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Keywords:

  • Csk-binding protein;
  • ganglioside;
  • Lyn;
  • rafts

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C-terminal src kinase)-binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent-resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti-ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over-expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314-phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314-phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP-43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.

Abbreviations used
CHO

Chinese hamster ovary

DRM

etergent-resistant membrane

GSL

glycosphingolipid

OG

octylglucoside

TX

Triton X-100

WT

wild-type

Gangliosides are glycosphingolipids (GSLs) containing sialic acid that are found in the outer leaflet of the plasma membrane of all vertebrate cells, and are thought to play functional roles in cellular interactions and the control of cell proliferation (Yamakawa and Nagai 1978; Hakomori 1981). GSLs are known to exist in clusters and form microdomains containing cholesterol at the plasma membrane called rafts (Simons and Gerl 2010). Rafts are insoluble in Triton X-100 (TX) and can be isolated from non-raft domains of the cell membrane (Brown and Rose 1992), and these are referred to as detergent-resistant membranes (DRMs). Rafts have been implicated in signal transduction because various signaling molecules, such as Src family kinases, are associated with them (Masserini et al. 1999). However, the precise functions of GSLs in rafts remain to be explored (Kasahara and Sanai 2000).

In the nervous system, where gangliosides are particularly abundant, the species and amounts of gangliosides undergo profound changes during development, suggesting that they play fundamental roles in this process (Tettamanti and Riboni 1993). Exogenously administered ganglioside stimulates the regeneration of dopaminergic neurons in vivo (Toffano et al. 1983). The addition of exogenous gangliosides to neuroblastoma cells in vitro stimulates cellular differentiation with neurite extension (Ledeen 1984). Biosynthesis of GSLs is required for embryonic development (Yamashita et al. 1999). The expression of the ganglioside GD3 synthase in neuroblastoma cells induces differentiation with neurite sprouting (Kojima et al. 1994). GD3 synthase-deficient mice exhibit thermal hyperalgesia and impairment in the regeneration of the lesioned hypoglossal nerves (Okada et al. 2002; Handa et al. 2005). Finally, ganglioside-deficient mice exhibit axonal degeneration (Yamashita et al. 2005). These data show that gangliosides are involved in neural differentiation and brain development. However, the molecular mechanisms underlying the ganglioside-dependent neural functions remain obscure.

We have been investigating the association of gangliosides with specific proteins in the central nervous system. We previously demonstrated that anti-ganglioside GD3 antibody (R24) coimmunoprecipitates phosphorylated proteins of 40, 53, 56, and 80 kDa from rat cerebellar neurons. Of these proteins, the 40-kDa protein was identified as the α–subunit of a heterotrimeric G protein Go (Yuyama et al. 2007). The 53- and 56-kDa proteins were identified as the Src family kinase Lyn (Kasahara et al. 1997). We have demonstrated that R24-mediated cross-linking of GD3 induces Lyn activation in the membrane rafts of primary cerebellar granule neurons.

In this study, we identified the 80-kDa phosphoprotein as the Csk (C-terminal src kinase)-binding protein (Cbp) or PAG (hereafter referred to as Cbp) (Brdicka et al. 2000; Kawabuchi et al. 2000) and demonstrated that the R24-mediated cross-linking of GD3 induces tyrosine phosphorylation of Cbp in cerebellar granule neurons.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Materials

The anti-Cbp rabbit polyclonal antibody and anti-pY314-Cbp polyclonal antibody, which recognizes phosphorylated Cbp on Tyr314, were prepared as described previously (Kawabuchi et al. 2000). The anti-ganglioside GD3 monoclonal antibody (R24), anti-ganglioside GD1b monoclonal antibody (GGR12), anti-Lyn monoclonal antibody (Lyn8), anti-Src family negative regulatory pY site phospho-specific rabbit polyclonal antibody, anti-Csk antibody (C-20), anti-growth-associated protein (GAP)-43 monoclonal antibody, and horseradish peroxidase-conjugated anti-phosphotyrosine antibody (PY20) were purchased from Signet Laboratories (Dedham, MA, USA), Seikagaku (Tokyo, Japan), Wako Chemicals (Osaka, Japan), BioSource International (Camarillo, CA, USA), Santa Cruz (CA, USA), Chemicon International (Billerica, MA, USA), and Transduction Laboratories (Lexington, KY, USA), respectively. The anti-Myc monoclonal antibody, anti-phospho-Src family (Tyr416) polyclonal antibody, and anti-p44/42 mitogen-activated protein kinase (MAPK) polyclonal IgG were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). An ECL detection kit (Amersham Pharmacia, Piscataway, NJ, USA) was used for protein visualization after immunoblotting. Complete Mini, a protease inhibitor cocktail, was purchased from Roche Diagnostics (Mannheim, Germany).

Immunoprecipitation and in vitro kinase assay

Membrane preparation from adult Wistar rat cerebella, immunoprecipitation with anti-ganglioside GD3 antibody (R24), and an in vitro kinase assay were performed as described previously (Kasahara et al. 1997). In brief, the membrane fractions were solubilized in TX lysis buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na3VO4, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 5 μg/mL leupeptin, and 5 μg/mL pepstatin A) at 4°C for 20 min. The supernatants were incubated with R24 and precipitated with protein G-Sepharose. Following immunoprecipitation, the in vitro kinase reaction was started by the addition of 5 μCi of [γ-32P] ATP (3000 Ci/mmol; NEN Life Science Products, Boston, MA, USA). Phosphorylation was stopped by the addition of Laemmli sample buffer, and the samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by autoradiography. In a reimmunoprecipitation experiment, following the kinase reaction, the samples were boiled for 5 min in lysis buffer with 1% SDS, diluted 10-fold with the lysis buffer, and then reimmunoprecipitated with anti-Cbp antibody.

Phosphoamino acid analysis

Phosphoamino acid analysis was performed as described (Kasahara et al. 1997). Phosphoprotein radiolabeled with 32Pi (p80) was eluted from a polyacrylamide gel and hydrolyzed in 6 M hydrochloric acid at 105°C for 2 h. The hydrolysate was evaporated and resuspended in 50 μL of carrier phosphoamino acid solution containing 1 mM phosphotyrosine, phosphothreonine, and phosphoserine. The solution was then subjected to cellulose thin layer chromatography with a developing solution consisting of 1-butanol, isopropyl alcohol, formic acid, and water (3 : 1 : 1 : 1). The plate was dried, sprayed with ninhydrin to determine the positions of phosphoamino acids, and then subjected to autoradiography.

Over-expression of Cbp in Chinese hamster ovary (CHO) cells

CST, a previously established CHO cell line expressing GD3 synthase as a GD3-positive line (Ogura, et al. 1996), or CHO cells were transfected with 4 μg of plasmid pCMV-Cbp for the expression of Myc-tagged Cbp using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Thirty hours after transfection, the cells were homogenized in TX lysis buffer or β-octylglucoside (OG) lysis buffer (2% OG, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na3VO4, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 5 μg/mL leupeptin, and 5 μg/mL pepstatin A) at 4°C. Transfected cell lysates were incubated with R24 or control mouse IgG. Immunoprecipitation was performed as described previously (Kasahara et al. 1997). The Myc-tagged Cbp protein in the immunoprecipitates was detected by immunoblotting with anti-Myc antibody.

Detection of phosphorylated Cbp in CHO cells

CHO cells were transfected with or without 2 μg of plasmid expressing wild-type (WT) or mutant Lyn, with or without 2 μg of plasmid expressing pCMV-Cbp as described above. The pME18S expression plasmids containing wild-type human Lyn, their active YF mutant, and negative KL mutant were previously described (Kasahara et al. 1997). Transfected cell lysates prepared from OG lysis buffer were subjected to immunoprecipitation by anti-Myc antibody followed by immunoblotting analysis using anti-phosphotyrosine antibody and/or anti-pY314-Cbp antibody.

Sucrose density gradient analysis

Sucrose gradient analysis with TX was performed according to a described method (Kasahara et al. 1997). Rat cerebella were homogenized using a Teflon glass homogenizer in 2 mL of TNE/TX buffer (1% Tx, 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 mM EGTA). The sucrose content in the homogenate was then adjusted to 40% by adding 80% sucrose. A linear sucrose gradient (5–30%) in 6 mL of TNE without TX was layered over the lysate. The gradients were centrifuged for 17 h at 200 000 g at 4°C in a Hitachi RPS40T rotor. Ten fractions were collected from the top of the gradient, followed by immunoblotting analysis using specific antibodies. If circumstances required, immunoprecipitation with a suitable antibody was carried out before immunoblotting analysis. The distribution of GD3 in the gradient fractions was observed by dot blotting with R24.

Primary culture

Rat cerebellar granule neurons were cultured according to the method of Gallo et al. 1986 with some modifications. Cerebella were dissected from killed 7-day-old rats and cerebellar neurons were prepared using a dissociation solution (Sumitomo Bakelite Co., Ltd., Tokyo, Japan). Then, the dissociated cells were plated onto 35-mm poly-l-lysine-coated plastic dishes (Becton Dickinson Labware, Franklin Lakes, NJ, USA) at a density of 2.5 × 106 cells in 2 mL of Neurobasal Medium (Gibco BRL, Rockville, MD, USA) containing 25 mM KCl, 2 mM glutamine, and B27 supplement (Gibco BRL). To prevent the proliferation of glial cells, 100 μM cytosine arabinoside was added 18 h after the seeding.

Antibody-mediated cross-linking of gangliosides in primary cerebellar cultures

Cerebellar granule cells (2.5 × 106) of 3 days in vitro were incubated with 20 μg/mL anti-GD3 antibody (R24), anti-GD1b antibody (GGR12), or mouse IgG at 37°C for 2 min on a dish. After washing with ice-cold phosphate-buffered saline (PBS) , the lysates were prepared in TX lysis buffer (100 μL) at 4°C. After centrifugation at 18 000 g for 3 min at 4°C, equal volumes of the supernatants were subjected to immunoblotting with anti-phosphotyrosine antibody PY20, or immunoprecipitation with anti-Cbp antibody, and immunoblotting with anti-pY314-Cbp antibody.

Growth cone preparation

The growth cone fraction of the rat cerebellum (post-natal day 7) was prepared by the method of Pfenninger et al. 1983 with minor modifications. Rat cerebella were homogenized at 4°C with six passes in a glass-teflon homogenizer in five volumes of 0.32 M sucrose, 1 mM Tris-HCl, pH 7.6, 1 mM MgCl2, and Complete Mini, a protease inhibitor mixture (Roche Applied Science). The crude cerebellar homogenate was centrifuged at 900 g for 10 min. The supernatant as a whole cerebellum fraction was layered over a step gradient of sucrose at 0.75 M and 1.0 M. The gradient was centrifuged at 250 000 g for 1 h, and the 0.32/0.75 M interface was collected. After centrifugation at 10 000 g for 30 min, the pellets were used as the growth cone fraction samples.

Electron microscopy

Ultrathin frozen sections were obtained by the procedures described (Suzuki et al. 1996). The fixed growth cones were sequentially immersed in 1 M sucrose in PBS for 60 min, 1.84 M sucrose in PBS for 2 h, and then 1.84 M sucrose and 20% polyvinylpyrrolidone (MW 10000; Sigma, St. Louis, MO, USA) in PBS overnight at 4°C. After freezing the growth cones in liquid nitrogen, ultrathin frozen sections were cut using an ultramicrotome (Ultracut, Reichert, Vienna, Austria) with a cryo-attachment (FC-4E; Reichert) at −90°C, and then the sections were mounted on nickel grids. The specimens were incubated with anti-Cbp rabbit polyclonal antibody and anti-GAP-43 mouse monoclonal antibody as the primary antibody for 60 min at 25°C. After rinsing with PBS five times, the specimens were incubated with goat anti-rabbit IgG coupled to 15-nm diameter colloidal gold and goat anti-mouse IgG coupled to 5-nm colloidal gold (BioCell Research Laboratories, Cardiff, UK) for 60 min at 25°C. The specimens were washed with DW five times, and analyzed using a JEM 1200EX transmission electron microscope (JEOL Ltd., Tokyo, Japan).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Association of ganglioside GD3 with the Cbp/PAG

The immunoprecipitates obtained with R24 from the TX extract of rat cerebellar membrane were analyzed for the presence of protein kinase activity. An in vitro kinase reaction resulted in the phosphorylation of several proteins of 40, 53, 56, and 80 kDa, as judged by SDS–PAGE (Fig. 1a, lane 1). We previously identified p40 and p53/56 as the heterotrimeric G protein Goα and src family kinase Lyn by sequential immunoprecipitation with anti-Goα antibody and anti-Lyn antibody, respectively (Kasahara et al. 1997; Yuyama et al. 2007). In this study, the same method was used for the identification of p80. Briefly, the in vitro kinase assay was conducted, after which the immune complexes were disrupted by boiling in SDS-containing buffer and subjected to a second immunoprecipitation with anti-Cbp antibody (Fig. 1a, lane 2) or anti-Lyn antibody (Fig. 1a, lane 3). As a result, the anti-Cbp antibody specifically precipitated p80 in reimmunoprecipitation experiments. The 32P-labeled p80 was eluted from SDS–PAGE and hydrolyzed with 6 M hydrochloric acid. The hydrolysate was separated by thin layer chromatography. Radioactivity was detected only in the position of phosphotyrosine (Fig. 1b). These results suggest that there is a specific association of tyrosine-phosphorylated Cbp with GD3 on the rat cerebellar cell membrane.

image

Figure 1. Detection of Cbp that is associated with ganglioside GD3 and phosphorylated in detergent-resistant membrane (DRM) rafts from a rat cerebellum. (a) The membrane fraction of rat cerebellum was solubilized in TX lysis buffer. Supernatants were immunoprecipitated with anti-GD3 antibody (R24). Immunoprecipitates were subjected to an in vitro kinase assay (lane 1). Reimmunoprecipitation was performed with an anti-Cbp antibody (lane 2) and an anti-Lyn monoclonal antibody (lane 3). Phosphorylations were visualized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography. (b) The phosphoamino acid of the p80 band in in vitro kinase assay was examined. The markers used were phosphotyrosine (P-Tyr), phosphothreonine (P-Thr), and phosphoserine (P-Ser). (c) The exogenous Myc-tagged Cbp was expressed in Chinese hamster ovary (CHO) cells (GD3 −) or CHO cells expressing GD3 synthase, CST (GD3 +). The cell lysates prepared using Triton X-100 or octylglucoside lysis buffer (indicated as TX or OG, respectively) were incubated with control mouse IgG (lanes 2, 5, 8, and 11) or R24 (lanes 3, 6, 9, and 12). The Myc-tagged Cbp protein in the immunoprecipitates was detected by immunoblotting with anti-Myc antibody. (d) Rat cerebellar lysates (post-natal day 4) in TX-containing buffer were subjected to linear sucrose gradient (5–30%) centrifugation. Ten individual fractions were collected from top to bottom, and equal volumes of different gradient fractions were subjected to SDS–PAGE followed by immunoblotting for Cbp, flotillin (Flt), calnexin (Cnx), and transferrin receptor (TfR), and dot blotting for ganglioside GD3. Lanes 3–5 and lanes 7–10 correspond to the DRM raft and non-raft fractions, respectively.

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Cbp was associated with GD3 in a cDNA expression system

The association of Cbp with GD3 was confirmed using a cDNA expression system in CHO cells (Fig. 1c). GM3 is the only ganglioside synthesized in CHO cells and is an enzymatic substrate of GD3 synthase. We previously established a CHO cell line, CST, constitutively expressing GD3 synthase and demonstrated that R24 coprecipitated Lyn from CST cells expressing Lyn (Kasahara et al. 1997), and R24 coprecipitated Goα from CST cells expressing Goα (Yuyama et al. 2007). Both CHO and CST cells were transfected transiently with an expression plasmid carrying Cbp cDNA. Cbp was immunoprecipitated by R24 from the TX extract of the CST cells, but not from that of the CHO cells (Fig. 1c, lanes 3 and 6). However, Cbp was not immunoprecipitated by R24 from OG lysates of the CST cells, suggesting that R24 precipitates GD3 rafts containing Cbp (Fig. 1c, lane 12).

Cbp was localized to DRM raft fraction of rat cerebellum

To confirm that cerebellar GD3 rafts contain Cbp, we examined the distribution of Cbp on a sucrose density gradient. The presence of GD3 and flotillin in the DRM raft fraction (lanes 3–5) and the exclusion of calnexin and transferrin receptor, a non-raft marker protein, from the DRM fraction, confirmed the quality of the fractionation. Cbp predominantly existed in the DRM raft fraction of post-natal day 4 rat cerebellum (Fig. 1d).

Lyn phosphorylates tyrosine residue 314 of Cbp

Cbp mediates the enzymatic inactivation of Src family kinase via Tyr314-phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Src family kinase) by Src family kinase. Therefore, we investigated whether Cbp is a substrate for Lyn using a cDNA expression system. Coexpression of exogenous wild-type Lyn, but not negative Lyn, and wild-type Cbp in CHO cells resulted in Cbp tyrosine phosphorylation (Fig. 2a). Coexpression of exogenous active Lyn and wild-type Cbp in CHO cells resulted in Cbp phosphorylation at tyrosine 314 (Fig. 2b). Previously, we demonstrated that treatment of rat primary cerebellar granule cells with R24 induced rapid Lyn activation and tyrosine phosphorylation of 80-kDa protein (Kasahara et al. 1997). Therefore, we investigated whether R24 induced tyrosine phosphorylation of Cbp. Treatment of rat primary cerebellar granule cells with R24 for 2 min induced Cbp phosphorylation of tyrosine residue 314 (Fig. 2c). Furthermore, treatment of rat primary cerebellar granule cells with anti-ganglioside GD1b antibody also induced tyrosine phosphorylation of 80-kDa protein (Fig. 3).

image

Figure 2. Lyn phosphorylates tyrosine residue 314 of Cbp. (a) Wild-type Lyn (W) and dominant-negative Lyn (N) were expressed with (+) or without (−) Cbp as Myc-tagged protein in Chinese hamster ovary (CHO) cells. Cbp was then immunoprecipitated (IP) with anti-Myc antibody and analyzed for phosphorylation status by immunoblotting (IB) with anti-phosphotyrosine antibody (top panel). The lysates were probed for Myc-tagged Cbp amount (middle panel) or Lyn amount (bottom panel). (b) Constitutively active Lyn (A), wild-type Lyn (W), or dominant-negative Lyn (N) was expressed with Myc-tagged Cbp. The immunoprecipitates on Myc-tagged Cbp were probed with anti-Myc antibody (top panel), anti-phosphotyrosine antibody (middle panel), and anti-pY314-Cbp antibody (bottom panel). (c) The rat primary cerebellar cells were incubated with 20 μg/mL mouse IgG (lane 1) or 20 μg/mL R24 (lane 2) at 37°C for 2 min. The lysates were subjected to immunoprecipitation with anti-Cbp antibody. The phosphorylation at Tyr314 of Cbp was detected by immunoblotting with anti-pY314-Cbp antibody.

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image

Figure 3. Anti-ganglioside GD1b antibody GGR12-induced tyrosine phosphorylation of 80-kDa protein in rat primary granule cells. Cells were incubated with 20 μg/mL mouse IgG (lane 1), anti-GD1b antibody GGR12 (lane 2), or anti-GD3 antibody R24 (lane 3) at 37°C for 2 min. After sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), tyrosine phosphorylation was detected by immunoblotting with anti-phosphotyrosine antibody PY20.

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Developmental change of Cbp protein and tyrosine phosphorylation in rat cerebellum

Src family kinases regulate multiple processes in brain development, such as differentiation, migration, and axonal pathfinding (Maness 1992). Cbp modulates src family kinase activity in brain maturation (Lindquist et al. 2011). Therefore, we tested the expression of Cbp and protein tyrosine phosphorylation at different stages in rat cerebellar development. Cbp protein and tyrosine phosphorylation were detected from post-natal day 1–20 in the external granule layer, molecular layer, and internal granule layer (Fig. 4). Cbp protein and tyrosine phosphorylation were much weaker in the adult cerebellum. However, Lyn is expressed throughout. Therefore, we compared Lyn activity between post-natal day 4 and adult.

image

Figure 4. Immunoreactivities of anti-Cbp, anti-Lyn, and anti-phosphotyrosine antibodies during rat cerebellar development. Sagittal sections from the rat cerebellum (post-natal days 1, 5, 7, 10, and 20, and adult) were immunostained with anti-Cbp antibody (top panels), anti-Lyn antibody (middle panels), and anti-phosphotyrosine antibody (bottom panels). Scale bar, 100 μm.

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Lyn activation and Cbp phosphorylation in membrane rafts of developing cerebella

Western blotting with anti-phosphotyrosine antibody also demonstrated that the total tyrosine phosphorylation level was higher in the post-natal day 4 developing cerebellum than that in the adult one, and the tyrosine-phosphorylated proteins were highly accumulated in the DRM raft fraction (lanes 3, 4, and 5) of the post-natal day 4 cerebellum (Fig. 5, top panel). Lyn protein was present in the DRM raft fraction of both post-natal day 4 and adult cerebellum (Fig. 5, middle panel). However, the active form of Lyn was highly accumulated in the DRM raft fraction prepared from the post-natal day 4 cerebellum compared with the DRM raft fraction of the adult one (Fig. 5, bottom panel). Tyrosine-phosphorylated 80-kDa protein was immunoprecipitated by the anti-Cbp antibody from the DRM raft fraction prepared from the post-natal day 4 cerebellum (Fig. 6a). Furthermore, Cbp and pY314-Cbp were highly accumulated in the DRM raft fraction prepared from the post-natal day 4 cerebellum (Fig. 6b). These observations suggested that Lyn is activated and Cbp is phosphorylated in membrane rafts of the developing cerebellum.

image

Figure 5. Lyn activation and tyrosine phosphorylation in the detergent-resistant membrane (DRM) raft fraction from developing cerebellum. Ten individual fractions were prepared from the rat cerebellum (post-natal day 4 and adult) as outlined in Fig. 1d. Equal volumes of different gradient fractions were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblotting with anti-phosphotyrosine antibody (top panels) and anti-Lyn antibody (middle panel). Lyn was immunoprecipitated from the individual fractions and analyzed for the activation by immunoblotting with anti-phospho-Src family kinase antibody that is recognizable for the active form of Lyn phosphorylated on Tyr416 (bottom panels).

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image

Figure 6. Tyrosine residues of Cbp are highly phosphorylated in the detergent-resistant membrane (DRM) raft fraction of developing cerebellum. (a) Ten individual fractions were prepared from the rat cerebellum (post-natal day 4) as outlined in Fig. 1d. Equal volumes of gradient fractions were subjected to immunoprecipitation using the anti-Cbp antibody followed by immunoblotting with anti-phosphotyrosine antibody. (b) Ten fractions from the rat cerebellum (post-natal day 4 and adult) were independently probed with anti-Cbp antibody (top panels) or anti-pY314-Cbp antibody (bottom panels).

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Cbp is concentrated in cerebellar growth cone membrane

Growth cones are specialized neuronal compartments that are transiently generated at the tips of growing neurites. They play important roles in the formation of neural circuits in a developing brain. Subcellular fractionation showed that the isolated growth cones (Fig. 7a, lane G) contained higher levels of Cbp and pY314-Cbp than the whole cerebellum (Fig. 7a, lane W). The abundance of GAP-43, a growth cone marker, in lane G confirms the quality of the fractionation. In contrast, MAP kinase, a cytosolic protein, was evenly distributed. Previously, we also demonstrated that ganglioside GD3 and GD3-binding protein (Lyn, Goα) were present in growth cone fractions of the rat cerebellum (Yuyama et al. 2007). Immunoelectron microscopy analysis with gold-labeled antibodies showed that Cbp and GAP-43, a growth cone marker, are localized in the same vesicles of the growth cone fraction (Fig. 7b). Taken together, these results suggest that the ganglioside GD3 raft-anchored Cbp is phosphorylated by Lyn in developing cerebellar growth cones.

image

Figure 7. Cbp is concentrated in the neuronal growth cone of rat cerebellum. (a) The growth cone fractions (G) from the rat cerebellum (post-natal day 7) and whole cerebellum (W) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting by specific antibodies (for Cbp, pY314-Cbp, GAP-43, and MAP kinase). A total of 10 μg of protein was loaded. (b) Immunoelectron microscopy of cerebellar growth cone membrane. The specimens were incubated with anti-Cbp rabbit polyclonal antibody and anti-GAP-43 mouse monoclonal antibody as primary antibody, and were incubated with goat anti-rabbit IgG coupled to 15-nm colloidal gold and goat anti-mouse IgG coupled to 5-nm colloidal gold. Scale bar, 100 nm.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

In this study, we demonstrated that a monoclonal antibody to ganglioside GD3, R24, coimmunoprecipitated Cbp from rat cerebellum, and R24 treatment of rat primary cerebellar granule cells induces tyrosine 314 phosphorylation of Cbp. Furthermore, Cbp, Lyn, and GD3 were exclusively detected in the DRM raft fraction of rat cerebellum. Immunohistochemical study showed that Cbp was present in the granular layer and molecular layer. We previously reported that R24 also coimmunoprecipitated Src family kinase Lyn, and R24 treatment of rat primary cerebellar granule cells induces Lyn activation (Kasahara et al. 1997). Lyn and GD3 were expressed in the granular layer (Umemori et al. 1992; Kotani et al. 1995) and in cerebellar granule cells (Kasahara et al. 2000, 2002; Loberto et al. 2005). Cbp is a transmembrane phosphoprotein that has been implicated in the regulation of Src family kinase through recruitment of Csk, a negative regulator of Src family kinase, to membrane rafts (Brdicka et al. 2000; Kawabuchi et al. 2000). The interaction of Cbp and Csk is dependent upon the phosphorylation of Cbp Tyr-314 by Src family kinases. Subpopulation of Csk and the inhibitory C-terminal tyrosine 508 phosphorylation of Lyn were detected in the DRM raft fraction of rat developing cerebellum (Figure S1a, b). Cbp is phosphorylated predominantly by Fyn in neonatal mouse brains (Lindquist et al. 2011). However, Lyn is the dominant Src family kinase in cerebellar granule cells (Umemori et al. 1992). R24 coimmunoprecipitates Lyn, but not Fyn, from rat cerebellum (Kasahara et al. 1997). Therefore, Cbp is a substrate of Lyn in GD3-enriched DRM rafts of cerebellar granule cells and may negatively regulate Lyn through recruitment of Csk to membrane rafts. Cbp is known to be also phosphorylated by Lyn in erythroid cells, mast cells, and B lymphomas (Ohtake et al. 2002; Ingley et al. 2006; Tauzin et al. 2008; Yerly et al. 2010).

Rat cerebellum expresses not only ganglioside GD3 but also ganglioside GM1, GD1a, GD1b, and GT1b. Developmental changes and differential distribution of gangliosides in post-natal rat cerebellar cortex suggest that a specific ganglioside may play an important role in cerebellar development (Kotani et al. 1995). GD3 is expressed intensely in the external granular layer at post-natal day1–10, and moderately in the external and internal granular layers at post-natal day 20. This finding suggests that GD3 is associated with premature granule cells during the early post-natal development. Rat cerebellar granule cells are known to proliferate at the external granular layer and migrate to the internal granular layer from post-natal day 1 to 20. In this study, Lyn was activated and Cbp tyrosine 314 was phosphorylated from post-natal day 1 to 20 cerebellum. Using the cDNA expression system, we demonstrated that Lyn phosphorylates Tyr314 of Cbp. Cbp and Tyr314-phosphorylated Cbp were concentrated in the growth cone fraction. Previously, we demonstrated that Lyn and GD3 are also concentrated in the growth cone fraction (Yuyama et al. 2007). Furthermore, degradation of cell-surface GD3 by endoglycoceramidase greatly reduced R24-mediated Lyn activation and tyrosine phosphorylation of 80-kDa protein, suggesting that GD3 is necessary for the R24-mediated signaling (Kasahara et al. 2000). Therefore, GD3-anchored Cbp may control Lyn in growth cone rafts of developing cerebellar granule cells. We demonstrated that treatment of rat primary cerebellar granule cells with anti-ganglioside GD1b monoclonal antibody also induced tyrosine phosphorylation, suggesting that not only GD3 but also other gangliosides are involved in the same signaling pathway.

We demonstrated that R24 induced paxillin tyrosine phosphorylation and filamentous actin assembly in the growth cone of cerebellar granule cells (Yuyama et al. 2011), suggesting that the downstream events of Lyn/Cbp signaling are paxillin tyrosine phosphorylation and actin cytoskeleton-dependent changes in cell morphology. What are the upstream molecules involved in Lyn/Cbp signaling of rat cerebellar granule cells? Previously, we found that R24 coimmunoprecipitated TAG-1, a glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule, and the antibody-mediated cross-linking of TAG-1 induced Lyn activation in rat cerebellar granule cells (Kasahara et al. 2000). Sucrose density gradient analysis showed that TAG-1 was present in DRM raft and growth cone fractions of the cerebellum (Kasahara et al. 2000; Palestini et al. 2002). Sonnino and his colleagues also reported that R24 coimmunoprecipitates TAG-1 and Lyn from rat cerebellar granule cells (Prinetti et al. 2001). Therefore, the putative upstream molecule is TAG-1 in Lyn/Cbp signaling of rat cerebellar granule cells. In conclusion, our results suggest that GD3 rafts are platforms of Lyn/Cbp signaling in developing cerebellar growth cones of the cerebellum.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

The authors declare no conflicts of interest. This work was supported by JSPS KAKENHI Grant Number 23570182 for Scientific Research (C).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
jnc12040-sup-0001-FigS1.pdfapplication/PDF1299KFigure S1. Distribution of Csk and pY508-Lyn on sucrose density gradient.

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