Professor Jens F Rehfeld
email: jens.f.rehfeld@rh.regionh.dk

Xiaorong Zhu

Christina Norrbom

Jens R Bundgaard

Anders H Johnsen

John E Nielsen

Jonas Vikesaa

Jeffrey Stein

Arunangsu Dey

Donald F Steiner

Xiaorong Zhu ++

Christina Norrbom ++

John E Nielsen +

Jeffrey Stein++

Arunangsu Dey++

Donald F Steiner ++

Lennart Friis-Hansen

Prohormone convertases 1/3 and 2 together orchestrate the site-specific cleavages

Keywords: biosynthesis, gastrin, peptide hormones, posttranslational processing, progastrin, prohormone convertases

come

INTRODUCTION

Gastrin is a gastrointestinal hormone that regulates gastric acid secretion and mucosal cell growth [for review, see ref. 1]. Most gastrin is synthesized in antral G-cells where progastrin through multiple steps is processed to α-amidated gastrins of different lengths (Figure 1). Of these, gastrin-34 and gastrin-17 predominate in normal tissue and circulation. Their synthesis requires cleavage of three evolutionary conserved dibasic sequences in progastrin: R 36 R 37 , K 53 K 54 and R 73 R 74 [2][3][4][5][6]. Outside antrum the gastrin gene is expressed at low levels in the small intestinal mucosa, in the fetal pancreas, in pituitary corticotrophs and melanotrophs, in hypothalamo-pituitary neurons and in spermatozoes [START_REF] Larsson | A peptide resembling the COOH-terminal tetrapeptide amide of gastrin from a new gastrointestinal endocrine cell type[END_REF][START_REF] Larsson | Pancreatic gastrin in foetal and neonatal rats[END_REF][START_REF] Rehfeld | Localisation of gastrins to neuro-and adenohypophysis[END_REF][START_REF] Rehfeld | Pituitary gastrin: Different processing in corticotrophs and melanotrophs[END_REF][START_REF] Schalling | Expression and localization of gastrin messenger RNA and peptide in spermatogenic cells[END_REF] as well as in a number of common cancers [for review, see ref . 12]. The extra-antral processing of progastrin is often cell-type specific. For instance, there is no cleavage of the K 53 K 54 site in neither pituitary corticotrophs nor ileal TG-cells [START_REF] Rehfeld | Accumulation of nonamidated preprogastrin and preprocholecystokinin products in porcine pituitary corticotrophs: Evidence of posttranlational control of cell differentiation[END_REF][START_REF] Friis-Hansen | Ileal expression of gastrin and cholecystokinin: In search of a related hormone[END_REF].

The G-cell maturation of progastrin requires several processing enzymes (Figure 1). While the involvement of tyrosyl protein sulfotransferases, carboxypeptidase E and the amidating enzyme complex (peptidylglycyl α-amidating monooxygenase (PAM)) appears well-established [START_REF] Hilsted | Alphacarboxyamidation of antral progastrin: Relation to other post-translational modifications[END_REF][START_REF] Eipper | Peptidylglycine alphaamidation monooxygenase: a multifunctional protein with catalytic, processing, and routing domains[END_REF][START_REF] Bundgaard | Tyrosine O-sulfation promotes proteolytic processing of progastrin[END_REF][START_REF] Lacourse | Disturbed progastrin processing in carboxypeptidase E-deficient fat mice[END_REF], the role of the individual prohormone convertase (PC) remains to be settled. Mammals express seven basic amino acid specific PCs of which PC1/3 and 2 are assumed to be involved primarily in endoproteolytic processing of neuroendocrine proproteins [for reviews, see refs. [START_REF] Zhou | Proteolytic processing in the secretory pathway[END_REF][START_REF] Müller | The cell biology of the prohormone convertases PC1 and PC2[END_REF][START_REF] Scamuffa | Proprotein convertases: Lessons from knockouts[END_REF]. Both PC1/3 and PC2 have been demonstrated by immunocytochemistry in human and rat antral G-cells [START_REF] Scopsi | Proprotein convertases (PC1/PC3 and PC2) in normal and neoplastic human tissues: Their use as markers of neuroendocrine differentiation[END_REF][START_REF] Macro | Regulation by gastric acid of the processing of progastrin-derived peptides in rat antral mucosa[END_REF] and, at mRNA level, in rat antral extracts [START_REF] Macro | Identification and expression of prohormoneconverting enzymes in the rat stomach[END_REF]. PC5/6, however, also appears to be neuroendocrine as it is expressed in gastrointestinal endocrine cells and cerebral neurons [START_REF] Macro | Regulation by gastric acid of the processing of progastrin-derived peptides in rat antral mucosa[END_REF][START_REF] Lusson | cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells[END_REF][START_REF] Villeneuve | Immunohistochemical distribution of the prohormone convertase PC5-A in rat brain[END_REF]. Accordingly, small amounts of PC5/6 mRNA were also detected in extracts of the rat antrum, and immunoreactive PC5/6 demonstrated in rat antral G-cells [START_REF] Macro | Regulation by gastric acid of the processing of progastrin-derived peptides in rat antral mucosa[END_REF][START_REF] Macro | Identification and expression of prohormoneconverting enzymes in the rat stomach[END_REF].

Recently, we showed in PC2 and 7B2 knockout mice that PC2 plays a distinct role limited to partial cleavage of the K 53 K 54 site of progastrin [START_REF] Rehfeld | Progastrin processing differs in 7B2 and PC2 knockout animals : a role for 7B2 independent of action on PC2[END_REF]. Hence, PC2 determines the concentrations and ratio between the two primary gastrins, gastrin-34 and gastrin-17. But PC2 was without effect on any of the two di-arginyl sites of which processing of the latter, R 73 R 74 , is decisive for production of bioactive gastrins (Figure 1). In the present study we have examined the roles of PC1/3 and PC5/6, which became possible with the development of mice lacking PC1/3 [START_REF] Zhu | Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects[END_REF], and the availability of active recombinant PC5/6. Taken together, the results should reflect the normal endoproteolytic profile of progastrin processing in antral G-cells since no other PCs are expressed in endocrine G-cells.

EXPERIMENTAL Mice

The PC1/3 null mutant mice were generated as described earlier [START_REF] Zhu | Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects[END_REF]. For the present study, tissue was obtained from 12-week-old PC1/3 null mice and wild-type (+/+) littermates of the same age. The care and treatment of all animals were in accordance with NIH and institutional guidelines.

Tissue isolation and extraction

Mice in groups of four were sacrificed by CO 2 inhalation, and stomach tissue was rapidly dissected and frozen on dry ice. The tissue was gently rinsed in phosphatebuffered saline on ice before freezing. Tissue extracts for radioimmunoassay (RIA) were prepared as previously described [START_REF] Rehfeld | Ontogeny of procholecystokinin maturation in rat duodenum, jejunum, and ileum[END_REF]. Briefly, frozen tissues were boiled in water (1 ml/mg) for 20 min, homogenized (polytron) and centrifuged for 30 min at 10,000 rpm. The supernatants were withdrawn and the pellets re-extracted in 0.5 M CH 3 COOH (1 ml/mg), rehomogenized, incubated at room temperature for 30 min and centrifuged. The neutral water and acid supernatants were stored at -20°C until RIA analysis. The acid extracts, however, turned out to contain only negligible amounts of progastrin and its products. Consequently they were not included in further examinations.

Chromatography

One or two ml of extracts were applied to Sephadex G-50 superfine columns (10 × 1,000 mm) which were eluted at 4°C with 0.02 M barbital buffer, pH 8.4, containing 0.1% bovine serum albumin. Fractions of 1.0 ml were collected at a rate of 4.0 ml/h. The columns were calibrated with [ 125 I]albumin (void volume), gastrin-34 and gastrin-17, and with 22 NaCl (total volume). The eluted fractions were assayed with a library of sequence-specific radioimmunoassays against different progastrin epitopes and fragments using antibodies nos. 2604, 2605, 8017, 2145, 5284, and 3208, as detailed below. The percentages of carboxyamidated gastrin were calculated by planimetry. Four different extracts from each of the two groups of mice (PC1/3 knockout, and corresponding wild-type controls) were subjected to gel chromatography.

Radioimmunoassays

A library of six sequence-specific antisera against progastrin and its fragments was used to measure the different forms of gastrin and progastrin processing intermediates (Figure 1). The sum of carboxyamidated gastrins was measured using the gastrin-specific antiserum no. 2604, [ 125 I]gastrin-17 as tracer and gastrin-17 as standard. Antibody no. 2604 binds the carboxyamidated gastrin-34 and gastrin-17 with equimolar affinity, irrespective of size and degree of tyrosyl O-sulfation [START_REF] Rehfeld | Production and evaluation of antibodies for the radioim-munoassay of gastrin[END_REF]. The crossreactivity with homologous cholecystokinin peptides is negligible [START_REF] Rehfeld | Production and evaluation of antibodies for the radioim-munoassay of gastrin[END_REF][START_REF] Rehfeld | Accurate measurement of cholecystokinin in plasma[END_REF]. Non-sulfated carboxyamidated gastrin-34 and -17 were measured in parallel using antibody no. 2605 [START_REF] Rehfeld | Sulfation of gastrin : effect on immunoreactivity[END_REF]. Glycine-extended processing intermediates of progastrin were measured using antisera nos. 5284 and 3208 with [ 125 I]glycine-extended gastrin-17 as tracer and glycine-extended gastrin-17 as standard [START_REF] Hilsted | Measurement of precursors for alpha-amidated hormones by radioimmunoassay of glycine-extended peptides after trypsin-carboxypeptidase B cleavage[END_REF]. Glycine-arginine-extended intermediate precursors were measured using antiserum nos. 5284 and 3208 following enzymatic pretreatment with only carboxypeptidase B [START_REF] Hilsted | Measurement of precursors for alpha-amidated hormones by radioimmunoassay of glycine-extended peptides after trypsin-carboxypeptidase B cleavage[END_REF]. To measure all precursor forms of gastrin, samples were pretreated with both trypsin and carboxypeptidase B followed by radioimmunoassay measurement using antiserum no. 5284, as previously described [START_REF] Hilsted | Measurement of precursors for alpha-amidated hormones by radioimmunoassay of glycine-extended peptides after trypsin-carboxypeptidase B cleavage[END_REF]. Carboxypeptidase B mimics the effect of carboxypeptidase E, while trypsin mimics the effects of PCs. Antiserum no. 2145 was used to measure fragment 38-52 of human progastrin, corresponding to the pyroglutaminated Nterminus of gastrin-34 using [ 125 I]gastrin-34 as tracer and gastrin-34 as standard [START_REF] Van Solinge | Radioimmunoassay for sequence 38-54 of human progastrin: increased diagnostic specificity of gastrin-cell diseases[END_REF]. Correspondingly, antiserum no. 8017 was used to measure the pyroglutaminated Nterminus of human gastrin-17 using [ 125 I]gastrin-17 as tracer and gastrin-17 as standard [35]. Due to species differences in the N-terminal sequences of mammalian gastrin-34 and -17, it was necessary to use antisera nos. 2145 and 8017 for characterization of possible progastrin cleavage products after incubation of human progastrin with recombinant PC5/6. To monitor the purification of recombinant human progastrin, we also used a radioimmunoassay based on antiserum no. 94023 directed against the very N-terminus of human progastrin together with the corresponding N-terminal decapeptide fragment tyrosylated at its C-terminus as tracer and standard [START_REF] Goetze | Antral content, secretion and metabolism of N-terminal progastrin fragments[END_REF].

In vitro cleavage of progastrin and progastrin fragments with recombinant PC5/6A

To produce recombinant human progastrin, an expression vector of human preprogastrin, as previously described [37], was transiently transfected into CHO cells using a standard Fugene6 transfection protocol (Roche Applied Science). After 48 h, culture media was recovered, separated by gel filtration on a 1000x 50 mm Superdex G50 Superfine column and eluted, as previously described [37]. Recovered fractions were analysed for progastrin using the radioimmunoassay specific to the N-terminus of human progastrin [START_REF] Goetze | Antral content, secretion and metabolism of N-terminal progastrin fragments[END_REF]. Fractions eluting at the position of intact progastrin were pooled and subjected to an in vitro PC5/6A cleavage assay. Half of the peptide pool was added 10 units of human PC5/6 (Phenoswitch Bioscience Inc., Sherbrooke, Canada) and reaction buffer to a concentration as suggested by Phenoswitch Bioscience (vide intra) and incubated for 4 hours at 37ºC. A control pool was only added reaction buffer and incubated like the reaction pool. Both pools were subjected to gel filtration and analysed using specific radioimmunoassays towards the N-terminals of progastrin, gastrin-34 and gastrin-17 as well as C-terminal assay against glycineextended gastrin [START_REF] Rehfeld | Production and evaluation of antibodies for the radioim-munoassay of gastrin[END_REF][START_REF] Hilsted | Measurement of precursors for alpha-amidated hormones by radioimmunoassay of glycine-extended peptides after trypsin-carboxypeptidase B cleavage[END_REF][START_REF] Van Solinge | Radioimmunoassay for sequence 38-54 of human progastrin: increased diagnostic specificity of gastrin-cell diseases[END_REF][START_REF] Goetze | Antral content, secretion and metabolism of N-terminal progastrin fragments[END_REF].

Gastrin-34 from Sigma and the peptides GPASHHRRQLGPQGP (progastrin cleavage site for gastrin-34 N-terminus) and PEELVHRVKRRADPDPMK (PC substrate [START_REF] Srour | TACE/ADAM-17 maturation and activation of scheddase activity require proprotein convertase activity[END_REF]) were custom synthesized by Schafer-N (Copenhagen, Denmark). Incubation of the peptides were performed as suggested by PhenoSwitch in a total volume of 100 µl 20 mM Bis-Tris buffer pH 6.5, containing 1 mM CaCl 2 , BSA (2 g/l), 100 µM peptide (substrate) and 10 U of PC5/6 for 2 h at 37°C. The reaction was terminated by addition of 10 µl 10% trifluoroacetic acid. The reaction mixture was separated by HPLC using a 2.1 × 150 mm C18 column (Vydac, Hisperia, USA) eluted with a gradient from 10-40% acetonitrile over 30 min, the elution buffers contained 0.1% trifluoroacetic acid. The chromatograms were monitored at 214 nm and peaks were collected manually. The peptides collected were analysed by MALDI-TOF mass spectrometry (AutoFlex II, Bruker, Bremen, Germany) using α-cyano-4 hydroxycinnamic acid as matrix.

Immunocytochemistry

To examine the cellular co-localization between gastrin and PC1/3 or PC2, the stomach was removed from three wild-type mice aged 3-4 months, cut open, washed, and immersion fixed overnight in 4% paraformaldehyde. Six µm traverse paraffin sections were cut, mounted on superfrosted glass slides and baked overnight at 60°C. Antigen retrieval was performed by boiling the slides in a microwave oven for 3 minutes at 800W, followed by 3 times 5 minutes at 400W in a 10mM Tris-buffered EGTA solution at pH 9.0. Preblocking was performed in 10% ovine serum in TBS for 20 minutes. The sections were incubated with PC1/3 or PC2 antisera diluted 1:10,000 overnight at 4°C [START_REF] Shen | Biosynthesis of the prohormone convertase PC2 in Chinese hamster ovary cells and in rat insulinoma cells[END_REF][START_REF] Hornby | Immunocytochemical localization of the neuropeptide-synthesizing enzyme PC1 in AtT-20 cells[END_REF]. The primary antiserum was detected with Envision+ (Dako, Glostrup, Denmark) for 1 hr followed by incubation with a Cy3 conjugated antihorseradish perioxidase antibody (Jackson, West Grove, Pennsylvania, USA) diluted 1:400 for 1 hr. Gastrin was detected with overnight incubation with antiserum no. 8007 (dilution 1:1000) at 4°C and subsequently reacted to Alexa 488 conjugated donkey-anti rabbit F(ab)-fragments (dilution 1:1000) (Molecular Probes, Inc., Eugene, Oregon, USA). Antiserum no. 8007 is specific to the caboxyamidated C-terminus of the gastrins [START_REF] Rehfeld | Accurate measurement of cholecystokinin in plasma[END_REF]. Cover slips were mounted with Aquamount (Dako). Analysis of the sections was performed with Zeiss LSM510 confocal microscope (Zeiss, Oberkochen, Germany).

In situ hybridisation

Serial sections were probed to the PC5/6 antisense probe, gastrin antiserum no. 8007 [START_REF] Rehfeld | Accurate measurement of cholecystokinin in plasma[END_REF] and PC5/6 sense probe.

In situ hybridisation was performed as previously described [START_REF] Nielsen | Germ cell differentiation-dependent and stage-specific expression of LANCL1 in rodent testis[END_REF]. In brief, mouse stomach was fixed in 4% paraformaldehyde, 8 µm sections were placed on Super-FrostRPlus slides (Menzel GmbH, Braunschweig Germany), deparaffinised and PFA (4%) fixed, and treated with proteinase K (P2308; Sigma, St. Louis, Missouri, USA), postfixed (4% PFA), prehybridised for 1h at 50°C, and incubated over night with the labelled PC5/6 probe (50°C), specific to nucleotide. Visualization was made with anti-digoxigenin-AP (Roche Diagnostics, Mannheim, Germany) and chromogens BCIP (Sigma B-8503) and NBT (Sigma N-6876).

RESULTS AND DISCUSSION

Progastrin processing in PC1/3 and PC2 knockout mice

As shown in Table 1 and Figure 2, the antral concentrations of progastrin and its products are grossly altered in PC1/3 knockout mice. The concentration of progastrin itself is increased nearly three-fold, whereas concentrations of the processing intermediates are reduced to one third, and the bioactive, carboxyamidated gastrins are reduced to one fourth of the concentrations in wild-type controls. The reductions contrast the earlier findings in PC2 knockout mice and the carboxypeptidase E-deficient (CPE fat ) mice, in which the antral concentrations of the bioactive, carboxyamidated gastrins are maintained at a normal level in spite of major disturbances at earlier steps in the processing pathway and in the ratio between gastrin-34 and gastrin-17 (Table 1, Figure 2 and [START_REF] Lacourse | Disturbed progastrin processing in carboxypeptidase E-deficient fat mice[END_REF][START_REF] Rehfeld | Progastrin processing differs in 7B2 and PC2 knockout animals : a role for 7B2 independent of action on PC2[END_REF]).

Gel chromatography of neutral antral extracts revealed an unchanged molecular pattern of carboxyamidated gastrins in the PC1/3 knockout mice, despite the marked difference in tissue concentrations (Figure 3). Hence, the ratios between gastrin-34 and gastrin-17, and between tyrosyl-sulfated and non-sulfated gastrins were as in the wildtype controls (Figure 3). The low concentrations ex- cluded chromatographic examination of the processing intermediates, but it was possible to study the progastrin immunoreactivity, which showed a significant difference between the null mice and their wild-type littermates (Figure 4). Thus, the progastrin molecule is only partly cleaved at the dibasic R 36 R 37 site and even less at the K 53 K 54 site in the PC1/3 null mice (Figure 4 upper panel), while progastrin in the controls is almost completely processed to shorter fragments (Figure 4 lower panel). The modest differences in antral concentrations of progastrin and its processing products between the three groups of wild-type controls (Table 1) supposedly reflect differences in mouse strains and age. Hence, the mice used in the present study are younger (3 months of age) than those used in the studies of carboxypeptidase Edeficiency and PC2 knockout that were up to 6 months of age [START_REF] Lacourse | Disturbed progastrin processing in carboxypeptidase E-deficient fat mice[END_REF][START_REF] Rehfeld | Progastrin processing differs in 7B2 and PC2 knockout animals : a role for 7B2 independent of action on PC2[END_REF]. As shown before, the antral concentration of progastrin declines with age in rodents [START_REF] Hilsted | Progastrin maturation during ontogenesis. Accumulation of glycine-extended gastrins in rat antrum at weaning[END_REF].

The processing observed in PC1/3 knockout mice indicates that PC1/3 initiates cleavage at the N-terminal di-arginyl site (R 36 R 37 ) at an early stage in the processing, since more progastrin and a large fragment corresponding to sequence 38-83 of progastrin were found in the antral extract of PC1/3 null mice (Figure 4). Subsequently, PC1/3 continues to cleave the C-terminal di-arginyl site R 73 R 74 . PC1/3, however, appears to be without effect on the di-lysyl site, K 53 K 54 (Figure 3). This endoproteolytic cleavage pattern of progastrin is opposite to that of PC2 (Table 1, Figure 2 and [START_REF] Rehfeld | Progastrin processing differs in 7B2 and PC2 knockout animals : a role for 7B2 independent of action on PC2[END_REF]). Thus, PC2 only cleaves the single di-lysyl site of progastrin, K 53 K 54 , the cleavage for half of which PC2 appears to be responsible [START_REF] Rehfeld | Progastrin processing differs in 7B2 and PC2 knockout animals : a role for 7B2 independent of action on PC2[END_REF]. The results hence support the contention that PC1/3 generally prefers arginine containing cleavage sites, while PC2 is activated later in the processing pathway to cleave mainly lysine containing dibasic sites at the lower pH characteristic of mature secretory granules [START_REF] Müller | The cell biology of the prohormone convertases PC1 and PC2[END_REF].

The present data support our earlier observation that pituitary corticotrophs which contain only PC1/3 and no PC2 cannot process the K 53 K 54 site of progastrin. Therefore, corticotrophs synthesize gastrin-34, but no gastrin-17 [START_REF] Rehfeld | Localisation of gastrins to neuro-and adenohypophysis[END_REF][START_REF] Rehfeld | Pituitary gastrin: Different processing in corticotrophs and melanotrophs[END_REF][START_REF] Rehfeld | Accumulation of nonamidated preprogastrin and preprocholecystokinin products in porcine pituitary corticotrophs: Evidence of posttranlational control of cell differentiation[END_REF]. This notion is also supported by Sawada el al. who found that PC2 transfected into AtT-20 cells but not PC1/3 can accomplish cleavage of the dilysyl site in progastrin [START_REF] Sawada | Diminished prohormone convertase 3 expression (PC1/PC3) inhibits progastrin post-translational processing[END_REF]. Also proopiomelanocortin (POMC) contains a KK site within β-endorphin which remains unprocessed in AtT-20 cells but can be processed upon contransfection with PC2 [START_REF] Zhou | The prohormone convertases PC1 and PC2 mediate distinct endoproteolytic cleavages in a strict temporal order during proopiomelanocortin biosynthetic processing[END_REF][START_REF] Zhou | Endoproteolytic processing of proopiomelanocortin and prohormone convertases 1 and 2 in neuroendocrine cells overexpressing prohormone convertases 1 or 2[END_REF]. However, cleavage of the KK site within proenkepalin is apparently largely mediated by PC1/3 since it is efficiently cleaved in AtT-20 cells [START_REF] Mathis | Posttranslational processing of proenkephalin in AtT-20 cells: evidence for cleavage at a Lys-Lys site[END_REF]. The primary structure of the three precursors (progastrin, POMC and proenkephalin) re-veals extensive differences in the vicinity of the KK sites, which could account for the differential ability of PC1/3 to recognize this type of processing site within different contexts.

A surprising finding in the present study was the increased concentration of progastrin in the antrum of PC1/3 null mice, while the concentrations of the processing products were drastically reduced (Table 1 and Figure 2). It is obvious that the lack of PC1/3 reduces the processing of progastrin severely. But it is an unsolved question why the total translational product in PC1/3 null mice is reduced, whereas it is increased in other processing enzyme deficiencies [START_REF] Lacourse | Disturbed progastrin processing in carboxypeptidase E-deficient fat mice[END_REF]. The C-terminal dibasic cleavage sites in progastrin are, however, part of the motif for sorting to the regulated secretory pathway [START_REF] Bundgaard | Progastrin is directed to the regulated secretory pathway by synergistically acting basic and acidic motifs[END_REF]. Moreover, PC1/3 has been shown to enhance the post-Golgi sorting of some prohormones [START_REF] Mulcahy | Prohormone-convertase 1 processing enhances post-Golgi sorting of prothyrotropin-releasing hormone-derived peptides[END_REF]. It is therefore possible that the lack of PC1/3 per se reduces the storage of progastrin and its products in the secretory granules of G-cells.

Progastrin cleavage by PC5/6

Since half of the KK site within progastrin is cleaved in the absence of PC2 [START_REF] Rehfeld | Progastrin processing differs in 7B2 and PC2 knockout animals : a role for 7B2 independent of action on PC2[END_REF], and since 30% of the two diarginyl sites are cleaved in the absence of PC1/3 (Table 1), other convertases might participate in the in vivo endoproteolytic processing of progastrin in normal Gcells. A candidate for this activity is PC5/6, which has been shown to be expressed in endocrine cells of the gastrointestinal tract [START_REF] Lusson | cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells[END_REF][START_REF] Nakagawa | Identification and functional expression of a new member of the mammalian Kex2-like processing endoprotease family: its striking structural similarity to PACE4[END_REF]. As shown here and in the rat, PC5/6 gene expression can be demonstrated in antral Gcells (Figure 7 and [START_REF] Macro | Regulation by gastric acid of the processing of progastrin-derived peptides in rat antral mucosa[END_REF]). PC5/6 is known to cleave basic processing sites [START_REF] Zhou | Proteolytic processing in the secretory pathway[END_REF][START_REF] Müller | The cell biology of the prohormone convertases PC1 and PC2[END_REF]. In order to examine possible effects of PC5/6, we first looked at the processing of progastrin in SK-N-MC cell line, since PC5/6 knockout mice were not available, and since SK-N-MC cells have been claimed to express the PC5A isoform but neither PC1/3 nor PC2 [START_REF] Cain | Neuronal cell lines expressing PC5, but not PC1 or PC2, process Pro-CCK into glycine-extended CCK 12 and 22[END_REF]. Resent re-examinations, however, have shown that SK-N-MC cells may indeed express PC1/3 at protein level [Bundgaard J, unpublished results] and also PC2, although without its chaperone 7B2 [Lindberg I, personal communication]. Consequently, we decided to exclude the results obtained of progastrin processing in this cell-line.

Instead we examined directly in vitro the substrate activity of PC5/6 against intact progastrin. Human progastrin was expressed in CHO cells, which do not cleave progastrin. Progastrin was subsequently purified by size exclusion gel chromatography, and recovered peptide was incubated in vitro with recombinant PC5/6A. Prod- ucts were subjected to repeated chromatography and analysed using radioimmunoassays that are specific to the dibasic cleavage sites of human progastrin [START_REF] Van Solinge | Radioimmunoassay for sequence 38-54 of human progastrin: increased diagnostic specificity of gastrin-cell diseases[END_REF]35]. Enzyme treated progastrin eluted identically to untreated control fractions (Figure 5), and the site-specific radioimmunoassays did not detect processing at any of the cleavage sites. Moreover, digestion with recombinant PC5/6 of gastrin-34 and the synthetic peptide spanning the N-terminus of gastrin-34 within progastrin resulted in no cleavage, while the general PC substrate [START_REF] Srour | TACE/ADAM-17 maturation and activation of scheddase activity require proprotein convertase activity[END_REF] used as control was cleaved as expected. Consequently, we conclude that PC5/6 is without effect on progastrin, and that the processing of progastrin originally observed in S-K-N-MC cells is due to other endoproteases than PC5/6.

Prohormone convertase expression in G-cells

As shown in Figure 6, both PC1/3 and PC2 are expressed in normal antral G-cells in mice. The immunocytochemical results are in accordance with earlier results in human and rat antrum [START_REF] Scopsi | Proprotein convertases (PC1/PC3 and PC2) in normal and neoplastic human tissues: Their use as markers of neuroendocrine differentiation[END_REF][START_REF] Macro | Regulation by gastric acid of the processing of progastrin-derived peptides in rat antral mucosa[END_REF]. In addition, as shown here by in situ hybridisation (Figure 7), also PC5/6 is expressed in murine antral G-cells.

The interplay of PC1/3 and PC2 and the role of PC5/6 in G-cells

Since no cleavage was found in the in vitro studies using PC5/6 against intact progastrin and progastrin fragments containing the dibasic cleavage sites, whereas the same PC5/6 was fully active in cleaving a control peptide. PC5/6 hardly plays a significant role in the G-cell maturation of progastrin. It is possible that PC5/6 is instead involved in the processing of other neuroendocrine G-cell products such as chromogranin A and neuropeptide W [START_REF] Helle | The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: Comparative and functional aspects[END_REF][START_REF] Mondal | Neuropeptide W is present in antral G cells of rat, mouse and human stomach[END_REF]. Thus, the endoproteolytic maturation of progastrin as seen in normal G-cells appears to require an interplay, primarily between PC1/3 and PC2. We believe that the processing begins with PC1/3, which is solely responsible for the cleavage of R 36 R 37 . But, subsequently, PC1/3 cleaves the crucial R 73 R 74 . Later, in secretory granules, PC2 performs the partial cleavage of K 53 K 54 to ensure the production of gastrin-17. earlier study [START_REF] Rehfeld | Progastrin processing differs in 7B2 and PC2 knockout animals : a role for 7B2 independent of action on PC2[END_REF]. A library of sequence-specific antibodies was used in combination with in vitro protease treatment to measure bioactive gastrin and various precursor peptides, as described in the text.

Figure 2 Average percentages of the processing parameters measured in antral extracts of PC1/3 knockout mice

The parameters (progastrin, gastrin-Gly-Arg, gastrin-Gly and bioactive gastrin-amide) are compared with those measured in PC2 knockout mice and in carboxypeptidase E deficient (CPE) mice [START_REF] Zhou | Proteolytic processing in the secretory pathway[END_REF][START_REF] Zhu | Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects[END_REF], as well as with their respective wild-type controls (broken line at 100%).

Figure 3. Gel chromatography on Sephadex G-50 superfine columns of antral extracts from PC1/3 knockout mice (left panels) and from corresponding wild-type control mice (WT, right panels)

The chromatographic elutions were monitored using antibody 2604 that binds carboxyamidated gastrins (the bioactive gastrins), irrespective of the degree of tyrosylsulfation (upper panels) and antibody 2605 that binds only non-sulfated (ns) gastrin (lower panels). Antral extracts from four different mice in each of the two groups (PC1/3 and controls) were subjected to chromatography. Extracts containing similar amounts of gastrin were applied to the columns. Those shown above are characteristic of each group. The chromatographic elutions were monitored using antibody 5284 that binds glycine-extended gastrins after trypsin and carboxypeptidase B treatment of each fraction. Antral extracts from four different mice in each of the two groups (PC1/3 and controls) were subjected to chromatography. Those shown are characteristic of each group.

Figure 5

Gel chromatography on Sephadex G-50 superfine columns of human progastrin before and after incubation with recombinant PC5/6

The chromatographic elution was monitored by a radioimmunoassay specific to the intact N-terminus of human progastrin using antiserum No. 94023 (ref. 36). The upper panel shows the elution without prior treatment with PC5/6. The lower panel shows the elution after treatment with PC5/6, which has been shown to cleave a relevant control peptide.

Figure 6 Gastrin and PC1/3 co-localization

Gastrin (green) co-localizes with PC1/3 (red) (Panel A). Gastrin (green) also co-localizes with PC2 (red) (Panel B). In contrast to PC1/3, which is mainly found in the antral G-cells, many other antral endocrine cells also express PC2 (Panel B). (Spacebar 50 µm). Gastrin was identified with antibody no. 8007.

Biochemical

  Journal Immediate Publication. Published on 13 Jun 2008 as manuscript BJ20080881 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080881 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

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Biochemical

  Journal Immediate Publication. Published on 13 Jun 2008 as manuscript BJ20080881 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080881 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 13 Jun 2008 as manuscript BJ20080881 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080881 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

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*

  products in antral mucosa of PC1/3 and PC2* null mice, carboxypeptidase E deficient mice** and corresponding wild-type mice (pmol/g tissue; mean ± SEM) data from ref. 27; ** data from ref. 18 Biochemical Journal Immediate Publication. Published on 13 Jun 2008 as manuscript BJ20080881 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080881 S t a g e 2 ( a ) P O S T -P R I N T

  Figure 1 (A) The mouse progastrin sequenceis given in single letter code with the three dibasic cleavage sites printed in bold and asterisk indicating the single tyrosyl-sulfation site. The bioactive tetrapeptide sequence in the gastrins is boxed.

Figure 1 (

 1 Figure 1 (B) Diagram of the co-and posttranslational modification of progastrin in antral GcellsActivation of the gastrin amidation site (-Phe71-Gly72-Arg73-Arg74-) occurs via a series of carboxyterminal cleavages and modifications. Endoproteolytic cleavage by prohormone convertases (PCs) produces the carboxypeptidase E substrate (-Phe-Gly-Arg). Carboxypeptidase E then acts in secretory granules to remove the Cterminal arginyl residue yielding glycine-extended gastrin (-Phe-Gly). Carboxyamidation by PAM subsequently results in the production of bioactive gastrin (-Phe-NH2). Concomitant N-terminal cleavage by PCs produces bioactive gastrins of varying size (e.g. gastrin-34 and gastrin-17). The indications of PC1/3, PC2 and PC5/6 in the diagram is based on the results of the present and an earlier study[START_REF] Rehfeld | Progastrin processing differs in 7B2 and PC2 knockout animals : a role for 7B2 independent of action on PC2[END_REF]. A library of sequence-specific antibodies was used in combination with in vitro protease treatment to measure bioactive gastrin and various precursor peptides, as described in the text.

Figure 4 .

 4 Figure 4. Gel chromatography on Sephadex G-50 superfine columns of antral extracts from PC1/3 knockout mice (KO, upper panel) and littermate wild-type controls (WT, lower panel)

Figure 7

 7 Figure 7Gastrin and PC5/6 co-localization Serial sections were examined for PC5/6 expression by in situ hybridisation (Panel A) and gastrin expression by immunohistochemistry (Panel B). Panel C shows the PC5/6 in situ using the sense probe. The black arrows point to cells that express both PC5/6 and gastrin (Panel A and B). The white arrow identifies a cell expressing PC5/6 but not gastrin (Panel A and B). (Spacebar 50 µm). Gastrin was identified using antibody no. 8007

Figure 1 BiochemicalFigure 3

 13 Figure 1

Figure 4 Biochemical

 4 Figure 4

Figure 5 Biochemical

 5 Figure 5

  Figure 6 G-CELLS

Table 2 The site-specific cleavages at dibasic sequences in progastrin by prohormone conver- tases (PCs) expressed in antral G-cells

 2

	Biochemical Journal Immediate Publication. Published on 13 Jun 2008 as manuscript BJ20080881
	Prohormone Convertase	R 36 R 37	K 53 K 54	R 73 R 74
	PC1/3	+ +	-	+ +
	PC2	-	+	-
	PC5/6	-	-	-

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Rehfeld, J.F. (1998) The new biology of gastrointestinal hormones. Physiol. Rev. 78, 1087-1108

2 Yoo, O.J., Powell, C.T., and Agarwal, K.L. (1982) Molecular cloning and nucleotide sequence of fulllength of cDNA coding for porcine gastrin. Proc. Natl. Acad. Sci. USA 79, 1049-

1053 3 Boel, E., Vuust, J., Norris, F., Norris, K., Wind, A., Rehfeld, J.F., and Marcker, K.A. (1983) Molecular cloning of human gastrin cDNA: evidence for evolution of gastrin by gene duplication. Proc. Natl. Acad. Sci. USA 80,

2866-2869 4 Gregory, R.A., and Tracy, H.J. (1964) The constitution and properties of two gastrins extracted from hog

antral mucosa. Gut 5, 103-117 5 Gregory, R.A., and Tracy, H.J. (1972) Isolation of two "big gastrins" from Zollinger-Ellison tumour tissue. Lancet 2, 797-799 6 Rehfeld, J.F., and Johnsen, A.H. (1994) Identification of gastrin component I as gastrin-71. The largest

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ACKNOWLEDGEMENTS

The skilful technical and secretarial assistance of Alice von der Lieth, Rikke Kröncke, Allan Kastrup, and Diana Skovgaard is gratefully acknowledged. The study was supported by grants from the Danish Biotechnology Program for Peptide Research; NIH grants DK-13914, DK-20595, and the Howard Hughes Medical Institute.


Libor Macurek

Eduarda Dráberová

Věra Richterová

Vadym Sulimenko

Tetyana Sulimenko

Lubica Dráberová

Vladimíra Marková

Dr Pavel Dráber
email: paveldra@img.cas.cz

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

Keywords: Fyn kinase, detergent-resistant membranes, phosphoinositide 3-kinase (PI3K), PI3K regulatory subunit (p85α), γ-tubulin, SH2-domain Ab, antibody, DRM, detergent-resistant membrane, GCP2, γ-tubulin complex protein 2, Fyn, protein tyrosine kinase p59 Fyn, mAb, mouse monoclonal antibody, MTOC, microtubule organizing center, p85α, regulatory p85α subunit of PI3K, PI3K, phosphoinositide

Molecular mechanisms controlling microtubule formation in cells with non-centrosomal microtubular arrays are not yet fully understood. The key component of microtubule nucleation is γ-tubulin. Although previous data suggested that tyrosine kinases might serve as regulators of γ-tubulin function, their exact roles remain enigmatic. Here we show that a pool of γ-tubulin associates with detergent-resistant membranes in differentiating P19 embryonal carcinoma cells which exhibit elevated expression of Src family kinase Fyn. Microtubule assembly assays demonstrated that membrane-associated γ-tubulin complexes are capable of initiating formation of microtubules. Pretreatment of the cells with Src family kinase inhibitors or wortmannin blocked the nucleation activity of γ-tubulin complexes. Immunoprecipitation experiments revealed that membrane-associated γ-tubulin forms complexes with Fyn kinase and phosphoinositide 3-kinase (PI3K). Furthermore, in vitro kinase assays showed that p85α regulatory subunit of PI3K serves as Fyn kinase substrate. Direct interaction of γ-tubulin with C-terminal Src homology 2 domain of p85α was determined by pull-down experiments and immunoprecipitation experiments with cells expressing truncated forms of p85α. The combined data suggest that Fyn kinase and PI3K might take a part in modulation of membrane-associated γ-tubulin activities.

INTRODUCTION

In most proliferating and migrating animal cells, centrosome is the main site for microtubule nucleation leading to the formation of radial microtubules. In contrast, differentiated cell types including muscle, epithelial and neuronal cells possess non-radial microtubules arranged in non-centrosomal arrays [START_REF] Bartolini | Generation of noncentrosomal microtubule arrays[END_REF]. In these cell types, the mechanism of microtubule nucleation is not fully understood. One of the key components required for microtubule organization is γ-tubulin [START_REF] Oakley | Identification of γ-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans[END_REF]. The γ-tubulin is associated with other proteins in cytoplasmic complexes. Large γ-tubulin ring complexes (γTuRCs) are formed by γ-tubulin small complexes (γTuSCs), comprising two molecules of γ-tubulin and one molecule each of γtubulin complex proteins (GCP)2 and GCP3, beside some other proteins [START_REF] Murphy | GCP5 and GCP6: two new members of the human γ-tubulin complex[END_REF]. These complexes are usually embedded into microtubule organizing centers (MTOCs), where they nucleate microtubules. We and others have demonstrated that γ-tubulin is also associated with cellular membranes [START_REF] Chabin-Brion | The Golgi complex is a microtubule-organizing organelle[END_REF][START_REF] Dryková | Plant γtubulin interacts with αβ-tubulin dimers and forms membrane-associated complexes[END_REF] and that protein tyrosin kinases form complexes with γ-tubulin [START_REF] Vogel | Phosphorylation of γ-tubulin regulates microtubule organization in budding yeast[END_REF][START_REF] Sulimenko | Regulation of microtubule formation in activated mast cells by complexes of gtubulin with Fyn and Syk kinases[END_REF]. Yet it remains to be clarified what are the regulatory mechanisms of microtubule nucleation from membranes.

Non-receptor protein tyrosin kinase p59 fyn (Fyn), a member of the Src kinases family, has been implicated in events regulating neuronal differentiation and function of neuronal cells. It is highly expressed in developing neurons, and the Fyn knock-out mice exhibit defects in neurite outgrowth in response to cell adhesion molecules. On the other hand, knock-outs of other kinases of Src family, the Src and Yes, do not lead to obvious defects in brain development [START_REF] Lowell | Knockouts of Src-family kinases: Stiff bones, wimpy T cells, and bad memories[END_REF]. Fyn therefore seems to play a key role in neuronal differentiation. It is recruited in neuronal cells to lipid microdomains rich in cholesterol and sphingolipids, and forms functional complexes with transmembrane proteins and other signaling molecules [START_REF] Tsui-Pierchala | Lipid rafts in neuronal signaling and function[END_REF].

It has been shown that Src and Lck from the family of Src kinases can also be involved in the regulation of phosphoinositide 3-kinase (PI3K)-dependent signaling pathways [START_REF] Chan | Small GTPases and tyrosine kinases coregulate a molecular switch in the phosphoinositide 3-kinase regulatory subunit[END_REF] required for neurite outgrowth [START_REF] Rodgers | Functions of PI 3-kinase in development of the nervous system[END_REF]. Class IA PI3Ks (hereafter referred to as PI3K) are heterodimers of a p110 catalytic subunit and a regulatory subunit. The most extensively studied regulatory subunit is the p85α. It consists of an inter-Src-homology 2 (SH2) domain flanked by two SH2 domains. The SH3 domain and RhoGAP domain reside in the N-terminal region of the molecule. Upon stimulation, the p85α binds to phosphorylated receptors or adaptor proteins, and the bond relieves basal inhibition of the catalytic subunit by p85α [START_REF] Engelman | The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism[END_REF]. Although complexes of p85α with γ-tubulin has been described [START_REF] Kapeller | Phosphoinositide 3-kinase binds constitutively to a/b-tubulin and binds to g-tubulin in response to insulin[END_REF][START_REF] Inukai | The N-terminal 34 residues of the 55 kDa regulatory subunits of phosphoinositide 3-kinase interact with tubulin[END_REF], the location of γtubulin binding site on p85α molecule as well as the type of this interaction are unknown. It is also unclear what is the biological relevance of γ-tubulin-p85α association.

Murine P19 embryonal carcinoma cells offer a well-characterized experimental model for in vitro neuronal differentiation induced by treatment with all-trans-retinoic acid (RA) [START_REF] Jones-Villeneuve | Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells[END_REF]. RA-stimulation induces changes in the expression signaling molecules [START_REF] Kukharskyy | Complexes of γ-tubulin with non-receptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells[END_REF] that make these cells suitable for biochemical studies of early stages of neuronal differentiation. We have previously shown that RA-induced differentiation of P19 cells results in an enhanced expression of Fyn, and that an association of γ-tubulin with detergent-resistant patches is detectable with immunofluorescence microscopy. Moreover, γ-tubulin forms complexes with Fyn and tyrosine-phosphorylated proteins [START_REF] Kukharskyy | Complexes of γ-tubulin with non-receptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells[END_REF]. To address the hypothesis that Src family kinases could be involved in the regulation of non-centrosomal microtubule formation, we characterized the membrane-associated γ-tubulin complexes in differentiating P19 cells.

MATERIAL AND METHODS

Materials

Protease-inhibitor cocktail tablets were from Roche. Src-family tyrosine kinase inhibitor PP2 and PP3 (negative control for PP2) were obtained from Calbiochem. RA, cytosine β-Darabinofuranoside, saponin, methyl-β-cyclodextrin, wortmannin and the Src-family tyrosine kinase inhibitor SU6656 were from Sigma. Imatinib mesylate, inhibitor of tyrosine kinases Bcr-Abl and c-kit was kindly donated by Novartis. 10 mM stock solutions of wortmannin, SU6656, PP2, PP3 and imatinib mesylate were prepared in DMSO. Recombinant PI3K (p110δ complexed to p85α) and Fyn were bought from Upstate Laboratories. Tubulin was prepared from porcine brain [START_REF] Sulimenko | Association of brain γ-tubulins with αβ-tubulin dimers[END_REF].

DNA constructs

Total RNA was isolated from BALB/c adult mouse brain using Trizol reagent (Gibco-BRL). Reverse transcription was performed with random hexamers and SuperScript III Reverse Transcriptase kit (Invitrogen). The fragment coding mouse GCP2 (GenBank NM_133755) was amplified by PCR using forward 5'-AGTCGGATCCAGCGAATTTCGGATTCAC-3' and reverse 5'-AAGCGTCGACTCAGCCAATCAGGAAATCTC-3' primers and total cDNA as template. Isolated fragment was ligated into pGEX-6P-1 vector (Amersham Biosciences) for preparation of GST-tagged fusion protein.

Full-length cDNA of human p85α was a gift of Dr. L. Rameh [START_REF] Rameh | Phosphatidylinositol (3,4,5)P3 interacts with SH2 domains and modulates PI-3-kinase association with tyrosine-phosphorylated proteins[END_REF]. Restriction sites were introduced by PCR, and p85α fragments were subcloned into pGEX-6P-1. The fragment encoding the cSH2 domain of p85α carrying R649L mutation in FLVR motif was amplified from p85-CT R649L construct provided by Dr. T. O. Chan [START_REF] Chan | Small GTPases and tyrosine kinases coregulate a molecular switch in the phosphoinositide 3-kinase regulatory subunit[END_REF] and subcloned into pGEX-6P-1 (cSH2-R649L). For in vivo experiments, p85α and its fragments were subcloned into pCI-NEO (Promega) expression vector. The p85α-CTwt construct encoding the C-terminal fragment of p85α was provided by Dr. T. O. Chan. Full length cDNA of mouse Fyn [START_REF] Gauen | P59Fyn tyrosine kinase associates with multiple T-cell receptor subunits through its unique amino-terminal domain[END_REF] was obtained from Dr. Pe. Dráber. Construct pFYN-EGFP was prepared by subcloning of Fyn into pEGFP-N3 vector (Clontech).

Full-length cDNA of human γ-tubulin was obtained from Dr. B. Oakley [START_REF] Wise | The γ-tubulin gene family in humans[END_REF]. Restriction sites EcoRI and XhoI were introduced by PCR, and full-length γ-tubulin was subcloned into pGEX-6P-1 vector for production of fusion protein. Constructs were verified by restriction mapping and sequencing. GST-tagged fusion proteins were expressed in Escherichia coli strain BL-21 after IPTG induction and purified on Glutathione Sepharose 4B Fast Flow beads (Amersham) according to manufacturer's directions.

Antibodies

Rabbit antibody (Ab) and mouse monoclonal Ab (mAb) to Fyn were from Santa Cruz Biotechnology and BD Transduction Laboratories, respectively. Ab to p85α and mAb 4G10 to phosphotyrosine conjugated with peroxidase (HRP) were from Upstate Laboratories. mAb to p85α was from US Biological. mAb GTU-88 and Abs (Cat. Nos. T5192 and T3195) to γtubulin, mAb TUB 2.1 to β-tubulin and TUB 2.1-FITC were from Sigma. Ab to GFP was from Clontech. mAbs TU-31 to γ-tubulin, TU-01 to α-tubulin, and NF-09 to neurofilament protein were specified previously [START_REF] Kukharskyy | Complexes of γ-tubulin with non-receptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells[END_REF]. mAb TU-30 to γ-tubulin conjugated with DY547, mAb Fyn-01 to Fyn and Ab for precipitation of GFP were from Exbio Praha. mAb NF-09 and Ab to nonmuscle myosin (Biomedical Technologies) served as negative controls in precipitation experiments. Abs to catalytic active (pY418) and inactive (pY530) forms of Src, recognizing homologous phosphorylation sites in Fyn, were from BioSource International. Ab to GST was provided by Dr. Pe. Dráber and mAb 20H5 to centrin by Dr. J. L. Salisbury [START_REF] Salisbury | The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells[END_REF]. Secondary HRP-conjugated Abs were from Promega. FITC-conjugated anti-mouse and indocarbocyanate (Cy3)-conjugated anti-rabbit Abs were from Jackson Immunoresearch Laboratories.

Rabbit Ab to GCP2 was prepared according to previously described procedure [START_REF] Dráber | Immunostaining of human spermatozoa with tubulin domain-specific monoclonal antibodies[END_REF] and immunoglobulins were affinity-purified on GST-GCP2 protein coupled to CNBr-activated Sepharose. Ab was eluted by 3.5 M MgCl 2 in 5% (v/v) dioxane and dialyzed against PBS, pH 7.5. The Ab specificity was verified by immunoblotting and immunofluorescence microscopy.

Cells

Mouse embryonal carcinoma P19 cells were cultured and subsequently differentiated by 1 μM RA for 9 days as described previously [START_REF] Kukharskyy | Complexes of γ-tubulin with non-receptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells[END_REF]. In some cases, cells before extraction were incubated for 60 min at 37°C with SU6656, PP2 and PP3 or with imatinib mesylate, an inhibitor of tyrosine kinases Bcr-Abl and c-kit, at concentrations 20 μM. Alternatively, cells were incubated for 60 min at 37°C with 200 nM wortmannin, an inhibitor of PI3K, or with 10 mM methyl-β-cyclodextrin for depletion of cholesterol. For immunofluorescence visualization on differentiated cells, a modified culturing procedure in the presence of cytosine β-D-arabinofuranoside was applied [START_REF] Kukharskyy | Complexes of γ-tubulin with non-receptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells[END_REF]. HEK293-FT cells (Promega) were transfected with 2-20 μg DNA per 6-cm tissue culture dish using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were harvested for experiment 48 h after transfection.

Preparation of cell extracts

All cell extracts were prepared at 4°C. For differential centrifugation, cells were rinsed with HEPES buffer (50 mM HEPES pH 7.6, 75 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA) and scraped into HEPES buffer supplemented with protease and phosphatase (1 mM Na 3 V0 4 , 1 mM NaF) inhibitors. Cells were disrupted by sonication (2 x 10 sec pulses, amplitude 20) using 500W Ultrasonic Homogenizer (Cole Parmer), and cell nuclei were removed by centrifugation at 1000 g for 10 min while post-nuclear supernatants were centrifuged at 20000 g for 15 min. Supernatants (S 20 ) were centrifuged at 55000 g for 30 min. Supernatants (S 55 ) were subsequently centrifuged at 100000 g for 1 h to obtain high-speed supernatants (S 100 ) and high-speed pellets (P 100 ). Alternatively, supernatants (S 20 ) were supplemented with buffer or with NP-40 to final concentration 0.5% (v/v), incubated for 30 min and centrifuged at 100000 g for 1 h. Supernatants without (S) or with NP-40 (SN) were mixed with 5x concentrated SDS-sample buffer, whereas the corresponding pellets (P, PN) were resuspended in 1x SDS-sample buffer in a volume which equalled that of the supernatants. In some experiments, pellets (P 100 ) were resuspended with HEPES buffer supplemented with one of the following detergents: Brij 98, NP-40 or Triton X-100 at concentration 1% (v/v). After 30 min of incubation the detergent-insoluble material was centrifuged at 55000 g for 30 min.

Cell extracts for GST pull-down assays [1% (v/v) NP-40 in HEPES buffer] were prepared as decribed [START_REF] Kukharskyy | Complexes of γ-tubulin with non-receptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells[END_REF].For precipitation from cytosolic fraction, supernatant (S 100 ) was adjusted with NP-40 to 1% (v/v) final concentration. For precipitation from microsomal fraction (P 100 ), hereafter referred to as membranes, the pelleted material was resuspended in HEPES buffer supplemented with 1% (v/v) NP-40. A volume equal to the corresponding supernatant was used for resuspension of the pellet. Suspension was homogenized by sonication (5 sec pulse, amplitude 10) and after 15 min incubation spun down at 20000 g for 15 min.

Density gradient centrifugation

For density gradient centrifugation, cells were extracted with ice-cold 1% (v/v) Triton X-100 in HEPES buffer supplemented with protease and phosphatase inhibitors for 10 min at 4°C and centrifuged at 20000 g for 15 min at 4°C. Where indicated, 0.2% (w/v) saponin was added to extraction buffer. 10-40% (w/v) step-density gradients in HEPES buffer were prepared in Ultra-Clear tubes (14 x 95 mm, Beckman). Extracts were adjusted to 40% (w/v) sucrose by adding equal volumes of 80% (w/v) sucrose in HEPES buffer. 3 ml were loaded into a tube, followed by 7 ml of 30% (w/v) sucrose and 2 ml of 10% (w/v) sucrose. Gradients were centrifuged at 35000 rev./min in Beckman SW40 rotor for 21 h at 4°C, and fractionated from the top into eleven fractions. The floating detergent-resistant membranes (DRM) were concentrated in fraction No. 2. To concentrate DRM, fraction No. 2 was diluted with 6 volumes of ice-cold water and centrifuged at 35000 rev./min in Beckman SW40 rotor for 1 h at 4°C. For SDS-PAGE pelleted DRM were mixed with SDS-sample buffer corresponding to 1/30 volume of the No. 2 fraction. Alternatively, pelleted DRM were resuspended in BRB80 buffer (80 mM PIPES pH 6.8, 1 mM MgCl 2 , 1 mM EGTA) and used for microtubule spindown assay.

Immunoprecipitation, kinase assay and immunoblotting

Immunoprecipitation was performed as described [START_REF] Kukharskyy | Complexes of γ-tubulin with non-receptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells[END_REF]. Beads of protein A were saturated with: (I) Ab to Fyn, (II) Ab to p85α, (III) Ab to GFP, (IV) negative control Ab to non-muscle myosin, (V) mAb TU-31 (IgG2b) against γ-tubulin, (VI) negative control mAb NF-09 (IgG2a), or (VII) immobilized protein A alone. For comparison of immunoprecipitation from unstimulated and stimulated cells, extracts were adjusted to the same protein concentration. Alternatively, beads with immunoprecipitated material were used for in vitro kinase assay as described [START_REF] Sulimenko | Regulation of microtubule formation in activated mast cells by complexes of gtubulin with Fyn and Syk kinases[END_REF]. The 32 P labelled-immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose, and the 32 P-labelled proteins were detected by bioimaging analyzer BAS-5000 (Fuji Photo Film). Details of immunoblotting are described elsewhere [START_REF] Dráberová | A microtubule-interacting protein involved in coalignment of vimentin intermediate filaments with microtubules[END_REF]. Anti-tubulin mAbs TU-01 (spent culture supernatant), TUB 2.1 and GTU-88 were diluted 1:50, 1:2000 and 1:5000, respectively. mAbs to Fyn, p85α, centrin and phosphotyrosine were diluted 1:300, 1:500, 1:1000 and 1:30000, respectively. Abs to GFP and GCP2 were diluted 1:500, while Ab to GST was diluted 1:30000. Abs to pY418 and pY530 were diluted 1:5000. Secondary HRPconjugated Abs were diluted 1:10000. Bound Abs were detected by chemiluminiscence reagents.

GST pull-down assay

GST pull-down assays with cell extracts were performed as described [START_REF] Kukharskyy | Complexes of γ-tubulin with non-receptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells[END_REF]. Alternatively, sedimented beads with immobilized GST-γ-tubulin were incubated with PI3K at concentration 0.4 μg/ml in TBS (10 mM Tris-HCl pH 7.4, 150 mM NaCl). After washing, bound proteins were eluted by boiling in SDS-sample buffer. In some experiments, 0.1 μg of active Fyn was added to beads with immobilized GST fusion proteins and subjected to kinase assay.

Immune complex PI3K assay

PI3K activity was determined essentially as described [START_REF] Dráberová | Exogenous administration of gangliosides inhibits Fcε RI-mediated mast cell degranulation by decreasing the activity of phospholipase Cγ[END_REF]. After immunoprecipitation, the beads were washed in kinase buffer and suspended in kinase buffer with or without 100 nM wortmannin. After 10 min incubation at 25°C, the assay was initiated by addition of kinase buffer containing sonicated phosphatidylinositol and [γ-32 P]ATP, and the reaction mixture was incubated for 30 min at 25°C.

Microtubule spin-down assay and immunofluorescence

Membranes were washed in BRB80 buffer and resuspended in BRB80 by sonication (5 sec pulse, amplitude 10). Spin-down assay was performed as described [START_REF] Evans | Influence of the centrosome on the structure of nucleated microtubules[END_REF]. Membranes were incubated with 10 μM tubulin in the presence of 1 mM GTP for 20-30 min at 37°C. After fixation with 1% (v/v) glutaraldehyde, membranes were centrifuged through 10% (v/v) glycerol cushion on glass coverslips, postfixed in cold methanol and used for immunostaining. Alternatively, concentrated DRM were used for the assay. In salt-washed experiments the membranes were first incubated in 2 M KCl for 1h on ice, then pelleted, extensively washed in BRB80 at 4°C and finally resuspended in BRB80 by sonication. In recovery experiments, salt-washed membranes were incubated for 1 h at room temperature with cytosol (S 100 ) preabsorbed with negative control Ab or with cytosol immunodepleted of γ-tubulin. After washing in BRB80, they were used for the assay. Immunodepletion of γtubulin was performed by incubation of 600 μl cytosol, diluted 1:5 in HEPES buffer, with protein A beads saturated with a mixture of polyclonal Abs to γ-tubulin. After 45 min incubation at room temperature, the beads were pelleted and supernatant was subjected to additional three subsequent absorbtion steps with new Ab-saturated beads. The same procedure was performed with negative control Ab.

Preparations after spin-down assay were stained for tubulin with TUB 2.1-FITC (1:50). TUB 2.1-FITC and anti-γ-tubulin mAb TU-30 conjugated with DY547 (1:500) were used for double-label staining. Alternatively, samples were stained with TUB 2.1-FITC and anti-Fyn (1:500) or anti-p85α (1:2000) Abs, followed by Cy3-conjugated anti-rabbit Ab (1:500).

Immunofluorescence microscopy on fixed cells was performed as described [START_REF] Dráberová | A microtubule-interacting protein involved in coalignment of vimentin intermediate filaments with microtubules[END_REF]. Cells were fixed in 3% (w/v) formaldehyde and extracted thereafter with 0.5% (v/v) Triton X-100. The samples were double-label stained with rabbit Ab to γ-tubulin (1:500) and mAb Fyn-01 (1:100) to Fyn, followed by Cy3-conjugated anti-rabbit Ab and FITC-conjugated anti-mouse Ab (1:200). The preparations were examined with Olympus A70 Provis microscope. Conjugates alone did not give any detectable staining.

RESULTS

Membrane-associated γ-tubulin in differentiated P19 cells

First, we determined the distribution of γ-tubulin in RA-differentiated P19 cells. Immunoblot analysis of fractions after differential centrifugation of cell homogenate revealed that γtubulin associated with both the pelleted materials (P 20 , P 55 , P 100 ) and the supernatants (S 20 , S 55 , S 100 ) On the other hand centrin, a marker of centrosomes, localized exclusively in lowspeed pellet (P 20 ) (Figure 1A).

To determine the relative distribution of tubulins and kinases, supernatants (S 20 ) were supplemented or not with 0.5% (v/v) NP-40 before centrifugation at 100000 g for 60 min. In the absence of detergent γ-tubulin was equally distributed between cytosolic (S 100 ) and membrane (P 100 ) fractions. In contrast, β-tubulin and p85α predominated in cytosolic fraction, while Fyn prevailed mainly in membrane fraction (Figure 1B). Distribution of α-tubulin resembled that of β-tubulin (not shown). γ-Tubulin associated with the detergent-resistant fraction (PN 100 ) even in the presence of 0. and PN 100 fractions resembled that of γ-tubulin (Figure 1C). When membrane-associated γtubulin (P 100 ) was extracted with lysis buffer containing 1% (v/v) Brij 98, 1% (v/v) NP-40 or 1% (v/v) Triton X-100, approximately 50%, 45% and 40%, respectively, of γ-tubulin was still associated with detergent-resistant material. These data indicate that the distribution patterns of γ-tubulin and αβ-tubulin dimers differ, and that a substantial part of γ-tubulin is present in cellular fraction containing Fyn. A part of γ-tubulin associated with microsomal fraction is resistant to treatment with non-ionic detergents.

Double-label immunostaining of differentiated cells with anti-γ-tubulin and anti-Fyn Abs revealed that both proteins were located in rounded cell bodies and in neurite projections. Colocalization of Fyn and γ-tubulin was observed in some regions of thicker processes (see Supplementary Figure 1S).

Nucleation of microtubules from membranes is dependent on γ-tubulin and kinases

In vitro microtubule assembly assay was performed to test whether or not the membraneassociated γ-tubulin is capable of nucleating microtubules. Membranes were incubated for 20 or 30 min in a buffer supplemented with 10 μM αβ-tubulin dimer and 1 mM GTP. The spindown assay revealed that microtubules grew from membranes (Figure 2A,a,c,e) that contained γ-tubulin (Figure 2A,b), Fyn (Figure 2A,d) as well as p85α (Figure 2A,f). On the other hand, centrin was not detected on membranes, suggesting that nucleation is not attributable to remnants of centrosomes. The growth of microtubules was time-dependent. We have noted that all membrane fragments stained for β-tubulin after incubation with tubulin dimers, similarly as it was reported for isolated Golgi membranes [START_REF] Chabin-Brion | The Golgi complex is a microtubule-organizing organelle[END_REF], but only some nucleated microtubules. To determine whether γ-tubulin is responsible for microtubule nucleation from membranes, isolated membranes were treated with 2M KCl. Under these conditions nucleation was not detected (Figure 2B,a). In recovery experiment, salt-treated membranes were incubated with cytosol preabsorbed with negative control Ab prior to in vitro microtubule assembly assay. In this case, nucleation of microtubules was restored (Figure 2B,b). When salt-treated membranes were incubated with cytosol immunodepleted of γ-tubulin, nucleation of microtubules was absent (Figure 2B,c). No spontaneous microtubule assembling took place when αβ-tubulin dimer was incubated in the absence of membranes (Figure 2B,d). Immunoblotting experiments confirmed a substantialy higher amount of γtubulin in untreated membranes when compared to salt-washed membranes. γ-Tubulin reoccurred after incubation of salt-washed membranes with cytosol preabsorbed with negative control Ab. However, when salt-treated membranes were incubated with cytosol immunodepleted of γ-tubulin, no increase in γ-tubulin was observed. The amount of Fyn on membranes did not substantially change after salt-treatment (see Supplementary Figure 2S).

When cells were pretreated with the Src family kinases inhibitors SU6556 or PP2 at 20 μM, microtubule nucleation was inhibited (Figure 2C,b). Nucleation was not affected when DMSO alone (Figure 2C, a) was used. It is known that PP2 also inhibits tyrosine kinases Kit and Bcr-Abl, in addition to Src family kinases [START_REF] Tatton | The Src-selective kinase inhibitor PP1 also inhibits Kit and Bcr-Abl tyrosine kinases[END_REF]. Nucleation experiments were therefore performed with membranes isolated from cells pretreated with 20 μM imatinib mesylate inhibiting kinases Kit and Bcr-Abl. In this case no inhibition was observed (not shown). Pretreatment of cells with 200 nM wortmannin also inhibited microtubule nucleation on isolated membranes (see Supplementary Figure 3S,A). Pretreatment with wortmannin or PP2 did not affect association of γ-tubulin with membranes (see Supplementary Figure 3S,B). These in vitro experiments suggest that γ-tubulin associated with microsomal fraction has the capability to nucleate microtubules, and that the process depends on activity of Src family kinases and PI3K.

Membrane-associated γ-tubulin interacts with Fyn and p85α

A series of precipitation experiments was performed to find out whether γ-tubulin and Fyn form complexes in membrane fractions of differentiated P19 cells. Anti-Fyn Ab coprecipitated γ-tubulin as well as p85α (Figure 3A). After precipitation with a negative control Ab, no specific staining was detected with Abs to Fyn, γ-tubulin and p85α. As Fyn formed complexes with p85α, we intended to find out whether p85α could serve in P19 cells as a substrate for Fyn. In vitro kinase assay revealed protein phosphorylation in Fyn membrane immunocomplexes isolated from both resting and differentiated cells. A protein corresponding to the p85α, as confirmed by immunoblotting with specific Ab, showed enhanced phosphorylation in differentiated cells (Figure 3B, 0 and 9). This phosphorylation was inhibited by pretreatment of the cells with PP2 (Figure 3B, + PP2). On the other hand treatment with PP3 was without effect (not shown). No kinase activity was detected after precipitation with the negative control Ab (Figure 3B,c).

Complexes of γ-tubulin with p85α have been previously described in cells overexpressing insulin receptor [START_REF] Kapeller | Phosphoinositide 3-kinase binds constitutively to a/b-tubulin and binds to g-tubulin in response to insulin[END_REF][START_REF] Inukai | The N-terminal 34 residues of the 55 kDa regulatory subunits of phosphoinositide 3-kinase interact with tubulin[END_REF]. Next we therefore examined the possibility that membrane-bound γtubulin associates with p85α in differentiated P19 cells. Ab to p85α coprecipitated γ-tubulin and its amount was higher in immunocomplexes from membrane fraction (Figure 3C). To corroborate the results of immunoblotting experiments, PI3K activity was also evaluated by immunocomplex kinase assay. PI3K activity associated with membrane-bound γ-tubulin was found in both resting and differentiated cells. However, a 1.82±0.24 (mean ± S.D., n=3) fold increase in PI3K activity was detected in differentiated cells. Pretreatment of immunocomplexes with 100 nM wortmannin completely inhibited the formation of 32 Plabelled PIP (Figure 3D). Summarily these data demostrate that membrane bound γ-tubulin in differentiated P19 cells forms complexes with both Fyn and active PI3K.

γ-Tubulin associates with detergent-resistant membranes

To determine whether γ-tubulin is also associated with DRM, cells were solubilized in 1% (v/v) Triton X-100 and lysates were fractionated by equilibrium density gradient centrifugation. To make the results comparable, the same protein amounts were loaded for centrifugation and the corresponding blots were exposed on the same autoradiography film. Fyn served as marker of DRM. Immunoblotting experiments revealed substantial differences in the distribution of γ-tubulin in the course of RA-induced differentitation. In undifferentiated cells (day 0) both Fyn and γ-tubulin were only found in high-density (HD) fractions, while in differentiated cells (day 9) they were detected in both HD and low-density (LD) fractions (Figure 4A). LD fractions correspond to floating DRM while HD fractions correspond to fractions not associated with DRM. Since DRM are rich in cholesterol, differentiated cells were extracted with buffer containing 1% (v/v) Triton X-100 and 0.2% (v/v) saponin, which is known to sequester cholesterol. Both Fyn and γ-tubulin were absent in LD fractions in saponin-treated samples (Figure 4A, Sap). Comparable effect was observed when cholesterol was depleted by preincubation of cells with methyl-β-cyclodextrin.

Complementary immunoblotting experiments were performed to compare the relative distribution of γ-tubulin and Fyn in total cell extracts and DRM. The amounts of proteins in whole-cell extract (lane 1 in Figure 4B) corresponded to those in concentrated DRM (lane 2 in Figure 4B). γ-Tubulin was present in DRM in a relatively high content in comparison with β-tubulin. Importantly, the GCP2 protein, a component of γ-tubulin complexes, was detected in DRM. As expected, Fyn was highly enriched with DRM. The microtubule nucleation capacity of concentrated DRM was demonstrated by spin-down assay. Microtubules grew from DRM (Figure 4C, a) that contained γ-tubulin (Figure 4C,b). Immunoprecipitation experiments with anti-Fyn Ab followed by immunobloting with Ab to γ-tubulin confirmed an association of γ-tubulin with Fyn in DRM (not shown). Collectively, these data indicate that a subpopulation of γ-tubulin is associated with DRM and forms there complexes with Fyn.

Association of Fyn with membrane is important for its interaction with γ-tubulin

To answer the question whether association of Fyn with membranes is important for its interaction with γ-tubulin, EGFP-tagged Fyn construct was prepared. The EGFP tag was added to the C-terminus of Fyn, and the distribution of Fyn-EGFP was determined in soluble and membrane fractions of transfected undifferentiated P19 cells. Overexpressed Fyn-EGFP was present in both soluble and membrane fractions, though a higher amount was associated with the latter. On the other hand EGFP, that served as control, was found exclusively in soluble fraction (Figure 5A, Load). Anti-γ-tubulin Ab precipitated γ-tubulin from both fractions, yet coprecipitated only membrane targeted Fyn-EGFP, but not cytosolic Fyn-EGFP or EGFP (Figure 5A, IP). Anti-phosphotyrosine Ab stained neither soluble nor membranebound γ-tubulin in immunoprecipitates (not shown). This suggests that γ-tubulin is not phosphorylated by Fyn. To rule out the possibility that cytosolic Fyn-EGFP is not folded properly and therefore not accessible to interaction with γ-tubulin, we have used in vitro kinase assay as a measure of proper folding. Membrane and cytosolic Fyn-EGFP were precipitated by Ab to GFP, and the immunoprecipitated material was used for in vitro kinase assay. Both membrane and cytosolic Fyn-EGFP precipitates were phosphorylated and contained an active form of Fyn as documented by autoradiography and by immunoblotting with Ab to the autophosphorylation site pY418 (Figure 5B). While Ab to the negative regulatory site pY530 also reacted with membrane and cytosolic form of Fyn-EGFP, nonactive Fyn-EGFP was not accumulated in cytosol (not shown). Combined data suggest that both membrane and cytosolic Fyn-EGFP contains a mixture of active and non-active forms of kinase. As γ-tubulin did not interact with Fyn-EGFP in cytosol, these two proteins apparently associate indirectly via adaptor protein(s) located in membranes.

γ-Tubulin binds directly to the C-terminal SH2 domain of p85α, outside the tyrosinephosphorylated protein-recognition site

Because γ-tubulin associated with p85α in differentiated P19 cells, we attempted to identify the region in p85α molecule responsible for interaction with γ-tubulin. The pull-down assays with GST-tagged p85α domains (Figure 6A) and extracts from differentiated cells revealed that γ-tubulin associated with all the fusion proteins containing the C-terminal SH2 (cSH2) domain. No interaction was observed with fusion proteins containing SH3-RhoGAP fragment, the N-terminal SH2 (nSH2) domain or GST alone (Figure 6B, γ-Tb). α-Tubulin interacted with the same fragments, except for the cSH2 domain (Figure 6B, α-Tb). The amounts of immobilized GST fusion proteins were similar, as evidenced by staining with anti-GST Ab (not shown). These results indicate that association of γ-tubulin with the cSH2 domain is independent of the presence of αβ-tubulin dimer. All SH2 domains contain a conserved F-L-V-R motif, which is essential for the phosphotyrosine binding. Mutation of the critical arginine residue in the F-L-V-R motif abolishes the capability of SH2 domain to bind phosphotyrosine proteins [START_REF] Kuriyan | Modular peptide recognition domains in eukaryotic signaling[END_REF]. When the pull-down assay was performed with fusion protein containing mutated arginine in cSH2 domain (GST-cSH2-R649L), the binding of γ-tubulin to this fusion protein was preserved (Figure 6B,γ with p85α directly, the GST pull-down assay was performed with GST-γ-tubulin and recombinant PI3K (p110δ complexed to p85α). Though PI3K did bind to GST-γ-tubulin, it failed to bind to GST fusion protein containing fragment of GCP2 (negative control) or GST alone (Figure 6C,p85α). The amounts of immobilized GST fusion proteins were similar (Figure 6C, GST).

To prove that the cSH2 domain is responsible for γ-tubulin binding in vivo, p85α and its truncated forms (Figure 7A) were transfected into HEK cells. All constructs were expressed in the cells (Figure 7B, Load, p85α) and did not alter the expression of γ-tubulin (Figure 7B, Load, γ-Tb). Cell extracts were precipitated with Ab to γ-tubulin and blots were probed with Ab to p85α. As expected, the amount of p85α in complex with γ-tubulin increased after overexpression of p85α. However, the truncated forms p85-ΔCT and p85-interΔCT without cSH2 domain were absent in γ-tubulin immunocomplexes. In contrast, p85α with deleted RhoGAP domain, but with intact cSH2 domain, was present there (Figure 7B, IP, p85α). When the C-terminal fragment of p85α containing cSH2 domain (CT) was expressed in cells, a dose-dependent decrease of p85α in γ-tubulin immunocomplexes was evident (Figure 7C, IP, p85α), while the expression level of p85α and γ-tubulin was unchanged (Figure 7C, Load). Collectively these data suggest that γ-tubulin directly interacts with the cSH2 domain and that this interaction is outside the tyrosine-phosphorylated protein-recognition site on the cSH2 domain of p85α.

DISCUSSION

It is known that the bulk of cytoplasmic γ-tubulin is not associated with MTOCs [START_REF] Moudjou | γ-Tubulin in mammalian cells: the centrosomal and the cytosolic forms[END_REF]. Here we show that γ-tubulin in RA-differentiated P19 cells is equally distributed to both soluble and membrane fractions. A fraction of membrane-associated γ-tubulin was resistant to nonionic detergents. On the other hand, αβ-tubulin dimers prevailed in soluble pool. Interestingly, γ-tubulin in differentiated cells was found not only in rough membrane fractions but also in DRM. Since γ-tubulin itself does not have a consensus membrane-binding motif, it is likely that other membrane-anchored proteins interacting with γ-tubulin mediate this association. In differentiated cells only a small fraction of αβ-tubulin dimers is present in DRM, contrary to γ-tubulin. Therefore it seems unlikely that tubulin dimers dock γ-tubulin to membranes. It is more probable that Src family kinases and/or adapter proteins accumulating in DRM [START_REF] Palestini | Developmental changes in the protein composition of sphingolipid-and cholesterol-enriched membrane domains of rat cerebellar granule cells[END_REF] take part in this docking.

Microtubules are essential for neurite formation, and Fyn kinase plays a key role in this process. We have previously demonstrated an enhanced interaction of γ-tubulin with Fyn in RA-differentiated cells [START_REF] Kukharskyy | Complexes of γ-tubulin with non-receptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells[END_REF]. Several lines of new evidence indicate that this interaction occurs predominantly in membrane fraction. First, Fyn was associated with γ-tubulin in immunoprecipitates from membranes. Second, complexes of both proteins were present in DRM that are known to accumulate Fyn. Third, γ-tubulin and Fyn were located on membranes from which microtubules were nucleated. Fourth, the nucleation activity of membrane-associated γ-tubulin was inhibited by pretreatment of cells with Src-family specific inihibitors; this implies that Src family kinases might play a role in modulation of microtubule nucleation from membranes. Finally, tests with EGFP-tagged Fyn showed that association of Fyn with membranes was required for interaction with γ-tubulin. Although this study was focused on Fyn, one cannot rule out that other members of the Src family could also modulate microtubule nucleation.

In differentiating cells, the enhanced interaction of γ-tubulin with Fyn was accompanied by increased activity of PI3K associated with γ-tubulin. Immunoprecipitation experiments confirmed the association of γ-tubulin with p85α. Although in vitro interactions between the γ-tubulin and p85α have been reported [START_REF] Kapeller | Phosphoinositide 3-kinase binds constitutively to a/b-tubulin and binds to g-tubulin in response to insulin[END_REF][START_REF] Inukai | The N-terminal 34 residues of the 55 kDa regulatory subunits of phosphoinositide 3-kinase interact with tubulin[END_REF], no conclusions were drawn on the exact location of γ-tubulin binding site on p85α molecule and the type of interaction between these two proteins. Our data from GST pull-down assays with p85α domains demonstrate that γtubulin binds to cSH2 domain and that phosphotyrosine-mediated binding is not required for this interaction. Moreover, GST pull-down assay with γ-tubulin fusion protein and recombinant PI3K confirms a direct interaction. Immunoprecipitation experiments from transfected cells, expressing truncated forms of p85α  or overexpressing cSH2 domain, demonstrate that the cSH2 domain is essential for interaction of p85α with γ-tubulin in vivo. The capability of the cSH2 domain of p85α to mediate phosphorylation-independent interaction has been reported for serine/threonine kinase A-Raf [START_REF] Fang | Two phosphorylationindependent sites on the p85 SH2 domains bind A-Raf kinase[END_REF], and the same may hold true for γ-tubulin.

It has been shown that the cSH2 domain of p85α regulates the activity of PI3K. This domain carries the Y688 that can be phosphorylated by cytoplasmic kinases [START_REF] Chan | Small GTPases and tyrosine kinases coregulate a molecular switch in the phosphoinositide 3-kinase regulatory subunit[END_REF]. Phosphorylation of this site then functionally blocks the inhibitory effect of p85α on the catalytic subunit. Neutralization of the inhibitory domain renders p85α responsive to PI3K activation signals that are transduced by small GTPases [START_REF] Chan | Small GTPases and tyrosine kinases coregulate a molecular switch in the phosphoinositide 3-kinase regulatory subunit[END_REF]. Interestingly, reduced PI3K activity was reported in activated mast cells isolated from Fyn knock-out mice [START_REF] Parravicini | Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation[END_REF]. Here we show that Fyn in P19 cells can phosphorylate p85α in vivo and that phosphorylation is enhanced in differentiated cells. The efficacy of Fyn in phosphorylating p85α was further confirmed by in vitro kinase assay with GST-cSH2 and purified active Fyn (see Supplementary Figure 4S). This indicates that Fyn in P19 cells phosphorylates the cSH2 of p85α and thus regulate the activity of catalytic subunit.

The results obtained by microtubule spin-down assay show that membrane-associated γtubulin complexes can nucleate microtubules from both rough membrane fractions and DRM. In this connection it should be noted that γ-tubulin associates with vesicular structures and Golgi-derived vesicles [START_REF] Chabin-Brion | The Golgi complex is a microtubule-organizing organelle[END_REF][START_REF] Dryková | Plant γtubulin interacts with αβ-tubulin dimers and forms membrane-associated complexes[END_REF] and it is essential for microtubule nucleation from Golgi membranes [START_REF] Chabin-Brion | The Golgi complex is a microtubule-organizing organelle[END_REF][START_REF] Rios | GMAP-210 recruits γ-tubulin complexes to cis-Golgi membranes and is required for Golgi ribbon formation[END_REF]. Although DRM are most abundant in plasma membrane, they first assemble in the Golgi apparatus [START_REF] Brown | Structure and origin of ordered lipid domains in biological membranes[END_REF]. It is therefore uncertain whether microtubule formation from DRM reflects nucleation from the plasma membrane or other membranous components.

Our data idicate that other microtubule nucleating sites associated with membraneassociated γ-tubulin may exist in addition to centrosomes in differentiating P19 cells. The process of microtubule nucleation on membranes could be regulated by Src family kinases and PI3K. This notion is supported by our data indicating that upregulation of Fyn leads to Fyn accumulation in the membranes and phosphorylation of p85α. It could lead to activation of p110 catalytic subunit and initiation of PI3K signaling pathway. Recruitment of p85α with bound γ-tubulin into membranes, probably through an adaptor protein, might contribute to membrane-associated microtubule nucleation. Since pretreatment of cells with wortmannin also inhibited microtubule nucleation on isolated membranes, PI3K itself, or some other proteins in the PI3K signaling pathway, could participate in the modulation of microtubule nucleation.

In conclusion, the presented data show that γ-tubulin in differentiating P19 cells is associated with membranes including DRM. Membrane-associated γ-tubulin is involved in nucleation of microtubules, and Src family kinases and PI3K play an important role in this process. Membrane-associated γ-tubulin forms complexes with Fyn and directly interacts with cSH2 domain of p85α. The findings suggest that proteins of the signaling cascades could modulate non-centrosomal microtubule nucleation by membrane-associated γ-tubulin. (A) Soluble (S) and membrane (P) fractions from differentiated cells were precipitated with Ab to Fyn and blots were probed with Abs to Fyn, γ-tubulin (γ-Tb) and p85α. Immobilized Ab not incubated with cell extract (C, control). White arrowheads point to heavy chain of IgG. (B) Kinase activity in Fyn membrane immunocomplexes isolated from resting (0) and differentiated (9) cells pretreated with (+) or without (-) PP2 inhibitor. Kinase activity after precipitation with negative control Ab (C). Precipitated Fyn was detected by immunoblotting. Positions of p85α and molecular mass marker (in kDa) are indicated on the left. (C) Soluble (S) and membrane (P) fractions from differentiated cells were precipitated with Ab to p85α and blots were probed with Abs against p85α and γ-tubulin (γ-Tb). Immobilized Ab not incubated with cell extract (C, control). White arrowhead points to heavy chain of IgG. (D) Activity of PI3K in γ-tubulin membrane immunocomplexes isolated from resting (0) and differentiated (9) cells. In labelled samples, the immunocomplexes were exposed to wortmannin (w). Precipitated γ-tubulin was detected by immunoblotting (γ-Tb). Migration of phosphatidylinositol phosphate (PIP) and starting point of thin layer chromatography (S) are denoted. proteins were precipitated with Ab to γ-tubulin and blots from extracts (Load) and precipitates (IP) were probed with Abs to GFP, Fyn and γ-tubulin (γ-Tb). (B) Soluble (S) and membrane (P) fractions from cell expressing EGFP and Fyn-EGFP proteins were precipitated with Ab to GFP. Kinase activity in GFP immunocomplexes ( 32 P), immunoblots with Ab to catalytic active form of Fyn (pY418) and with Ab to GFP. To compare the relative distribution of proteins, pelleted material was resuspended in a volume equal to that of the corresponding supernatant.

Figure 6 Interaction of γ-tubulin with p85α domains in GST pull-down assay

(A) Schematic diagram of GST-p85α (a) and its truncated forms (b-e) used in pull-down assays. (B) Lysates from differentiated cells (Load) were incubated with GST-fusion proteins or GST alone immobilized on Glutathion Sepharose beads. Blots of bound proteins were probed with Abs to γ-tubulin (γ-Tb) and α-tubulin (α-Tb). (C) Binding of PI3K to GST-γtubulin. PI3K (p110δ complexed to p85α) was incubated with immobilized GST-γ-tubulin, GST alone and GST-GCP2 fragment. Alternatively, PI3K was incubated with beads without GST-fusion proteins (carrier alone). Blots of bound proteins were probed with Abs to p85α (p85α) or GST. Molecular-mass markers (in kDa) are indicated on the left.

  5 % (v/v) NP-40. Under such conditions β-tubulin prevailed mainly in detergent-soluble fraction (SN 100 ). Distributions of Fyn and p85α in SN 100 7 Biochemical Journal Immediate Publication. Published on 17 Jul 2008 as manuscript BJ20080909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080909 S t a g e 2 ( a ) P O S T -P R I N T

  -Tb). To decide whether γ-tubulin interacts 10 Biochemical Journal Immediate Publication. Published on 17 Jul 2008 as manuscript BJ20080909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080909 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1

 1 Figure 1 Distribution of γ-tubulin and kinases in RA-differentiated P19 cells

Figure 2

 2 Figure 2 Assembly of microtubules from membranes

Figure 3

 3 Figure 3 Coimmunoprecipitation of membrane-bound γ-tubulin with Fyn and p85α

Figure 4

 4 Figure 4 Distribution of Fyn and γ-tubulin during density gradient centrifugation of lysates from resting and RA-differentiated P19 cells

Figure 5

 5 Figure 5 Association of γ-tubulin with Fyn in cells expressing EGFP-tagged Fyn

Figure 7

 7 Figure 7 Interaction of γ-tubulin with p85α truncated forms expressed in cells

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080909 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 17 Jul 2008 as manuscript BJ20080909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080909 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

We thank Dr. L. Rameh (Boston Biomedical Research Institute, Boston, USA) for providing cDNA of p85α, Dr. T.O. Chan (Thomas Jefferson University, Philadelphia, USA) for providing p85-CTwt and p85-CT R649L constructs, and Dr. B. Oakley (Ohio State University, Columbus, USA) for cDNA of γ-tubulin. We thank Dr. Petr Dráber (Institute of Molecular Genetics, Prague, Czech Republic) for cDNA of Fyn, Ab against GST and for critical reading of the manuscript. We thank Dr. J. L. Salisbury (Mayo Clinic, Rochester, USA) for Ab against centrin. This work was suported in part by Grants 304/04/1273 and 204/05/2375 from the Grant Agency of Czech Republic, Grants LC545 and IM6837805001 from Ministry of Education, Youth and Sports of the Czech Republic, and by the Institutional Research Support (AVOZ 50520514).