Differential expression of human γ-tubulin isotypes during neuronal development and oxidative stress points to a γ-tubulin-2 prosurvival function.

γ-Tubulins are highly conserved members of the tubulin superfamily essential for microtubule nucleation. Humans possess 2 γ-tubulin genes. It is thought that γ-tubulin-1 represents a ubiquitous isotype, whereas γ-tubulin-2 is found predominantly in the brain, where it may be endowed with divergent functions beyond microtubule nucleation. The molecular basis of the purported functional differences between γ-tubulins is unknown. We report discrimination of human γ-tubulins according to their electrophoretic and immunochemical properties. In vitro mutagenesis revealed that the differences in electrophoretic mobility originate in the C-terminal regions of the γ-tubulins. Using epitope mapping, we discovered mouse monoclonal antibodies that can discriminate between human γ-tubulin isotypes. Real time quantitative RT-PCR and 2-dimensional-PAGE showed that γ-tubulin-1 is the dominant isotype in fetal neurons. Although γ-tubulin-2 accumulates in the adult brain, γ-tubulin-1 remains the major isotype in various brain regions. Localization of γ-tubulin-1 in mature neurons was confirmed by immunohistochemistry and immunofluorescence microscopy on clinical samples and tissue microarrays. Differentiation of SH-SY5Y human neuroblastoma cells by all-trans retinoic acid, or oxidative stress induced by mitochondrial inhibitors, resulted in upregulation of γ-tubulin-2, whereas the expression of γ-tubulin-1 was unchanged. Fractionation experiments and immunoelectron microscopy revealed an association of γ-tubulins with mitochondrial membranes. These data indicate that in the face of predominant γ-tubulin-1 expression, the accumulation of γ-tubulin-2 in mature neurons and neuroblastoma cells during oxidative stress may denote a prosurvival role of γ-tubulin-2 in neurons.-Dráberová, E., Sulimenko, V., Vinopal, S., Sulimenko, T., Sládková, V., D'Agostino, L., Sobol, M., Hozák, P., Kren, L., Katsetos, C. D., Dráber, P. Differential expression of human γ-tubulin isotypes during neuronal development and oxidative stress points to γ-tubulin-2 prosurvival function.

GIT1/ßPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.

Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (ßPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, ßPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of ßPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and ßPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and ßPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, ßPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and ßPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of ßPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/ßPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells.

Microtubule nucleation in mouse bone marrow-derived mast cells is regulated by the concerted action of GIT1/ßPIX proteins and calcium.

Ag-mediated activation of mast cells initiates signaling events leading to Ca(2+) response, release of allergic mediators from cytoplasmic granules, and synthesis of cytokines and chemokines. Although microtubule rearrangement during activation has been described, the molecular mechanisms that control their remodeling are largely unknown. Microtubule nucleation is mediated by complexes that are formed by γ-tubulin and γ-tubulin complex proteins. In this study, we report that, in bone marrow-derived mast cells (BMMCs), γ-tubulin interacts with p21-activated kinase interacting exchange factor ß (ßPIX) and G protein-coupled receptor kinase-interacting protein (GIT)1. Microtubule regrowth experiments showed that the depletion of ßPIX in BMMCs stimulated microtubule nucleation, whereas depletion of GIT1 led to the inhibition of nucleation compared with control cells. Phenotypic rescue experiments confirmed that ßPIX and GIT1 represent negative and positive regulators of microtubule nucleation in BMMCs, respectively. Live-cell imaging disclosed that both proteins are associated with centrosomes. Immunoprecipitation and pull-down experiments revealed that an enhanced level of free cytosolic Ca(2+) affects γ-tubulin properties and stimulates the association of GIT1 and γ-tubulin complex proteins with γ-tubulin. Microtubule nucleation also was affected by Ca(2+) level. Moreover, in activated BMMCs, γ-tubulin formed complexes with tyrosine-phosphorylated GIT1. Further experiments showed that GIT1 and ßPIX are involved in the regulation of such important physiological processes as Ag-induced chemotaxis and degranulation. Our study provides for the first time, to our knowledge, a possible mechanism for the concerted action of tyrosine kinases, GIT1/ßPIX proteins, and Ca(2+) in the propagation of signals leading to the regulation of microtubule nucleation in activated mast cells.

Molecular events accompanying rous sarcoma virus rescue from rodent cells and the role of viral gene complementation.

UNLABELLED: Transformation of rodent cells with avian Rous sarcoma virus (RSV) opened new ways to studying virus integration and expression in nonpermissive cells. We were interested in (i) the molecular changes accompanying fusion of RSV-transformed mammalian cells with avian cells leading to virus rescue and (ii) enhancement of this process by retroviral gene products. The RSV-transformed hamster RSCh cell line was characterized as producing only a marginal amount of env mRNA, no envelope glycoprotein, and a small amount of unprocessed Gag protein. Egress of viral unspliced genomic RNA from the nucleus was hampered, and its stability decreased. Cell fusion of the chicken DF-1 cell line with RSCh cells led to production of env mRNA, envelope glycoprotein, and processed Gag and virus-like particle formation. Proteosynthesis inhibition in DF-1 cells suppressed steps leading to virus rescue. Furthermore, new aberrantly spliced env mRNA species were found in the RSCh cells. Finally, we demonstrated that virus rescue efficiency can be significantly increased by complementation with the env gene and the highly expressed gag gene and can be increased the most by a helper virus infection. In summary, Env and Gag synthesis is increased after RSV-transformed hamster cell fusion with chicken fibroblasts, and both proteins provided in trans enhance RSV rescue. We conclude that the chicken fibroblast yields some factor(s) needed for RSV replication, particularly Env and Gag synthesis, in nonpermissive rodent cells. IMPORTANCE: One of the important issues in retrovirus heterotransmission is related to cellular factors that prevent virus replication. Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, is able to infect and transform mammalian cells; however, such transformed cells do not produce infectious virus particles. Using the well-defined model of RSV-transformed rodent cells, we established that the lack of virus replication is due to the absence of chicken factor(s), which can be supplemented by cell fusion. Cell fusion with permissive chicken cells led to an increase in RNA splicing and nuclear export of specific viral mRNAs, as well as synthesis of respective viral proteins and production of virus-like particles. RSV rescue by cell fusion can be potentiated by in trans expression of viral genes in chicken cells. We conclude that rodent cells lack some chicken factor(s) required for proper viral RNA processing and viral protein synthesis.

Regulation of microtubule nucleation in mouse bone marrow-derived mast cells by ARF GTPase-activating protein GIT2.

Aggregation of high-affinity IgE receptors (FcϵRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcϵRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.

STIM1-directed reorganization of microtubules in activated mast cells.

Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca(2+) controlled by store-operated Ca(2+) entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca(2+). Changes in cytosolic Ca(2+)concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with knockdown or reexpression of STIM1, the key regulator of SOCE, confirmed the important role of STIM1 in the formation of microtubule protrusions. Although STIM1 in activated cells formed puncta associated with microtubules in protrusions, relocation of STIM1 to a close proximity of cell membrane was independent of growing microtubules. In accordance with the inhibition of Ag-induced Ca(2+) response and decreased formation of microtubule protrusions in BMMCs with reduced STIM1, the cells also exhibited impaired chemotactic response to Ag. We propose that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca(2+) plays an important role in the formation of microtubule protrusions in BMMCs.

Nuclear γ-tubulin associates with nucleoli and interacts with tumor suppressor protein C53.

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).

γ-Tubulin in microtubule nucleation and beyond.

Microtubules composed of αß-tubulin dimers are dynamic cytoskeletal polymers that play key roles in essential cellular processes such as cell division, organelle positioning, intracellular transport, and cell migration. γ-Tubulin is a highly conserved member of the tubulin family that is required for microtubule nucleation. γ-Tubulin, together with its associated proteins, forms the γ-tubulin ring complex (γ-TuRC), that templates microtubules. Here we review recent advances in the structure of γ-TuRC, its activation, and centrosomal recruitment. This provides new mechanistic insights into the molecular mechanism of microtubule nucleation. Accumulating data suggest that γ-tubulin also has other, less well understood functions. We discuss emerging evidence that γ-tubulin can form oligomers and filaments, has specific nuclear functions, and might be involved in centrosomal cross-talk between microtubules and microfilaments.

C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress.

ER distribution depends on microtubules, and ER homeostasis disturbance activates the unfolded protein response resulting in ER remodeling. CDK5RAP3 (C53) implicated in various signaling pathways interacts with UFM1-protein ligase 1 (UFL1), which mediates the ufmylation of proteins in response to ER stress. Here we find that UFL1 and C53 associate with γ-tubulin ring complex proteins. Knockout of UFL1 or C53 in human osteosarcoma cells induces ER stress and boosts centrosomal microtubule nucleation accompanied by γ-tubulin accumulation, microtubule formation, and ER expansion. C53, which is stabilized by UFL1, associates with the centrosome and rescues microtubule nucleation in cells lacking UFL1. Pharmacological induction of ER stress by tunicamycin also leads to increased microtubule nucleation and ER expansion. Furthermore, tunicamycin suppresses the association of C53 with the centrosome. These findings point to a novel mechanism for the relief of ER stress by stimulation of centrosomal microtubule nucleation.

Dysregulation of Microtubule Nucleating Proteins in Cancer Cells.

In cells, microtubules typically nucleate from microtubule organizing centers, such as centrosomes. γ-Tubulin, which forms multiprotein complexes, is essential for nucleation. The γ-tubulin ring complex (γ-TuRC) is an efficient microtubule nucleator that requires additional centrosomal proteins for its activation and targeting. Evidence suggests that there is a dysfunction of centrosomal microtubule nucleation in cancer cells. Despite decades of molecular analysis of γ-TuRC and its interacting factors, the mechanisms of microtubule nucleation in normal and cancer cells remains obscure. Here, we review recent work on the high-resolution structure of γ-TuRC, which brings new insight into the mechanism of microtubule nucleation. We discuss the effects of γ-TuRC protein dysregulation on cancer cell behavior and new compounds targeting γ-tubulin. Drugs inhibiting γ-TuRC functions could represent an alternative to microtubule targeting agents in cancer chemotherapy.

Stabilization of Proteins by Freeze-Drying in the Presence of Trehalose: A Case Study of Tubulin.

Microtubules, polymers of the heterodimeric protein αß-tubulin, are indispensable for many cellular activities such as maintenance of cell shape, division, migration, and ordered vesicle transport. In vitro assays to study microtubule functions and their regulation by associated proteins require the availability of assembly-competent purified tubulin. However, tubulin is a thermolabile protein that rapidly converts into a nonpolymerizing state. For this reason, it is usually stored at -80 °C or liquid nitrogen to preserve its conformation and polymerization properties. In this chapter, we describe a method for freeze-drying of assembly-competent tubulin in the presence of nonreducing sugar trehalose, and methods enabling the evaluation of tubulin functions in rehydrated samples.

The actin regulator profilin 1 is functionally associated with the mammalian centrosome.

Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.

Multimodal fluorescently labeled polymer-coated GdF3 nanoparticles inhibit degranulation in mast cells.

Multimodal gadolinium fluoride nanoparticles belong to potential contrast agents useful for bimodal optical fluorescence and magnetic resonance imaging. However, the metallic nature of the nanoparticles, similarly to some paramagnetic iron oxides, might induce allergic and anaphylactic reactions in patients after administration. A reduction of these adverse side effects is a priority for the safe application of the nanoparticles. Herein, we prepared paramagnetic poly(4-styrenesulfonic acid-co-maleic acid) (PSSMA)-stabilized GdF3 nanoparticles with surface modified by Atto 488-labeled poly(styrene-grad-2-dimethylaminoethyl acrylate)-block-poly(2-dimethylaminoethyl acrylate) (PSDA-A488) with reactive amino groups for introduction of an additional imaging (luminescence) modality and possible targeting of anticancer drugs. The saturation magnetization of GdF3@PSSMA particles according to SQUID magnetometry reached 157 Am2 kg-1 at 2 K and magnetic field of 7 T. GdF3@PSSMA-PSDA-A488 nanoparticles were well tolerated by human cervical adenocarcinoma (HeLa), mouse bone marrow-derived mast cells (BMMC), and rat basophilic mast cells (RBL-2H3); the particles also affected cell morphology and protein tyrosine phosphorylation in mast cells. Moreover, the nanoparticles interfered with the activation of mast cells by multivalent antigens and inhibited calcium mobilization and cell degranulation. These findings show that the new multimodal GdF3-based nanoparticles possess properties useful for various imaging methods and might minimize mast cell degranulation incurred after future nanoparticle diagnostic administration.

Differential expression and cellular distribution of gamma-tubulin and betaIII-tubulin in medulloblastomas and human medulloblastoma cell lines.

In previous studies, we have shown overexpression and ectopic subcellular distribution of gamma-tubulin and betaIII-tubulin in human glioblastomas and glioblastoma cell lines (Katsetos et al., 2006, J Neuropathol Exp Neurol 65:455-467; Katsetos et al., 2007, Neurochem Res 32:1387-1398). Here we determined the expression of gamma-tubulin in surgically excised medulloblastomas (n = 20) and in the human medulloblastoma cell lines D283 Med and DAOY. In clinical tissue samples, the immunohistochemical distribution of gamma-tubulin labeling was pervasive and inversely related to neuritogenesis. Overexpression of gamma-tubulin was widespread in poorly differentiated, proliferating tumor cells but was significantly diminished in quiescent differentiating tumor cells undergoing neuritogenesis, highlighted by betaIII-tubulin immunolabeling. By quantitative real-time PCR, gamma-tubulin transcripts for TUBG1, TUBG2, and TUBB3 genes were detected in both cell lines but expression was less prominent when compared with the human glioblastoma cell lines. Immunoblotting revealed comparable amounts of gamma-tubulin and betaIII-tubulin in different phases of cell cycle; however, a larger amount of gamma-tubulin was detected in D283 Med when compared with DAOY cells. Interphase D283 Med cells exhibited predominantly diffuse cytoplasmic gamma-tubulin localization, in addition to the expected centrosome-associated distribution. Robust betaIII-tubulin immunoreactivity was detected in mitotic spindles of DAOY cells. Our data indicate that overexpression of gamma-tubulin may be linked to phenotypic dedifferentiation (anaplasia) and tumor progression in medulloblastomas and may potentially serve as a promising tumor marker.

Recovery of tubulin functions after freeze-drying in the presence of trehalose.

Microtubules represent cytoplasmic structures that are indispensable for the maintenance of cell morphology and motility generation. Due to their regular structural organization, microtubules have become of great interest for preparation of in vitro nanotransport systems. However, tubulin, the major building protein of microtubules, is a thermolabile protein and is usually stored at -80 degrees C to preserve its conformation and polymerization properties. Here we describe a novel method for freeze-drying of assembly-competent tubulin in the presence of a nonreducing sugar trehalose. Even after prolonged storage at ambient temperature, rehydrated tubulin is capable of binding antimitotic drugs and assembling to microtubules that bind microtubule-associated proteins in the usual way. Electron microscopy confirmed that rehydrated tubulin assembles into normal microtubules that are able to generate motility by interaction with the motor protein kinesin in a cell-free environment. Freeze-drying also preserved preformed microtubules. Rehydrated tubulin and microtubules can be used for preparation of diverse in vitro and in vivo assays as well as for preparation of bionanodevices.

Microtubule Cytoskeleton Remodeling by Nanosecond Pulsed Electric Fields.

Remodeling of nanoscopic structures is not just crucial for cell biology, but it is also at the core of bioinspired materials. While the microtubule cytoskeleton in cells undergoes fast adaptation, adaptive materials still face this remodeling challenge. Moreover, the guided reorganization of the microtubule network and the correction of its abnormalities is still a major aim. This work reports new findings for externally triggered microtubule network remodeling by nanosecond electropulses (nsEPs). At first, a wide range of nsEP parameters, applied in a low conductivity buffer, is explored to find out the minimal nsEP dosage needed to disturb microtubules in various cell types. The time course of apoptosis and microtubule recovery in the culture medium is thereafter assessed. Application of nsEPs to cells in culture media result in modulation of microtubule binding properties to end-binding (EB1) protein, quantified by newly developed image processing techniques. The microtubules in nsEP-treated cells in the culture medium have longer EB1 comets but their density is lower than that of the control. The nsEP treatment represents a strategy for microtubule remodeling-based nano-biotechnological applications, such as engineering of self-healing materials, and as a manipulation tool for the evaluation of microtubule remodeling mechanisms during various biological processes in health and disease.

Tubulin targets in the pathobiology and therapy of glioblastoma multiforme. II. gamma-Tubulin.

Glioblastoma multiforme (GBM) is the most common and deadliest form of primary brain cancer in adults. Despite advances in molecular biology and genetics of cancer there is no currently available treatment for these tumors. Aberrant patterns of gamma-tubulin expression and compartmentalization in GBM have been reported lending credence to the assertion that these changes might underlie perturbations in microtubule nucleation and mitosis associated with glioma tumorigenesis and tumor progression. This minireview focuses on the role of gamma-tubulin in the pathobiology of GBM in the light of emerging concepts concerning the function of gamma-tubulin and its potential role in tumorigenesis putting forward the concept that gamma-tubulin might serve as a novel marker of anaplastic change in gliomas.

Tubulin targets in the pathobiology and therapy of glioblastoma multiforme. I. Class III beta-tubulin.

Glioblastoma multiforme (GBM) is the most common and deadliest form of primary brain cancer in adults. Despite advances in molecular biology and genetics of gliomas currently there is no effective treatment or promising molecularly targeted experimental therapeutic strategies for these tumors. In previous studies we have shown aberrant overexpression of the class III beta-tubulin isotype (betaIII-tubulin) in GBM and have proposed that this change may reflect perturbations in microtubule dynamics associated with glioma tumorigenesis, tumor progression and malignant transformation into GBM. This minireview focuses on microtubules and tubulin as emerging targets in potential therapy of GBM using a new class of betaIII-tubulin-targeted drugs in the light of recent developments concerning the function and potential role of this isotype in clinically aggressive tumor behavior, cancer stem cells, tumor hypoxia and chemoresistance to tubulin binding agents, principally taxanes.

Regulation of microtubule nucleation from membranes by complexes of membrane-bound gamma-tubulin with Fyn kinase and phosphoinositide 3-kinase.

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.