A critical role of conventional protein kinase C in morphological changes of rodent mast cells

In mast cells, crosslinking the high-affinity IgE receptor (Fc epsilon RI) results in a dynamic reorganization of the actin cytoskeleton that is associated with membrane ruffling. Although the signaling involved in degranulation has been well described, it is less understood in morphological changes. In this study, we investigated the specific role of conventional protein kinase C (cPKC), a crucial signal for degranulation, in antigen-induced membrane ruffling of mast cells. In RBL-2H3 mast cells, antigen induced a long-lasting membrane ruffling, which was blocked with late-added Go6976, a specific cPKC inhibitor, indicating that sustained activation of cPKC is required for maintaining the reaction. Immunofluorescence staining of endogenous PKC alpha/beta and real-time imaging of transfected green fluorescent protein-tagged PKC alpha/beta demonstrated that in response to antigen both PKC alpha and PKC beta I quickly translocated to the plasma membrane and were colocalized with actin filaments at the ruffling sites. These reactions were blocked by expression of kinase-negative PKC beta I, but not kinase-negative PKC alpha, and by treatment with a specific PKC beta inhibitor, LY333531. The adhesion, spreading and membrane ruffling of mouse bone marrow-derived mast cells (BMMCs), which are mostly nonadhesive, were promoted by both antigen and thymeleatoxin. Treatment with Go6976 abolished all these reactions. Antigen-mediated migration of BMMC was also sensitive to Go6076 and LY333531. In addition, BMMC adhesion by and migration toward stem cell factor were shown to be dependent on cPKC. Thus, cPKC, at least PKC beta subtype, may be critical for the dynamic morphological changes that lead to the migration of mast cells. Immunology and Cell Biology (2011) 89, 149-159; doi:10.1038/icb.2010.67; published online 25 May 2010