Showing papers by "Andre E. Nel published in 1991"

Protein kinase C plays a role in the induction of tyrosine phosphorylation of lymphoid microtubule-associated protein-2 kinase. Evidence for a CD3-associated cascade that includes pp56lck and that is defective in HPB-ALL.

Abstract: Ligation of the CD3 receptor induces multiple signal transduction events that modify the activation state of the T cell. We have compared two lines that express biologically active CD3 receptors but differ in their biochemical activation pathways during ligation of this receptor. Jurkat cells respond to anti-CD3 with Ca2+ mobilization, PKC activation, induction of protein tyrosine phosphorylation, and activation of newly characterized lymphoid microtubule associated protein-2 kinase (MAP-2K). MAP-2K itself is a 43-kDa phosphoprotein that requires tyrosine phosphorylation for activation. Although ligation of the CD3 receptor in HPB-ALL could stimulate tyrosine phosphorylation of a 59- kDa substrate, there was no associated induction of [Ca2+]i flux, PKC, or MAP-2K activation. A specific PKC agonist, PMA, which bypasses the CD3 receptor, could, however, activate MAP-2K in HPB-ALL cells. This implies that defective stimulation of PKC by the CD3 receptor is responsible for its failure to activate MAP-2K in HPB-ALL. The defect in PKC activation is likely distal to the CD3 receptor as A1F14- failed to activate MAP-2K in HPB-ALL but was effective in Jurkat cells. The stimulatory effect of PMA on MAP-2K activity in HPB-ALL was accompanied by tyrosine phosphorylation of this kinase which implies that PKC may, in some way, regulate tyrosine phosphorylation of MAP-2K. A candidate for this role is pp56lck which underwent posttranslational modification (seen as mobility change on SDS-PAGE) during anti-CD3 and PMA stimulation in Jurkat or PMA treatment in HPB-ALL. There was, in fact, exact coincidence between induction of PKC activity, posttranslational modification of lck and tyrosine phosphorylation/activation of MAP-2K. Lck kinase activity in an immune complex kinase assay was unchanged during PMA treatment. An alternative explanation is that modification of lck may alter its substrate profile. We therefore looked at the previously documented ability of PKC to dissociate lck from the CD4 receptor and found that PMA could reduce the stoichiometry of the lck interaction with CD4 in HPB-ALL and to a lesser extent in Jurkat cells. These results imply the existence of a kinase cascade that is initiated by PKC and, in the course of which, lck and MAP-2K may interact.

Activation of MAP-2 kinase activity by the CD2 receptor in Jurkat T cells can be reversed by CD45 phosphatase.

Abstract: We have recently characterized a serine kinase in T lymphocytes which phosphorylates microtubule-associated protein-2 (MAP-2) in vitro. This kinase is activated in a rapidly reversible fashion during ligation of CD3/Ti by a process which involves tyrosine phosphorylation of the enzyme itself. We show that the stimulatory anti-CD2 mAb combination, anti-(T11(2) + T11(3), stimulates MAP-2K activity in Jurkat cells with kinetics that are more prolonged than during anti-CD3 treatment. The principal difference is not in the rate of response induction, but in the decline of the response beyond the peak, to which end anti-CD2 stimulation resembles the sustained phytohaemagglutin (PHA) response. Parallel immunoblotting, utilizing anti-phosphotyrosine antibodies, also revealed differences in the rate at which tyrosine phosphorylation of pp43 (MAP-2K) disappears after induction. In spite of these differences, CD2 was absolutely dependent on the presence of CD3 for inducing a MAP-2K response in Jurkat cells. These results indicate that, even though CD2 and CD3 are using a common signalling pathway in Jurkat cells, additional differences such as the involvement of a tyrosine phosphatase may have to be considered in response generation. We also demonstrate that the common CD45 isoform, when cross-linked to CD2 by mAb, could inhibit the MAP-2K response during both induction as well as the disappearing phase of the response.

Evidence for involvement of glycoprotein-CD45 phosphatase in reversing glycoprotein-CD3-induced microtubule-associated protein-2 kinase activity in Jurkat T-cells

Abstract: Ligation of CD3/TCR on T-cells induces transient activation of lymphoid MAP-2 kinase (MAP-2K), a 43 kDa serine kinase which itself is a substrate of an unidentified tyrosine kinase (pp43). The reversibility of the MAP-2K response agrees with removal of tyrosine phosphates from pp43. Since both activity as well as tyrosine phosphorylation of MAP-2K could be prolonged by Na3VO4, a phosphotyrosine phosphatase inhibitor, we studied the effect of the common CD45 isoform, which is a member of the CD45 phosphatase family, on MAP-2K activity in vivo and in vitro. We demonstrate the ability of purified CD45 phosphatase to remove tyrosine phosphates from partially purified lymphoid MAP-2K. Utilizing the approach of heterologous receptor aggregation, we also showed that CD45 could inhibit the induction of MAP-2K activity in intact Jurkat cells during CD3 or CD3 + CD4 stimulation. We therefore suggest that this phosphatase may control the activity of lymphoid MAP-2K in vivo.