Protein tyrosine kinase p53/p56(lyn) forms complexes with gamma-tubulin in rat basophilic leukemia cells.
The aggregation of receptors with high affinity for IgE (FcepsilonRI) on the surface of mast cells and basophils initiates a chain of biochemical events 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 alphabeta-tubulin dimers ( approximately 70%) and gamma-tubulin ( approximately 85%) are found in a soluble pool which was released from the cells after permeabilization with saponin, or extraction with non-ionic detergents. Soluble tubulins were found in large complexes with other molecules. Complexes of soluble gamma-tubulin released from activated cells contained tyrosine-phosphorylated proteins of relative mol. wt approximately 25, 50, 53, 56, 60, 75, 80, 97, 115 and 200 kDa. Increased tyrosine phosphorylation of proteins associated with the cytoskeleton, i.e. around centrosomes, was detected by immunofluorescence microscopy. In vitro kinase assays revealed increased tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from activated cells. Two of the tyrosine phosphorylated proteins in these complexes were identified as the p53/56(lyn) kinase. Furthermore, gamma-tubulin bound to the N-terminal fragment of recombinant Lyn kinase and its binding was slightly enhanced in activated cells. Pretreatment of the cells with Src family-selective tyrosine kinase inhibitor, PP1, decreased the amount of tyrosine 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.
Dráberová
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L
Department of Mammalian Gene Expression and Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic.
Dráberová
E
E
Surviladze
Z
Z
Dráber
P
P
Dráber
P
P
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001491:Basophils / Q000276:immunology*; D002499:Centrifugation, Density Gradient; D005455:Fluorescent Antibody Technique; D015471:Leukemia, Basophilic, Acute / Q000276:immunology; D008213:Lymphocyte Activation; D010766:Phosphorylation; D051381:Rats; D014404:Tubulin / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D014404:Tubulin; D014443:Tyrosine; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases