Aggregation of the high-affinity IgE receptors (FcεRIs) on the surface of granulated mast cells initiates a chain of signaling events culminating in the release of allergy mediators. Although microtubules are involved in mast cell degranulation, the molecular mechanism that controls microtubule rearrangement after FcεRI triggering is poorly understood. In this study, we show that the activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate leads to a rapid polymerization of microtubules. This polymerization was not dependent on the presence of Lyn kinase as determined by experiments with BMMCs isolated from Lyn-negative mice. One of the key regulators of microtubule polymerization is γ-tubulin. Immunoprecipitation experiments revealed that γ-tubulin from activated cells formed complexes with Fyn and Syk protein tyrosine kinases and several tyrosine phosphorylated proteins from both wild-type and Lyn−/− BMMCs. Pretreatment of the cells with Src-family or Syk-family selective tyrosine kinase inhibitors, PP2 or piceatannol, respectively, inhibited the formation of microtubules and reduced the amount of tyrosine phosphorylated proteins in γ-tubulin complexes, suggesting that Src and Syk family kinases are involved in the initial stages of microtubule formation. This notion was corroborated by pull-down experiments in which γ-tubulin complex bounds to the recombinant Src homology 2 and Src homology 3 domains of Fyn kinase. We propose that Fyn and Syk kinases are involved in the regulation of binding properties of γ-tubulin and/or its associated proteins, and thus modulate the microtubule nucleation in activated mast cells.

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γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ-tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γ-tubulin antagonized the inhibitory effect of C53 on DNA damage G(2) /M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin may also have nuclear-specific function(s).

The molecular mechanisms controlling microtubule formation in cells with non-centrosomal microtubular arrays are not yet fully understood. The key component of microtubule nucleation is gamma-tubulin. Although previous results suggested that tyrosine kinases might serve as regulators of gamma-tubulin function, their exact roles remain enigmatic. In the present study, we show that a pool of gamma-tubulin associates with detergent-resistant membranes in differentiating P19 embryonal carcinoma cells, which exhibit elevated expression of the Src family kinase Fyn (protein tyrosine kinase p59(Fyn)). Microtubule-assembly assays demonstrated that membrane-associated gamma-tubulin complexes are capable of initiating the formation of microtubules. Pretreatment of the cells with Src family kinase inhibitors or wortmannin blocked the nucleation activity of the gamma-tubulin complexes. Immunoprecipitation experiments revealed that membrane-associated gamma-tubulin forms complexes with Fyn and PI3K (phosphoinositide 3-kinase). Furthermore, in vitro kinase assays showed that p85alpha (regulatory p85alpha subunit of PI3K) serves as a Fyn substrate. Direct interaction of gamma-tubulin with the C-terminal Src homology 2 domain of p85alpha was determined by pull-down experiments and immunoprecipitation experiments with cells expressing truncated forms of p85alpha. The combined results suggest that Fyn and PI3K might take part in the modulation of membrane-associated gamma-tubulin activities.