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The 8 isoform of protein kinase C is phosphorylated on tyrosine in response to antigen activation of the high-affinity receptor for immunoglobulin E. While protein kinase C-6 associates with and phosphorylates this receptor, immunoprecipitation of the receptor revealed that little, if any, tyrosine-phosphorylated protein kinase C-S is receptor associated. In vitro kinase assays with immunoprecipitated tyrosine-phosphorylated protein kinase C-S showed that the modified enzyme had diminished activity toward the receptor y-chain peptide as a substrate but not toward histones or myelin basic protein peptide. We propose a model in which the tyrosine phosphorylation of protein kinase C-S regulates the kinase specificity toward a given substrate. This may represent a general mechanism by which in vivo protein kinase activities are regulated in response to external stimuli.The activation of the mucosal mast cell model, rat basophilic leukemia (RBL) cells, by the interaction of antigen with cell-bound immunoglobulin E (IgE) initiates the rapid translocation and increased activity of protein kinase C (PKC) (1, 2). This PKC activation is critical to the effector function of the mast cell, namely exocytosis of mediators of the allergic response (3,4). Protein kinase C-8 associates with and phosphorylates the high-affinity receptor for IgE FcsRI (5) on these cells, while other PKC isozymes regulate phosphoinositide hydrolysis (6) and induce protooncogene expression (7).Recent studies have begun to suggest that PKC may regulate tyrosine kinases and is itself regulated by phosphorylation interactions with these kinases (8-10). Yao et al. (10) have shown a PKC-mediated inactivation of Bruton tyrosine kinase (BTK), an enzyme mutated in chromosome X-linked agammaglobulinemia patients. Moreover, BTK was found to associate with the f3I isoform of PKC in mast cells. Phosphorylation of serine/threonine kinases by tyrosine kinase activity has also been described (9, 11). Denning et al. (9) showed that PKC-8 was tyrosine phosphorylated in cells expressing oncogenic ras. Li et al. (12) reported that 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment of PKC-8 transfectants also led to tyrosine phosphorylation of this isozyme. More recent evidence from the latter group suggests that the tyrosine phosphorylation of PKC-8 can occur in response to platelet-derived growth factor receptor stimulation in PKC-8 transfected cells (13). Collectively, the latter studies document the phosphorylation of a serine/threonine kinase by a tyrosine kinase activity in response to a stimulus, although the physiological significance of tyrosine phosphorylation of PKC-8 is unclear.In the present study, we investigate whether tyrosine phosphorylation of PKC-8 occurs in response to FceRI aggregation and whether the tyrosine-phosphorylated form of PKC-8 is preferentially receptor associated. We also analyze the effect of tyrosine phosphorylation on the activity of PKC-8 toward various substrates, including the FcsRI y chain. MATERIALS AND METHODSCell Cu...

Adhesion of RBL-2H3 mucosal mast cells to fibronectin-coated surfaces has been linked to changes in secretion and tyrosine kinase activity. We now show that adhesion affects the sensitivity of RBL cells to the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). In suspended cells, PMA inhibited antigen-induced calcium influx (as measured by manganese influx) and changes in intracellular free calcium and had complex effects on antigen-stimulated secretion. However, in adherent cells PMA had little effect on these responses. Suspended cells only secreted in response to thapsigargin if they were co-treated with PMA, while adherent cells secreted in response to thapsigargin alone. The thapsigargin-induced secretion in adherent cells was inhibited by protein kinase C down-regulation and by the protein kinase C inhibitor GF 109203X, but not by calphostin C. We suggest that protein kinase C is constitutively activated in adherent cells, possibly due to modification of the regulatory domain of the enzyme.