Protein tyrosine kinase p53/p56lyn forms complexes with {gamma}-tubulin in rat basophilic leukemia cells

Lubica Dráberová1, Eduarda Dráberová2, Zurab Surviladze1, Petr Dráber1 and Pavel Dráber2

1 Department of Mammalian Gene Expression and
2 Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic

Correspondence to: L Dráberová


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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 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 {alpha}ß-tubulin dimers (~70%) and {gamma}-tubulin (~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 ~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 p53/56lyn 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.

Keywords: cytoskeleton, IgE receptor, Lyn kinase, mast cell, rat basophilic leukemia cell


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mast cells, basophils and their tumor derivatives, including rat basophilic leukemia (RBL) cells, express on their surfaces receptors with a high affinity for IgE (Fc{varepsilon}RI). An aggregation of the Fc{varepsilon}RI by multivalent IgE–antigen complexes or anti-Fc{varepsilon}RI antibodies triggers a series of biochemical events culminating in the secretion of inflammatory mediators and cytokines that have an important role in allergic responses. Fc{varepsilon}RI is a tetrameric complex composed of one IgE binding {alpha} subunit, one ß subunit and a homodimer of {gamma} subunits (1). The intracellular part of the ß subunit seems to be constitutively associated with the Src-family kinase p53/p56lyn (Lyn) which becomes rapidly activated after Fc{varepsilon}RI aggregation. The stoichiometry and affinity of association of Lyn kinase with Fc{varepsilon}RI are low in unstimulated cells, but are increased after antigen-mediated Fc{varepsilon}RI aggregation (2). The activated Lyn phosphorylates ß and {gamma} subunits of Fc{varepsilon}RI, and contributes to further propagation of the activation signal which entails the binding of Syk kinase to Fc{varepsilon}RI {gamma} subunit and its activation, stimulation of phosphatidylinositol hydrolysis, elevation of cytoplasmic Ca2+, activation of protein tyrosine kinases, and other biochemical pathways (1,3). Similar activation events, albeit less extensive, are also observed after an aggregation of glycosylphosphatidylinositol-anchored Thy-1 glycoprotein which is expressed in large amounts on the surface of rat mast cells and RBL cells (4,5).

Morphologically, the cells cultured as monolayers and activated via Fc{varepsilon}RI become flattened and spread, and exhibit an increased assembly of actin, increased fluid pinocytosis and membrane ruffling (6,7). Interestingly, the rate and extent of the mediator release from Fc{varepsilon}RI-activated cells are greater in attached than suspended cells (8), indicating that changes in cell shape and/or cell–substrate adhesion modulate the secretion. These and some other findings suggest that certain events, which take place during the process of cell activation, are in part controlled by cytoskeleton.

The cytoskeleton is a filamentous network of microfilaments, microtubules and intermediate filaments composed of respectively actin, tubulin and one of several classes of intermediate filament proteins. The filaments of cytoskeleton form a continuous dynamic connection between cellular structures, and constitute an enormous surface area whereon proteins and other cytoplasmic components can dock. Several lines of evidence suggested that the microtubules might play a role in intracellular signaling. Thus in CD3 antibody-stimulated Jurkat T cells {alpha}-tubulin but not ß-tubulin was phosphorylated on tyrosine (9). In B cells activated via B cell antigen receptor, p72syk kinase reacted with soluble {alpha}-tubulin (10). Furthermore, an analysis of HL-60 cells exposed to tetradecanoyl phorbol acetate revealed that phosphorylation of tubulins by Src family kinases, Lyn and Fyn, altered the tubulin assembly and consequently monocyte differentiation (11). Of the three major tubulin types in eukaryotic cells, {alpha}- and ß-tubulin form heterodimeric complexes which associate head to tail into protofilaments and then laterally to make up the wall of the cylindrical microtubules. The third isotype, {gamma}-tubulin, appears in the cytosol and in microtubule-organizing centers (MTOC) where it nucleates microtubules (12). Although previous experiments indicated that microtubule-disrupting drugs inhibited the degranulation in mast cells and RBL cells (13,14), the molecular basis of the interaction of signal-transduction molecules with various tubulins is not known. In this study we analyzed the solubility and other properties of {alpha}ß-tubulin dimers and {gamma}-tubulin in resting and activated RBL cells, and their association with signal transduction molecules.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies and reagents
The following purified mAb were used: MRCOX7 (OX7, IgG1), specific for Thy-1.1 (15), Lyn-01/Pr (IgG1), specific for protein tyrosine kinase p53/p56lyn (16), HTF-14 (IgG1), specific for human transferrin (17), TU-31 (IgG2b) and TU-32 (IgG1) recognizing {gamma}-tubulin (18), and TU-01 (IgG1) (19) and TU-16 (IgM) (20) specific for {alpha}-tubulin. mAb TEC-11 (IgG1) (21) and TEC-01 (IgM) (22) served as negative controls. mAb IGEL b4 1 (IgE), specific for TNP (23), was used for cell sensitizing. Microtubular structures were detected with an affinity-purified rabbit antibody against {alpha}ß-tubulin dimer (24). The position of MTOC was identified using affinity-purified rabbit antibody M8 against pericentrin (25). For some experiments, purified OX7 and TU-01 mAb were biotinylated with Immuno-Pure NHS-LC-biotin (Pierce, Rockford, IL) according to the manufacturer's instructions. Isolated anti-phosphotyrosine mAb PY-20, horseradish peroxidase (HRP)-conjugated mAb PY-20 and HRP-conjugated goat anti-mouse IgG were purchased from Transduction Laboratories (Lexington, KY). Lissamine rhodamine-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG were from Jackson ImmunoResearch (West Grove, PA). ECL Western blotting detection reagents and [{gamma}-32P]ATP were from Amersham (Little Chalfont, UK). Streptavidin was from Promega (Madison, WI) and Sepharose 4B from Pharmacia (Uppsala, Sweden). HRP-conjugated streptavidin and UltraLink immobilized Protein A on 3M Emphaze were obtained from Pierce. Triton X-100 and NP-40 were from Fluka (Buckingham, UK), glutathione agarose from Molecular Probes (Eugene, OR), non-fat dry milk from Super G (Landover, MD), polyethylene glycol from Koch-Light (Haverhill, UK), and Mowiol 4-88 from Calbiochem-Novabiochem (La Jolla, CA). Glutathione-S-transferase (GST) fused to N-terminal domains of Lyn and GST alone were prepared as previously described (26). Src family-selective tyrosine kinase inhibitor, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) was obtained from Pfizer (Central Research Division, Groton, CT). All other reagents were from Sigma (St Louis, MO).

Cell activation and cell extraction
RBL-2H3 cells were cultured and activated as described (5). Briefly, after harvesting, the cells were resuspended in culture medium and sensitized in suspension with IgE specific for TNP (IGEL b4 1 ascites, diluted 1:1000) or biotinylated OX7 (10 µg/ml) for 20 min at 37°C. The sensitized cells were washed in Tyrode's buffer (10 mM HEPES, pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose and 0.1% BSA) and activated for 15 min with the corresponding cross-linking reagents at a final concentration of 1 µg/ml TNP-BSA or 10 µg/ml of streptavidin. Alternatively, the cells were exposed to freshly prepared pervanadate solution for 15 min as described (26). For immunofluorescence studies the cells were activated by adding pervanadate solution directly to the cells grown on coverslips. Cell activation was stopped by transferring the tubes on ice and pelleting the cells by a brief centrifugation; cells grown on coverslips were washed in PBS and fixed (see below). Sedimented cells were lysed by adding ice-cold lysis buffer containing 25 mM Tris–HCl, pH 8.0, 140 mM NaCl, protease and phosphatase inhibitors (2 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 5 µg/ml aprotinin and 2 µg/ml leupeptin) and supplemented with 1% NP-40 or 1% Triton X-100. After 30 min incubation on ice the tubes were centrifuged for 5 min at 12,000 g at 4°C, and postnuclear supernatants and nuclear pellets were collected. To release soluble material from the cells, the cells were first exposed to 0.2% saponin in PBS supplemented with 5 mM MgCl2. After 10 min incubation on ice the cells were briefly centrifuged and supernatant collected (SS). The pellet was resuspended in ice-cold 1% NP-40-containing lysis buffer and extracted as above (SNP). In some experiments the cells were also solubilized at 37°C.

Gel chromatography and sucrose density-gradient ultracentrifugation
Chromatography on Sepharose 4B gel minicolumns was performed as previously described (27). Briefly, postnuclear supernatants from activated or control cells (300 µl) were applied onto the top of 5 ml disposable columns (Pierce) filled with Sepharose 4B and equilibrated with lysis buffer containing 1% NP-40. The fractions (300 µl) were collected at 8 min intervals. Distribution of proteins in fractions 3–10, as determined by the BCA kit (Pierce), was as follows (% of total): 0, 5, 10, 16, 19, 24, 17 and 9. Sucrose density-gradient ultracentrifugation was carried out as described before (28). Cells were lysed for 15 min with sucrose-gradient lysis buffer, containing 25 mM MES, pH 6.5, 150 mM NaCl, protease and phosphatase inhibitors, and 1% Triton X-100, followed by homogenizing by passing the suspension 10 times through a 27-gauge needle. Sucrose gradient was prepared in MES buffer (25 mM MES, pH 6.5 and 150 mM NaCl) and was formed by the following concentrations (w/v) of sucrose layers: 60 (bottom), 40 (containing the cell lysate), 35, 30, 25, 20, 15 and 10% (top). The gradient was ultracentrifuged in SW 55 rotor (Beckman, Palo Alto, CA) at 210,000 g for 15 h at 4°C and fractionated into 0.5 ml aliquots withdrawn from the top.

Distribution of proteins in fractions 1 (top)–10 (bottom) was as follow (% of total): <1, <1, <1, <1, <1, 4, 16, 25, 34 and 20.

Immunoprecipitation
Postnuclear supernatants from 107 cells per sample were incubated for 2 h at 4°C with beads of (i) Protein A saturated with anti-{gamma}-tubulin mAb (TU-31), (ii) Sepharose 4B with covalently bound goat anti-mouse IgM and saturated with anti-{alpha}-tubulin mAb (TU-16) or TEC-01 mAb (negative control), (iii) Protein G saturated with anti-Lyn mAb (Lyn-01/Pr) or irrelevant mAb of the same isotype (negative control), and (iv) glutathione agarose with bound GST–Lyn fusion protein or GST protein alone. The beads were washed 3 times with ice-cold lysis buffer and the bound proteins were eluted by boiling in SDS–PAGE sample buffer [62.5 mM Tris–HCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 0.00125 % (w/v) bromophenol blue] supplemented or not with 2.5% (v/v) 2-mercaptoethanol.

In vitro kinase assay and reprecipitation
Cells were solubilized in 1% NP-40 lysis buffer and postnuclear supernatants were size-fractionated by Sepharose 4B gel chromatography. The fractions were analyzed by the immunocomplex kinase assay as described (27). Briefly, F-bottom, Immulon II plates (Dynatech, Chantilly, VA) were covered with antibodies and the antigen was bound to the immunosorbent for 12 h on ice. The immunocomplex kinase assay was performed directly in the wells by incubating the `immunoprecipitates' for 25 min at room temperature with 50 µl of kinase buffer (25 mM HEPES-Na, pH 7.2, 3 mM MnCl2 and 0.1% NP-40) supplemented with 0.2 µCi of [{gamma}-32P]ATP. The reaction was stopped by washing the wells 5 times with kinase buffer. The labeled immunocomplexes were solubilized by warm SDS–PAGE sample buffer, and analyzed by SDS–PAGE and autoradiography. For reprecipitation experiments the immunocomplexes were solubilized by 1% SDS, diluted 1:10 in 1% NP-40 lysis buffer and Lyn was reprecipitated by anti-Lyn antibody. 32P-labeled Lyn was visualized by SDS–PAGE and autoradiography.

Immunoblotting
Immunoblotting was performed with a Mini-Protean II transblot module (BioRad, Hercules, CA) according to the manufacturer's instructions. The nitrocellulose replicas were blocked for 1 h in TNT blocking buffer (10 mM Tris–HCl, pH 7.5, 100 mM NaCl and 0.1% Tween 20) supplemented with 1% BSA and the proteins immunostained by sequential incubation with mAb (ascitic fluid containing TU-32 or Lyn-01/Pr and diluted 1:1000 in TNT with 1% BSA) followed by anti-mouse IgG–HRP conjugate diluted 1:8000 in TNT supplemented with 5% non-fat dry milk. Biotinylated antibody (diluted 1:2000) were detected by streptavidin–HRP conjugate and phosphotyrosine residues were detected with 1:5000 diluted PY-20–HRP conjugate. The ECL Western blotting detection reagent was used according to manufacturer's instructions to visualize the labeled proteins. Densitometric evaluation of the bands on films was done using a gel documentation system (UVP; The Science Park, Cambridge, UK).

Immunofluorescence
Fixed cytoskeletons were immunostained as described (19) with some modifications. Cells grown on coverslips were fixed for 10 min in methanol at –20°C followed by 6 min in acetone at –20°C. Alternatively, cells were extracted for 1 min with 0.2% Triton X-100 in microtubule stabilizing buffer (MSB), containing 100 mM MES (adjusted to pH 6.9 with KOH), 2 mM EGTA, 2 mM MgCl2 and 4% polyethylene glycol 6000 at 37°C, and fixed for 20 min in 3% formaldehyde in MSB at the same temperature. For multiple-labeled fluorescence the coverslips were incubated simultaneously with the primary antibodies and after washing incubated simultaneously with the secondary fluorochrome-conjugated antibodies. TU-31 mAb was used as undiluted hybridoma supernatant, Lyn-01/Pr was used as ascitic fluid diluted 1:100 and PY-20 was used at a concentration of 2.5 µg/ml. Affinity-purified antibody against {alpha}ß-tubulin dimer and antibody against pericentrin were diluted 1:5 and 1:200 respectively. The preparations were mounted in Mowiol 4-88 supplemented with 1 µg/ml of 4,6-diamidino-2-phenylindole and n-propylgalate, and examined with an Olympus A70 Provis microscope equipped with a x60 water-immersion objective. For epi-illumination the following filter combination cubes were used: NIBA, WIG and NUA. Photographs were taken using Kodak Tri-Xpan film. Neither the control antibody HTF-14 nor the conjugate alone gave any detectable staining.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Tubulins in resting RBL cells
In the first series of experiments we analyzed the solubility and cytoplasmic distribution of {alpha}ß-tubulin dimers and {gamma}-tubulin in RBL cells, using antibodies specific for the corresponding tubulins. Figure 1Go(A) shows that polyclonal antibody against {alpha}ß-tubulin dimer stained a typical network of microtubules originating from the perinuclear region and ending at the cell periphery. A different picture was obtained when mAb specific for {gamma}-tubulin was used (Fig. 1BGo). The mAb stained {gamma}-tubulin not just in MTOC in the perinuclear region but also in distinct spots throughout the cell cytoplasm. It should be noted that the pattern of {gamma}-tubulin distribution as presented in Fig. 1Go(B) requires that cold methanol/acetone fixation be used.



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Fig. 1. Immunofluorescence localization of {alpha}ß-tubulin dimers and {gamma}-tubulin in fixed RBL cells. The cells were grown on a coverslip and fixed with cold methanol/acetone before triple labeling with polyclonal antibody against {alpha}ß-tubulin dimers (A), TU-31 mAb specific for {gamma}-tubulin (B) and DNA-binding dye to visualize nuclei (C). Bar, 10 µm.

 
A characteristic feature of the {alpha}- and ß-tubulins is that they exist in the form of polymers (microtubules) resistant to the extraction with non-ionic detergents, as well as in the form of soluble {alpha}ß-tubulin dimers. When the cells are extracted at low temperature, the majority of microtubules are depolymerized and only cold-resistant microtubules remain intact. To determine which fraction of {alpha}ß-tubulin dimers and {gamma}-tubulin can be found in detergent-soluble and -insoluble forms in cells extracted with non-ionic detergent at low temperature, non-activated (control) cells were exposed to non-ionic detergent NP-40 for 30 min and quickly fractionated by sedimentation at 12,000 g for 5 min. Both the material remaining in the supernatant and that associated with the insoluble pellet were analyzed by immunoblotting with mAb TU-01 (against {alpha}-tubulin) and mAb TU-32 (against {gamma}-tubulin). Previous experiments showed that both antibodies reacted in whole cell lysate of RBL cells exclusively with the corresponding proteins and no cross-reactivity with other proteins was observed (not shown). The data presented in Fig. 2Go(A; Control+, S and P) indicate that ~70% of the {alpha}ß-tubulin dimers and 85% of {gamma}-tubulin were released into the supernatant (they did not sediment during 5 min centrifugation). Similar results were obtained when the cells were extracted in 1% Triton X-100 (not shown). These results suggested that a large fraction of tubulins was present in soluble form. This conclusion was supported by another set of experiments in which the cells were fractionated by a two-step procedure (16). In the first step the cells were exposed to ice-cold 0.2% saponin and the soluble material released from the permeabilized cells was collected in supernatant after low-speed centrifugation (saponin soluble fraction, SS). In the second step the cell ghosts were solubilized by ice-cold 1% NP-40 lysis buffer and the supernatant, after removal of nuclei by low-speed centrifugation, was again gathered (saponin/NP-40-soluble fraction, SNP). Using this fractionation procedure we found the most of the {alpha}ß-tubulin dimers and {gamma}-tubulin were found in the SS fraction (Fig. 2BGo, Control+, SS) and only a smaller amount was released into the SNP fraction (Fig. 2BGo, Control+, SNP). This confirms that only a small fraction of tubulins is present in cold- and detergent-resistant (polymerized) form.



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Fig. 2. Solubility of {alpha}ß-tubulin dimers and {gamma}-tubulin in resting and activated cells. (A) The cells were activated in suspension by Fc{varepsilon}RI-cross-linking (IgE), Thy-1-cross-linking (OX7) or by pervanadate (Pv) for 15 min followed by lysis in ice-cold buffer with 1% NP-40 and fractionated into postnuclear supernatant (S) and insoluble pellet (P). Control: non-activated cells. (B) The cells were activated as above followed by exposure for 10 min to ice-cold 0.2% saponin in PBS, supplemented with 5 mM MgCl2; the material released from the cells into supernatant was collected after low-speed centrifugation (SS). Saponin-resistant cell ghosts were lysed in a lysis buffer with 1% NP-40 and supernatant was again collected (SNP). All fractions were analyzed by SDS–PAGE (under non-reducing conditions) and immunoblotting with mAb TU-01–biotin conjugate (specific for {alpha}-tubulin) or with mAb TU-32 (specific for {gamma}-tubulin), followed by second layer detection reagents and ECL. Positions of {alpha}- and {gamma}-tubulins are indicated by arrows.

 
To determine whether solubility of {gamma}-tubulin is dependent on temperature, we performed another set of experiments in which the cells were lysed at 4 or 37°C in a buffer containing 0.1% Triton X-100. Data presented in Fig. 3Go indicate that the amount of {gamma}-tubulin in postnuclear supernatants from cells lysed at these two different temperatures was similar, suggesting that most of {gamma}-tubulin is soluble at physiological conditions.



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Fig. 3. Release of {gamma}-tubulin from resting or activated cells solubilized on ice or at 37°C. Non-activated or antigen-activated (15 min) cells were lysed in a lysis buffer, containing 0.1% Triton X-100 at 4 or 37°C, and fractionated into postnuclear supernatant (S) or insoluble pellet (P). The fractions were analyzed by SDS–PAGE and immunoblotting with TU-32 mAb, followed by HRP-conjugated goat anti-mouse IgG and ECL. Intensity of bands corresponding to {gamma}-tubulin was analyzed by densitometry. Means ± SE were calculated from three experiments.

 
Tubulins in activated RBL cells
The cells were activated by (i) aggregation of surface Fc{varepsilon}RI via IgE–antigen complexes, (ii) OX7 mAb-mediated aggregation of surface Thy-1 or (iii) by exposure to pervanadate. The data presented in Fig. 2Go(A and B; IgE+, OX7+, Pv+) indicate that the proportions of {alpha}ß-tubulin dimers and {gamma}-tubulin in NP-40- or saponin-soluble and -insoluble materials are not significantly affected by activation of the cells via different routes. When the cells were solubilized in the presence of 0.1% Triton X-100 at 4°C, only a small decrease in the solubility of {gamma}-tubulin was observed in cells activated via Fc{varepsilon}RI (Fig. 3Go).

In further experiments we analyzed size of the {gamma}-tubulin complexes by means of Sepharose 4B gel chromatography (Fig. 4AGo). It should be noted that although only postnuclear supernatants from NP-40-extracted cells were used for these experiments, the `soluble' forms of tubulins exhibited size heterogeneity ranging from very large complexes (>40 MDa, eluted in the void volume of the Sepharose 4B column, fraction 4) to small complexes or single molecules (eluted in fractions 9 and 10). Furthermore, there were no dramatic changes in the density of complexes of tubulins in antigen-activated cells as determined by density gradient ultracentrifugation (Fig. 4BGo). It is interesting, however, that in cells activated by aggregation of surface Thy-1 a small fraction of {alpha}ß-tubulin dimers (~5%) and still a smaller proportion of {gamma}-tubulin migrated into light-density fractions containing membrane microdomains. The tubulins did not appear in these fractions if the cells had been pre-treated with saponin (not shown), implicating cholesterol as a possible important factor in the formation of these complexes.



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Fig. 4. Size and density distribution of {alpha}ß-tubulin dimers and {gamma}-tubulins in resting and activated cells. The cells were activated in suspension by Fc{varepsilon}RI cross-linking (IgE), Thy-1 cross-linking (OX7) or pervanadate (Pv) for 15 min. Control: non-activated cells. The cells were solubilized in NP-40 lysis buffer and postnuclear supernatants were size-fractionated by Sepharose 4B gel chromatography (A). Alternatively, the cells were lysed in 1% Triton X-100 lysis buffer and density-fractionated by ultracentrifugation in a sucrose gradient (B). Fractions were analyzed by SDS–PAGE (under non-reducing conditions) and immunoblotting with mAb TU-01–biotin (specific for {alpha}-tubulin) and TU-32 (specific for {gamma}-tubulin). Positions of {alpha}- and {gamma}-tubulins are indicated by arrows.

 
Tyrosine phosphorylation of tubulin-associated proteins in activated cells
Tubulin immunocomplexes from both control and activated cells were analyzed in order to determine whether or not the proteins forming complexes with tubulins are tyrosine phosphorylated during the cell activation. The cells in suspension were activated via Fc{varepsilon}RI, Thy-1 or by exposure to pervanadate and tubulins, and their associated proteins in SS and SNP were isolated by immunoprecipitation. Figure 5Go(A) shows the patterns of {gamma}-tubulin-associated proteins phosphorylated on tyrosine. In non-activated cells {gamma}-tubulin in SS associated with tyrosine phosphorylated proteins of 25, 50, 53, 56, 60, 75 and 97 kDa. A slightly different profile was observed in SNP, i.e. the appearance of new bands of ~80 and 115 kDa. In IgE + antigen-activated cells an increase in tyrosine-phosphorylated proteins in the region of 50–60 kDa bands was observed in SS as well as in SNP, and an 85 kDa band in SNP; a new tyrosine phosphorylated protein of ~200 kDa newly appeared in SNP. This pattern of tyrosine phosphorylated proteins in {gamma}-tubulin complexes was preserved when SS and SNP were prepared from cells activated in adherent cultures (not shown). Even more extensive tyrosine phosphorylation was observed in Thy-1-activated cells, although the pattern was similar to that seen with IgE + antigen-activated cells (Fig. 5AGo). Interestingly, in pervanadate pretreated cells showing large numbers of proteins phosphorylated on tyrosine (26) only a fraction of them bound to {gamma}-tubulin, confirming specificity of the reaction. When anti-{alpha}-tubulin antibody was used for immunoprecipitation, only a slightly different pattern of proteins phosphorylated on tyrosine was observed (Fig. 5BGo). If isotype-matched negative control antibodies were employed, no tyrosine phosphorylated proteins were detected (not shown), indicating a high degree of specificity of the observed reactivity.



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Fig. 5. Tyrosine phosphorylated proteins associated with {alpha}ß-tubulin dimers and {gamma}-tubulins in resting and activated cells. The cells were activated in suspension by Fc{varepsilon}RI cross-linking (IgE), Thy-1 cross-linking (OX7) or pervanadate (Pv) for 15 min followed by exposure for 10 min to 0.2% saponin. Control: non-activated cells. Material released from the cells was collected in supernatant after low-speed centrifugation (SS). Cell ghosts in the pellet were extracted with a lysis buffer containing 1% NP-40 and supernatant was again collected (SNP). The material was immunoprecipitated (IP) with mAb specific for {gamma}-tubulin (TU-31) bound to Protein A beads (A) or {alpha}-tubulin (TU-16) bound to Sepharose 4B via rabbit anti-mouse IgM (B). Immunoprecipitated material was analyzed by immunoblotting with PY-20–HRP conjugate, specific for phosphotyrosine and ECL. Molecular mass standards on the left are in kDa.

 
A substantial increase in the amount of proteins phosphorylated on tyrosine in activated cells was also confirmed by immunofluorescence. In non-activated cells extracted with 0.2% Triton X-100, fixed and stained with PY-20 mAb, tyrosine phosphorylated proteins were distributed diffusely in the cytoplasm and slightly prevailing in the perinuclear region where the pericentrin, a typical MTOC protein, is located (Fig. 6A and DGo). After a 20 min exposure to pervanadate the amount of detergent-resistant phosphotyrosine residues was markedly increased, especially in the perinuclear region (Fig. 6B and EGo). In contrast, the amount of p53/p56lyn kinase bound to detergent-resistant structures did not substantially differ between non-activated (not shown) and pervanadateactivated cells (Fig. 6CGo). A punctuate staining pattern of p53/p56lyn kinase was found in the cytoplasm and was more prominent on the cell periphery. The kinase also accumulated in the perinuclear region as detected by anti-pericentrin antibody (Fig. 6FGo). Microtubule structures were preserved (not shown) under the conditions used for the preparation of samples for immunofluorescence staining. Accumulation of tyrosine phosphorylated proteins and Lyn kinase in perinuclear region was also observed in cells fixed with paraformaldehyde before extraction with 0.2% Triton X-100 (not shown).



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Fig. 6. Immunofluorescence localization of tyrosine phosphorylated proteins and Lyn kinase in resting (A and D) and pervanadate-activated (B, C, E and F) cells. The cells were stained with mAb PY-20, specific for phosphotyrosine (A and B), mAb Lyn-01/Pr, specific for p53/p56lyn kinase (C) and with polyclonal antibody against pericentrin (D–F). Each pair (A–D, B–E and C–F) represents the same cells. Arrows indicate the same position in each pair. Bar, 10 µm.

 
The observed enhanced tyrosine phosphorylation might possibly reflect an increased activity of the tubulin-associated tyrosine kinases. In further experiments we therefore employed an immunocomplex kinase assay to test the hypothesis. To increase the sensitivity of the assay, cell extracts were size-fractionated by Sepharose 4B gel chromatography prior to immunoprecipitation. As shown in Fig. 7Go(A), {gamma}-tubulin in the extract from unstimulated cells was associated with several kinase substrates. The molecular mass of major 32P-labeled proteins in large complexes, eluted in the void volume of the column (>40 MDa), was about 55 kDa, whereas smaller size complexes eluted at the elution peak of IgG (fraction 7) contained {gamma}-tubulin associates with dominant 115 and 80 kDa 32P-labeled proteins. Cells activated by Thy-1 aggregation were characterized by an increased kinase activity, although the pattern of 32P-labeled proteins remained similar as that seen in control cells (Fig. 7BGo). A different profile of 32P-labeled proteins was observed when {alpha}-tubulin immunocomplexes were assayed (Fig. 7CGo). There were more 32P-labeled proteins in very large complexes, i.e. of 20 and 32–38 kDa. {alpha}-Tubulin in lower size complexes (eluted at fraction 6 and 7) was associated mainly with 90 and 150 kDa phosphorylated proteins. A band of ~55 kDa was observed in all fractions but its extent was smaller than in {gamma}-tubulin complexes. Cells activated by aggregation of Thy-1 displayed an increased activity of kinases, although the dominant 32P-labeled proteins were similar to those in non-activated cells (Fig. 7DGo). Reprecipitation analysis revealed that the 32P-labeled band of ~55 kDa associated with {gamma}-tubulin from control as well as Thy-1-activated cells was attributable to Lyn kinase (not shown).



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Fig. 7. Kinase activity of immunocomplexes containing {gamma}-tubulin- or {alpha}ß-tubulin dimers. The cells were activated in suspension by Thy-1 (OX7) cross-linking for 15 min and solubilized in 1% NP-40 lysis buffer. Control: non-activated cells. Postnuclear supernatants were size-fractionated on Sepharose 4B gel chromatography. Fractions 3–10 were immunoprecipitated (IP) with mAb specific for {gamma}-tubulin (TU-31; A and B) or {alpha}-tubulin (TU-16; C and D). Immunocomplexes were subjected to an in vitro kinase assay, separated by SDS–PAGE under reducing conditions and visualized by autoradiography. Elution peaks of erythrocytes, IgM and albumin were in fractions 4, 5 and 8 respectively. Molecular mass standards on the left are in kDa.

 
Binding of tubulins to Lyn kinase
To get more insight into the interaction of tubulins with Lyn kinase, another series of experiments was performed in which recombinant N-terminal fragment of Lyn, encompassing SH2 and SH3 domains, was used. GST–Lyn or GST alone, which served as a negative control, were immobilized on glutathione–agarose beads and the binding of tubulins from non-activated or activated cells was analyzed by immunoblotting. As shown in Fig. 8Go, {gamma}-tubulin from resting cells (control) bound to Lyn–GST but not to GST alone, suggesting that the binding was specific for Lyn. When extracts from cells activated via Fc{varepsilon}RI, pervanadate (Fig. 8Go) or Thy-1 (not shown) were used, a slightly higher binding (~30%) was reproducibly observed in three independent experiments. When the same ECL exposure times were used for {alpha}-tubulin, no signal was detected. However, longer exposure times revealed that some {alpha}-tubulin also bound to GST–Lyn but not to GST alone (not shown). The binding of {gamma}-tubulin to GST–Lyn was not inhibited with relatively high concentrations of phenylphosphate (up to 4 mM, not shown), even though they did inhibit the SH2-mediated binding of Lyn to Syk (26); these data suggest that tyrosine-phosphorylated residues are not involved in these interactions.



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Fig. 8. Binding of {gamma}-tubulins to GST–Lyn fusion protein. The cells were activated in suspension by Fc{varepsilon}RI cross-linking (IgE) or by pervanadate (Pv) for 15 min and solubilized in 1% NP-40 lysis buffer. Control: non-activated cells. Postnuclear supernatants were incubated with GST–Lyn fusion protein or GST alone (negative control) immobilized to glutathione beads. The proteins were eluted with SDS sample buffer, fractionated by SDS–PAGE and {gamma}-tubulin was detected by immunoblotting with mAb TU-32. Molecular mass standards (in kDa) and position of {gamma}-tubulin (arrow) are indicated on the left.

 
Role of Lyn and/or other Src-kinases in formation of {gamma}-tubulin complexes
To determine whether Lyn and/or other Src-family kinases are involved in formation of {gamma}-tubulin complexes, we used for further analysis a Src family-selective tyrosine kinase inhibitor, PP1. Complexes of {gamma}-tubulin and Lyn kinase were isolated by immunoprecipitation from non-activated or antigen-activated cells which were pretreated for 10 min before activation with 10 µm PP1. As a control, PP1 untreated cells were used. Immunoblotting analysis with HRP-labeled PY-20 antibody indicated that PP1 decreased the extent of tyrosine phosphorylation of p55 kDa protein (presumably Lyn) in {gamma}-tubulin complexes in non-activated cells (Fig. 9AGo). In antigen-activated cells, more tyrosine phosphorylated proteins were found in the complexes (see also Fig. 5AGo) and most of them were dependent on the kinase activity of Src kinases as revealed by their sensitivity to pretreatment with PP1 (Fig. 9AGo). As detected by analysis of the same blot with {gamma}-tubulin-specific antibody, this PP1-mediated inhibition was not due to decreased immunoprecipitation of {gamma}-tubulin (Fig. 9BGo).



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Fig. 9. Inhibitory effect of PP1 on formation of {gamma}-tubulin complexes. IgE-primed cells were treated with PP1 inhibitor for 10 min before exposure to antigen (IgE+) or Tyrode's solution alone (Control+). After 15 min activation the cells were lysed and {gamma}-tubulin (A and B) or Lyn kinase (C and D) from postnuclear supernatants were immunoprecipitated. The immunoprecipitates were analyzed by immunoblotting using antibodies specific for phosphotyrosine (A; PY-20–HRP conjugate), {gamma}-tubulin (B and C; TU-31) or Lyn kinase (D; Lyn-01/Pr). Molecular mass standards on the left, in (A), are in kDa.

 
These data were also supported by our finding that pretreatment with PP1 decreased the amount of {gamma}-tubulin in Lyn immunocomplexes isolated from both non-activated and antigen-activated cells (Fig. 9CGo); and, again, this decrease was not due to decreased amount of Lyn kinase in the immunoprecipitates (Fig. 9DGo).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Upon aggregation of Fc{varepsilon}RI by IgE–antigen complexes, RBL cells undergo rapid morphological and cytoskeletal changes which may have an important role in intracellular signaling and degranulation (6,2931). Although most of the previous studies were directed towards analysis of microfilaments, microtubules seem to have a key role in changes of the cell morphology and large-scale movement of cytoplasmic organelles (32). Data presented in this paper show that in RBL cells extracted with cold non-ionic detergents (NP-40 or Triton X-100), significant fractions of {alpha}ß-tubulin dimers (~70%) and even more {gamma}-tubulin (~85%) appear in the pool of non-polymerized tubulins. Importantly, solubility of {gamma}-tubulin was not significantly changed if the cells were extracted at 37°C, indicating that most {gamma}-tubulin is soluble at physiological temperature.

Several lines of evidence indicated that tubulins extracted from RBL cells with non-ionic detergents formed complexes with signal transduction molecules. First, Sepharose 4B gel chromatography showed that tubulins from postnuclear supernatant were eluted in numerous fractions, including the void volume of the column. This implies that the complexes are heterogeneous in size and that some of them are very large (>40 MDa). Second, several tyrosine-phosphorylated proteins were found to be associated with immunoprecipitated tubulins. The number of the tubulin-associated proteins and/or the intensity of tyrosine phosphorylation increased in activated cells. Third, the immunocomplex kinase assays revealed that tubulins were physically associated with kinases and their substrates. The pattern of 32P-labeled tubulin-associated molecules depended on the tubulin analyzed and the size of the complexes. Very large complexes of tubulins were associated with kinases and/or their substrates, which were different from those associated with smaller size complexes. An increased tyrosine phosphorylation of proteins associated with different tubulins was observed in activated cells. Fourth, 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. Finally, upon activation of RBL cells with anti-Thy-1 mAb and their fractionation (after lysis in Triton X-100) by sucrose density-gradient ultracentrifugation, a small proportion of {alpha}ß-tubulin dimers (<5%) and a still smaller proportion of {gamma}-tubulin were found in light-density fractions. Light density usually reflects an association of the studied proteins with membrane vesicles (rafts) enriched in lipids and cholesterol (33). Further support for the association with membrane rafts of tubulins from Thy-1-activated cells can be seen in our own finding that the cholesterol-sequestering agent saponin, known to solubilize otherwise detergent-insoluble membrane microdomains (16,34), abolished the migration of tubulins into the light-density fractions. Thus, membrane microdomains which are enriched in signal transduction molecules could be directly involved in an interaction with tubulins in the course of cell activation.

Our data also indicate, for the first time, that {gamma}-tubulin and to a lesser extent also {alpha}ß-tubulin dimers form complexes with Lyn kinase. Thus, a tyrosine-phosphorylated protein of 53/56 kDa was detected in immunoprecipitated tubulin complexes and proteins of the same relative mol. wt were also found among the 32P-labeled proteins in the {gamma}-tubulin immunocomplex kinase assay. Direct evidence that Lyn is indeed present in tubulin complexes was obtained by reprecipitation experiments and also by the discovery of tubulins in material bound to recombinant GST–Lyn. Until now it has not been clear whether Lyn interacts with tubulin directly or indirectly via adaptor-like molecules. Lyn interacts with several targets, including Fc{varepsilon}RI and Syk kinase from activated RBL cells, via the SH2 domain which recognizes phosphotyrosine in a consensus amino acid sequence tyrosine–hydrophilic– hydrophilic–hydrophobic (26,35,36). However, amino acid sequence analysis indicates that such consensus sequence motif is not present in {gamma}-tubulin and therefore Lyn is very unlikely to interact with phosphotyrosine of {gamma}-tubulin through a typical SH2-mediated interaction. This conclusion is also strengthened by our finding that relatively high concentrations of phenylphosphate had no effect on GST–Lyn–{gamma}-tubulin interaction and that only a slight increase in {gamma}-tubulin binding to GST–Lyn was observed in activated cells. One possible explanation for the association of Lyn with tubulins presumes the formation of detergent-insoluble membrane microdomains (rafts) in which signal-transduction molecules are kept together by lipids; in RBL cells >50% of total cellular Lyn, and Thy-1 as well, are present in such domains (28). This explanation was, however, rendered unlikely because in non-activated cells tubulin was excluded from membrane rafts, as determined by sucrose gradient ultracentrifugation, and because the complexes, in contrast to membrane rafts (28), were resistant to a pre-treatment with saponin before NP-40 extraction. However, tubulins in Thy-1-activated cells might interact with the membrane rafts (see below).

The formation of complexes of {gamma}-tubulin with other cellular proteins has recently been described in extracts from Xenopus eggs (37,38), Saccharomyces cerevisiae (39) as well as mammalian cell lines (4042). However, the major focus of these studies was on yeast spindle pole body components Spc97p and Spc98p, and their mammalian homologues, which seem to play a role in the function of MTOC and no kinases have so far been described in such complexes. Furthermore, it has been reported that phosphoinositide 3-kinase binds to {gamma}-tubulin in response to insulin, whereas the binding to {alpha}ß-tubulin dimers was constitutive (43). Some other data point to the interaction of signal-transduction molecules with microtubules in immunoreceptor-activated cells. In T cells, activation led to a rapid tyrosine phosphorylation of {alpha}-tubulin (9,44) and its association with p59fyn kinase. In addition, Syk and Zap-70 were able to associate with and phosphorylate the C-terminus of {alpha}-tubulin, which is known to coincide with the binding site for microtubule-associated proteins (MAP) (10,45). Importantly, phosphorylated {alpha}-tubulin was not incorporated into microtubules, but remained in the soluble, unpolymerized tubulin pool. These and other experiments led to the suggestion that immunoreceptor-induced activation of Syk/Zap-70 kinases leads to phosphorylation of {alpha}-tubulin and subsequent dissociation of MAP. These changes could destabilize the existing microtubules and reduce the pool of tubulins available for polymerization. If these changes are located on one side of the cell, the result could be a repositioning of MTOC. It was suggested that repositioning of MTOC could form a biochemical basis of restructuralization of microtubules in antigen-activated cells (46). Several findings presented in this report support the role of Lyn kinase in biochemical events leading to morphological changes in activated cells: (i) increased tyrosine phosphorylation in activated cells is found in the perinuclear region where a typical MTOC protein, pericentrin, is located, (ii) Lyn kinase is also concentrated in the perinuclear region and (iii) Lyn kinase interacts directly or indirectly with {gamma}-tubulin. Because we failed to find any Syk kinase in precipitates of soluble tubulin (L. Dráberová, unpublished results), it is possible that in RBL cells it is Lyn kinase, rather than Syk kinase, that has a role in microtubule formation. The presence of a large cytosolic pool of {gamma}-tubulin contrasts with the fact that microtubule nucleation reaction is restricted to the centrosome. However, it is possible that regulatory events, initiated by cell activation, are able to turn on and off the nucleation reaction in MTOC. Alternatively, a critical concentration of {gamma}-tubulin and {gamma}-tubulin-interacting proteins could be capable of initiating the microtubule nucleation in other cytoplasmic sites and thus contribute to a more rapid restructuralization of microtubule network.

It has been previously found that a significant fraction of Lyn kinase is located in membrane microdomains resistant to non-ionic detergents (27,47). When Fc{varepsilon}RI or Thy-1 are aggregated by cross-linking they accumulate in these microdomains where Lyn could initiate a signaling cascade by transphosphorylation followed by the engagement of other signaling components. In RBL cells activated by antibody-mediated Thy-1 aggregation, we observed a movement of {alpha}ß-tubulin dimers, and to a lesser extent also {gamma}-tubulin, into light-density membrane microdomains where Lyn, Thy-1 and some other signaling proteins are located (28). This association could lead to tyrosine phosphorylation of tubulins with the same consequences as tyrosine phosphorylation mediated by Syk/Zap kinases (see above). Interestingly, the movement of {alpha}ß-tubulin dimers into light-density fractions was not observed in cells activated by IgE–antigen complexes. This could correspond with previous findings that movement of Fc{varepsilon}RI into membrane microdomains required strong cross-linking of Fc{varepsilon}RI by complexes of biotinylated IgE and streptavidin; antigen-mediated cross-linking was much less efficient (48).

In summary, our results indicate that {gamma}-tubulin form complexes with Lyn and other signal transduction molecules involved in activation of RBL cells. The role of these complexes in early stages of mast cell activation as well as in activation-mediated changes in cell morphology remain to be elucidated.


    Acknowledgments
 
We thank S. J. Doxsey (University of Massachusetts Medical Center, Worcester, MA) for antibody against pericentrin. This work was supported in part by grants A5052704 and A5052605 from the Grant Agency of the Academy of Sciences of the Czech Republic, and by grants 301/96/0897, 312/96/K205, 310/97/0237 and 204/97/0239 from the Grant Agency of the Czech Republic. The research of Petr. D. was supported in part by an International Research Scholar's award from Howard Hughes Medical Institute.


    Abbreviations
 
GST glutathione-S-transferase
HRP horseradish peroxidase
MAP microtubule-associated protein
MSB microtubule stabilizing buffer
MTOC microtubule-organizing center
PP1 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine
RBL rat basophilic leukemia
SS supernatant from saponin-treated cells
SNP supernatant from NP-40-extracted saponin-insoluble cell ghosts

    Notes
 
Transmitting editor: I. Pecht

Received 4 April 1999, accepted 27 July 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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