of PKC (and possibly pleckstrin) from the platelets, whereas secretion stimulated by Ca2+ or by GTP[S] and Ca2+ utilizes membrane-associated Ca2+- and GTP-binding proteins and occurs independently of PKC activation.

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

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