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Antigen activation of mitogen-activated protein kinase in mast cells through protein kinase C-dependent and
C Zhang1, N Hirasawa, M A Beaven
1Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract:
We demonstrate discrete pathways for activation of mitogen-activated protein (MAP) kinase in cultured RBL-2H3 mast cells through protein kinase C (PKC), cytosolic calcium, and a third pathway that provides sustained signals for activation in Ag-stimulated cells. Thus, p42 MAP kinase was activated by increasing intracellular free Ca2+ with thapsigargin or by stimulating PKC with PMA. The latter stimulation was selectively blocked by the protein kinase C inhibitor, Ro31-7549. Stimulation of p42 MAP kinase by Ag resulted in relatively sustained activation of MAP kinase which was only partially suppressed by Ro31-7549. Kinetic studies revealed two components of the MAP kinase response to Ag: a rapid but transient component that was Ro31-7549 sensitive and presumably PKC dependent; and a more sustained component that was Ro31-7549 resistant and presumably PKC independent. Similarly, Ro31-7549 inhibited the early but not late release of arachidonic acid, a finding that was consistent with the known regulation of phospholipase A2 by MAP kinase. Early tyrosine phosphorylation events which were thought to be essential for Ag-induced activation of p42 MAP kinase and release of arachidonic acid were unaffected by Ro31-7549. The findings suggested that release of arachidonic acid was regulated primarily through MAP kinase but that PKC may transiently influence this release, either directly or indirectly through MAP kinase.
Insights
Mitogen-activated protein (MAP) kinase activation in mast cells involves distinct pathways. Protein kinase C (PKC) and calcium mediate early activation, while a sustained pathway exists for antigen stimulation.
Area of Science:
- Cellular signaling
- Immunology
- Biochemistry
Background:
- Mitogen-activated protein (MAP) kinases are crucial signaling molecules in cellular responses.
- Protein kinase C (PKC) and intracellular calcium are known regulators of cellular activation.
- Mast cells play a key role in allergic and inflammatory responses.
Purpose of the Study:
- To elucidate the distinct pathways involved in MAP kinase activation in RBL-2H3 mast cells.
- To investigate the roles of PKC and cytosolic calcium in MAP kinase signaling.
- To characterize the components of MAP kinase activation following antigen stimulation.
Main Methods:
- Utilized RBL-2H3 mast cells for in vitro studies.
- Stimulated cells with thapsigargin (to increase Ca2+), phorbol 12-myristate 13-acetate (PMA), and antigen (Ag).
- Employed the PKC inhibitor Ro31-7549 to differentiate signaling pathways.
Main Results:
- p42 MAP kinase activation was induced by increased intracellular Ca2+ or PKC stimulation.
- Antigen stimulation resulted in sustained p42 MAP kinase activation, with both transient (PKC-dependent) and sustained (PKC-independent) components.
- Ro31-7549 selectively inhibited early arachidonic acid release but not early tyrosine phosphorylation events.
Conclusions:
- MAP kinase activation in mast cells occurs through discrete pathways involving PKC and calcium.
- A sustained MAP kinase activation component, independent of PKC, is observed upon antigen stimulation.
- MAP kinase primarily regulates arachidonic acid release, with a potential transient influence from PKC.