Protein tyrosine kinase p53/p56(lyn) forms complexes with gamma-tubulin inrat basophilic leukemia cells

The aggregation of receptors with high affinity for IgE (Fc epsilon RI) on the surface of mast cells and basophils initiates a chain of biochemical ev ents culminating in the release of allergy mediators. Although microtubules have been implicated in the activation process, the molecular mechanism of their interactions with signal transduction molecules is poorly understood . Here we show that in rat basophilic leukemia cells large amounts of alpha beta-tubulin dimers (similar to 70%) and gamma-tubulin (similar to 85%) ar e found in a soluble pool which was released from the cells after permeabil ization with saponin, or extraction with non-ionic detergents. Soluble tubu lins were found in large complexes with other molecules, Complexes of solub le gamma-tubulin released from activated cells contained tyrosine-phosphory lated proteins of relative mel. wt similar to 25, 50, 53, 56, 60, 75, 80, 9 7, 115 and 200 kDa, Increased tyrosine phosphorylation of proteins associat ed with the cytoskeleton, i.e. around centrosomes, was detected by immunofl uorescence microscopy. In vitro kinase assays revealed increased tyrosine p hosphorylation of proteins in gamma-tubulin complexes isolated from activat ed coils, Two of the tyrosine phosphorylated proteins in these complexes we re identified as the p53/56(lyn) kinase, Furthermore, gamma-tubulin bound t o the N-terminal fragment of recombinant Lyn kinase and its binding was sli ghtly enhanced in activated cells. Pretreatment of the cells with Src famil y-selective tyrosine kinase inhibitor, PP1, decreased the amount of tyrosin e phosphorylated proteins in gamma-tubulin complexes, as well as the amount of gamma-tubulin in Lyn kinase immunocomplexes, The combined data suggest that gamma-tubulin is involved in early stages of mast cell activation.