Rottlerin-independent attenuation of pervanadate-induced tyrosine phosphorylation events by protein kinase C-δ in hemopoietic cells

Abstract

The understanding and control of many pathophysiological conditions is based on knowledge of subtly regulated intracellular signaling networks. We have found that in pervanadate (PV)-treated J558L myeloma cells, amongst other signaling proteins, protein kinase C (PKC)-δ and src homology 2-containing inositol phosphatase (SHIP) are tyrosine phosphorylated on expression of the B cell receptor, suggesting a role for these proteins in the preformed B cell receptor transducer complex. Rottlerin, a widely used PKC-δ-specific inhibitor, efficiently blocks these PV-induced tyrosine phosphorylation events. Furthermore, PV treatment of bone marrow-derived mast cells (BMMC) also results in tyrosine phosphorylation of PKC-δ SHIP, and additional proteins. Rottlerin also inhibits these responses, indicating that PKC-δ might play an important enhancing role in the propagation of phosphotyrosine signals in B cells and mast cells and hence in the regulation of function of both cell types. Therefore, BMMC from PKC-δ -/- mice were generated by in vitro differentiation and assayed for tyrosine phosphorylation events in response to PV. Intriguingly, and opposite to the Rottlerin data, PKC-δ -/- BMMC show a stronger response to PV than wild-type cells, suggesting an attenuating role for PKC-δ. This response can be inhibited equally well by Rottlerin, indicating clearly that Rottlerin is not specific for PKC-δ in vivo. A comparison between Rottlerin and the panspecific PKC inhibitor bisindolylmaleimide suggests that Rottlerin also targets kinases beyond the PKC family. Moreover, Ser473 phosphorylation of protein kinase B (PKB) after PV treatment is blocked by Rottlerin as efficiently as by the phosphatidylinositol 3-kinase inhibitor LY294002. In this report, we provide evidence that PKC-δ constitutes a crucial attenuating factor in B cell and mast cell signal transduction and suggest that PKC-δ is important for the regulation of physiological B and mast cell functions as well as for their pathophysiology. Furthermore, dominant PKC-δ-independent effects of Rottlerin are presented, indicating restrictions of this inhibitor for use in signal transduction research.