Fibroblast Growth Factor-2-induced Signaling through Lipid Raft-associated Fibroblast Growth Factor Receptor Substrate 2 (FRS2)*

Marc S. RidyardDagger and Stephen M. Robbins§

From the Departments of Oncology and Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Received for publication, October 7, 2002, and in revised form, February 4, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The plasma membrane is not homogenous but contains specific subcompartments characterized by their unique lipid and protein composition. Based on their enrichment in various signaling molecules, these membrane microdomains are recognized to be sites of localized signal transduction for a number of extracellular stimuli. We have previously shown that fibroblast growth factor-2 (FGF2) induced a specific signaling response within a lipid raft membrane microdomain in human neuroblastoma cells characterized by the tyrosine phosphorylation of a p80 phosphoprotein. Herein, we show that this protein is the signaling adaptor FRS2 and that it is localized exclusively to lipid rafts in vitro and in vivo. We have examined how the tyrosine phosphorylation and serine-threonine phosphorylation of FRS2 within lipid rafts affect the response of cells to FGF2 signaling. Our data suggest that activation of protein kinase C, Src family kinases, and MEK1/2 are involved in regulating serine-threonine phosphorylation of FRS2, which can indirectly affect FRS2 phosphotyrosine levels. We also show that Grb2 is recruited to lipid rafts during signaling events and that activation of MEK1/2 by different mechanisms within lipid rafts may lead to different cellular responses. This work suggests that compartmentalized signaling within lipid rafts may provide a level of specificity for growth factor signaling.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fibroblast growth factors (FGFs)1 constitute a large family of peptide hormones that influence a wide variety of biological processes such as angiogenesis, embryogenesis, differentiation, and proliferation depending on the cell type (1, 2). In the nervous system, FGFs have been shown to stimulate both the differentiation and survival of post-mitotic cells as well as being proliferative factors for non-differentiated cells (3). FGFs induce their biological effects by binding to and activating a family of trans-membrane receptor tyrosine kinases (FGFR1-4) (2, 4). The activation of the FGFRs occurs by dimerization of the trans-membrane receptors upon the binding of the FGF ligand followed by autophosphorylation of a number of tyrosine residues, some of which can act as recruitment sites for various downstream effectors (2, 4-6).

One relatively unique aspect of FGF signaling is the recruitment of signaling molecules to FGFRs through the adaptor protein FGF receptor substrate 2 (FRS2, a.k.a. Suc1-associated neurotrophic factor target or SNT) (7, 8). FRS2 associates directly with FGFRs and shows increased tyrosine phosphorylation upon FGFR signaling and functions to recruit SH2 domain-containing proteins such as Grb2 that link the FGFR to a variety of downstream pathways (7, 9, 10). FRS2 also exhibits a high level of serine-threonine as well as tyrosine phosphorylation (7, 10). The function of the serine-threonine phosphorylation on FRS2 is presently unclear, although it may provide another level of specificity to the FGFR signaling by regulating pathways distinct from those associated with the tyrosine phosphorylation of FRS2. The importance of FRS2 in FGF signaling is reflected in the embryonic lethal phenotype observed during mouse development after disruption of the FRS2 gene. In addition, FRS2 null cell lines derived from these embryos show an impairment of FGF-induced migration, proliferation, and MAPK activation (11).

FGFR signaling also leads to activation of members of the Src protein tyrosine kinase family (12). Studies on Xenopus laevis have demonstrated that Laloo, a novel member of the Src family of tyrosine kinases, is required for mesoderm induction in response to FGF (13). The cooperation of FRS2 and Laloo in this pathway is suggested by the observed association of FRS2 with Laloo (14, 15).

Although many receptor tyrosine kinases activate similar downstream pathways such as the MAPK pathway, they induce specific phenotypic responses from the cell. Specificity can be achieved temporally by the developmentally regulated expression of certain components of the pathways. For instance, the expression of different FGFR types is regulated during oligodendrocyte maturation, providing a molecular basis for the developmentally varying response of cells to a common ligand (16). Specificity can also originate from the type of receptor expressed, because all of the FGFRs do not appear to have the same efficiency at activating various downstream pathways (17-19). In addition, the location of the receptor or downstream events within the cell may add another level of specificity in the signaling response.

Microdomains within the plasma membrane such as lipid rafts function to sequester signaling molecules and act as sites of signal transduction that can regulate cell physiology (20-22). Lipid rafts are characterized by their enrichment in glycosphingolipids and cholesterol as well as by their unique protein composition (20, 22). On the inner leaflet of the plasma membrane, proteins such as G-proteins and members of the Src-family of protein tyrosine kinases are found associated with these membrane microdomains (23-25). There are at least two different microcompartments that can be distinguished by their shape and protein composition (22). Caveolae are one such compartment characterized by the presence of caveolin-1, a 22-kDa protein known as the structural component of these small flask-shaped caves (26-29). Originally, caveolae were thought to function in receptor-mediated potocytosis (30). However, their functions are rapidly expanding to include events such as an alternative route of receptor endocytosis, trans-cytosis, regulation of calcium homeostasis, a site of clustering of glycosylphosphatidylinositol-linked proteins, and more recently, a localized site for signal transduction (20, 30-34).

Previously, we had observed an FGF2-induced signaling event that was localized to membrane microdomains that we termed caveolae-like domains (35). Herein, we have identified the FGF2-responsive protein FRS2 as a lipid raft-associated protein, both in vitro and in the developing mouse brain. We have also examined FGF2 signaling through FRS2 within lipid rafts and show that serine-threonine phosphorylation of FRS2 is dependent on a pathway involving protein kinase C (PKC), Src family kinases, and MEK1/2 and that FGF2 signaling through MEK1/2 may have different phenotypic consequences in the cell depending upon the pathway used for activation of MEK1/2.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antibodies-- Anti-FRS2 polyclonal antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); anti-Grb2 and anti-PKC monoclonal antibodies were obtained from Transduction Laboratories (Lexington, KY); 4G10 anti-phosphotyrosine was from Upstate Biotechnology Inc. (Lake Placid, NY); anti-active MAPK polyclonal was purchased from Promega (Madison, WI); and anti-MAPK was from Zymed Laboratories Inc. (San Francisco, CA).

Cell Culture-- LAN-1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% cosmic calf serum (Hyclone) and 1% penicillin/streptomycin at 37 °C in 5% CO2. Prior to growth factor treatment, cells were serum-starved in Dulbecco's modified Eagle's medium with 0.5% cosmic calf serum for 18 h. Cells were stimulated with 20 ng/ml FGF2, 50 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), or 100 nM thymeleatoxin for the times indicated in experiments. Inhibitor studies were done with the addition of 10 µM PP2 or PP3, 1 µM bisindolylmaleimide (Bis) I or V, or 10 µM U0126 to the cells for 30 min prior to cell stimulation.

Immunoprecipitation and Western Blotting-- Cells were rinsed once with phosphate-buffered saline, pH 7.4, and lysed in 1% Nonidet P-40 lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Nonidet P-40 (protein grade; Calbiochem), 1 mM PMSF (Roche Molecular Biochemicals), 1 mM sodium orthovanadate, and 10 µg/ml each of aprotinin and leupeptin (Sigma)). Insoluble material was removed by centrifugation at 12000 × g for 15 min at 4 °C. Cell-free lysates were incubated with 1 µg/ml FRS2 polyclonal antibody for 1 h at 4 °C. Protein A-Sepharose was added, and the mixtures were incubated for an additional 30 min at 4 °C. The immunocomplexes were washed several times with 1% Nonidet P-40 lysis buffer, and the samples were boiled in 2× SDS Laemmli's sample buffer prior to separation on 10% SDS-polyacrylamide gels. Following electrophoresis, the proteins were transferred to nitrocellulose membranes (Schleicher & Schuell). Membranes were dried and blocked in Tris-buffered saline (5 mM Tris, 135 mM NaCl, and 5 mM KCl) containing 0.1% Nonidet P-40, 0.1% Tween 20, and 5% bovine serum albumin for 20 min. Membranes were then incubated for 1 h with a 1:1000 dilution of the appropriate antibodies in their respective blocking buffers. The membranes were washed extensively in Tris-buffered saline containing 0.1% Nonidet P-40 and 0.1% Tween 20 or 0.5% Nonidet P-40 and 0.1% Tween 20 when alpha -phosphotyrosine antibody was used. This was followed by incubation for 20 min with appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences), each diluted 1:10000 in blocking buffer. The membranes were washed as described above and developed using an ECL substrate.

Isolation of Detergent-insoluble Low Density Fractions-- Cells were grown to confluence on 150-mm-diameter dishes and stimulated as described above. Cells were rinsed in ice-cold phosphate-buffered saline and lysed in 1% Triton X-100 buffer. The cells were fractionated on sucrose gradients, and low density soluble and pelleted fractions were isolated as described in detail previously (23).

Preparation of Mouse Brain Homogenate-- CD1 mouse embryos were recovered at stages E11, E16, and P1. Whole brain tissue was isolated and homogenized in 1% Triton X-100 buffer before fractionation on sucrose gradient and isolation of low density fractions as described above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of FRS2 as a Lipid Raft-associated Protein-- Previous work from our laboratory has shown that FGF2 was unique from other growth factors (epidermal growth factor and platelet-derived growth factor) and neurotrophins in that it was able to induce a compartmentalized signaling response in a human neuroblastoma cell line, LAN-1 (35). In our previous work, we referred to these membrane microdomains as caveolae-like domains, because the LAN-1 cells expressed both caveolin-1 and -2. However, since we have only characterized this membrane fraction based on its detergent insolubility and low buoyant density fractionation, it is more appropriate to refer to these domains as lipid rafts, consistent with recent nomenclature (36).

We observed that FGF2 was able to induce an increase in tyrosine phosphorylation of several proteins within the lipid raft fraction including an unknown protein migrating between 75-80 kDa that bound to glutathione S-transferase fusion proteins containing p13suc1 or Src-SH2 domains (35). Association with p13suc1 or SH2 domains is a feature of the 80-kDa FGFR-associated adaptor protein, FRS2, and related family members (7, 8, 10). Therefore, in an attempt to characterize and identify the p75-80 protein, we used antibodies directed to FRS2 to probe fractionated LAN-1 cell lysates that had been treated with 20 ng/ml FGF2 for 5 min. Western blotting showed that FRS2 is localized exclusively to lipid raft fractions and correlates with the FGF2-induced phosphotyrosine seen at 80 kDa in the lipid raft fraction (Fig. 1A). A 90-kDa band was also observed in the Triton X-100-soluble and pelleted fractions when Western blotting for FRS2 (Fig. 1A). This band was never observed to be tyrosine-phosphorylated in response to FGF2, so it is probably a nonspecific band or possibly a FGF non-responsive FRS2-related protein. Immunoprecipitation of FRS2 from whole cell lysates confirmed that the 80-kDa FRS2 was tyrosine-phosphorylated in response to FGF2 (Fig. 1B). Therefore, we have identified FRS2 as a FGF2-responsive protein that is localized specifically to the lipid raft fraction of LAN-1 cells.


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Fig. 1.   Localization of FRS2 to lipid rafts. Cells were lysed in 1% Triton X-100 buffer and fractionated on sucrose gradients to allow isolation of lipid raft fractions along with Triton X-100-soluble and insoluble fractions. A, LAN-1 cells were treated with 20 ng/ml FGF2 for 5 min prior to lysis and fractionation. Blotting for phosphotyrosine showed an 80-kDa protein that was phosphorylated in response to FGF2 that occurs specifically in the lipid raft fraction. Antibodies to FRS2 showed that FRS2 is also localized specifically to lipid rafts and corresponds to the phosphorylated protein at 80 kDa. B, LAN-1 cells were treated with 20 ng/ml FGF2 for 5 min, and the cell-free lysate was used for immunoprecipitation with antibodies to FRS2. Immunoprecipitated FRS2 was tyrosine-phosphorylated in response to FGF2. C, embryonic mouse brain was isolated and lysed prior to fractionation on sucrose gradients. Blotting for FRS2 showed FRS2 localization to lipid raft fractions but not Triton X-100-soluble fractions of the mouse brain.

To establish that this localization was not unique to established cell lines, we also examined the expression and localization of FRS2 at various time points in the developing mouse brain. Whole mouse brain lysates were fractionated on sucrose gradients and probed by Western blotting for FRS2 expression. We found that FRS2 was expressed at each of the stages of development examined and that in all stages FRS2 was specifically localized to the lipid raft fractions (Fig. 1C).

Serine-Threonine Phosphorylation of FRS2 Is Regulated by Protein Kinase C, Src Family Kinase, and MEK1/2 Activity-- The FRS2 response to FGF2 stimulation is characterized by an increased tyrosine phosphorylation as well as a change in electrophoretic mobility on SDS-PAGE gels that has been attributed to the action of serine-threonine kinases toward FRS2 (10). Therefore, we have used the mobility shift as an indicator of FRS2 serine-threonine levels. Because FGFRs have been shown to activate phospholipase Cgamma (38), which can lead to the activation of PKC, we examined the possibility that PKC was involved in serine-threonine phosphorylation of FRS2.

Using a 5-min stimulation of 20 ng/ml FGF2, we could visualize the shift in electrophoretic mobility of FRS2, indicative of its serine-threonine phosphorylation (Fig. 2A). The effects of FGF2 on FRS2 could be blocked by PP2, an inhibitor of the Src family kinases, or the PKC inhibitor Bis I, suggesting that PKC and Src family kinase activity are required for FGF2-induced serine-threonine phosphorylation of FRS2 (Fig. 2A). FGF2-induced serine-threonine phosphorylation of FRS2 was also completely blocked by the MEK1/2 inhibitor U0126, suggesting that activation of the MAPK cascade is also required for serine-threonine phosphorylation of FRS2. The electrophoretic shift in mobility in FRS2 using FGF2 treatment could be replicated using 50 ng/ml TPA, a diacyl glycerol analog that activates conventional and novel PKC isoforms (Fig. 2A), as well as when cells were stimulated with thymeleatoxin, which specifically activates conventional isoforms of PKC (data not shown) (39). The electrophoretic mobility shift of FRS2 induced by TPA could also be abrogated in the presence of PP2, Bis I, or U0126 inhibitors. These data confirmed that direct activation of PKC could lead to serine-threonine phosphorylation of FRS2 and that it was also dependent on downstream activation of Src family kinases and MEK1/2 as observed for FGF2 stimulation. FRS2 was only detected in lipid raft fractions and not in the Triton X-100-soluble fraction of the cells (Fig. 2A).


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Fig. 2.   Characterization of FRS2 serine-threonine and tyrosine phosphorylation-dependent signaling in lipid rafts. LAN-1 cells were stimulated with either 20 ng/ml FGF2 or 50 ng/ml TPA and then lysed in 1% Triton X-100 buffer and fractionated on sucrose gradients to allow isolation of lipid raft fractions along with Triton X-100-soluble fractions. A, Western blotting for FRS2 shows that FGF2 induced serine-threonine phosphorylation of FRS2 that was blocked by pretreatment of the cells with PP2, Bis I, or U0126. TPA also induced the serine-threonine phosphorylation of FRS2 and was blocked by pretreatment of the cells with PP2, Bis I, or U0126. Examination of phosphotyrosine levels showed that FGF2 treatment lead to tyrosine phosphorylation of FRS2, which was enhanced by PP2, Bis I, or U0126 treatment. TPA treatment did not stimulate tyrosine phosphorylation of FRS2. Analysis of Grb2 levels showed that Grb2 was present in the soluble fractions but not in the raft fractions and was recruited to lipid rafts upon FGF2 stimulation. The recruitment of Grb2 to lipid rafts was enhanced by treatment with PP2 or U0126. Caveolin levels confirm equal loading of lipid raft fractions. B, LAN-1 cells fractionated on sucrose gradients were blotted for expression of PKC isoforms. PKCalpha , PKCgamma , and PKCiota were all expressed, but only PKCgamma showed strong localization to lipid raft fractions.

An analysis of Western blots showing phosphotyrosine levels in lipid rafts revealed that FGF2 treatment induced tyrosine phosphorylation of FRS2, which was not blocked by pretreatment of the cells with PP2, Bis I, or U0126 (inhibitors that blocked serine-threonine phosphorylation of FRS2). Rather, under these conditions, tyrosine phosphorylation of FRS2 appeared to be enhanced (Fig. 2A). Unlike FGF2, treatment of the cells with TPA did not result in the increased tyrosine phosphorylation of FRS2 (Fig. 2A). These results suggest that serine-threonine phosphorylation of FRS2 is not required for tyrosine phosphorylation of FRS2 to occur and that blocking the serine-threonine phosphorylation of FRS2 can lead to enhanced tyrosine phosphorylation.

Tyrosine-phosphorylated FRS2 has been shown to directly recruit Grb2 and activate the MAPK pathway in response to FGF2 (40, 41). We also confirmed that Grb2 associates in a complex with FRS2 in LAN-1 cells as determined by immunoprecipitation of FRS2 (data not shown). Because FRS2 was associated exclusively with lipid rafts, we examined the level of Grb2 recruitment to lipid rafts after cell stimulation. Grb2 was present in the Triton X-100-soluble cell fraction under all of the conditions examined (Fig. 2A). Under control conditions, Grb2 was either not present in lipid rafts or at extremely low levels. FGF2 stimulation induced the recruitment of Grb2 to lipid rafts, and this was not blocked by PP2, Bis I, or U0126, which blocked serine-threonine phosphorylation of FRS2. As was observed for tyrosine phosphorylation of FRS2, it appeared that Grb2 recruitment to lipid rafts was actually enhanced under conditions that inhibit serine-threonine phosphorylation of FRS2 (Fig. 2A). TPA treatment did not induce Grb2 recruitment to lipid rafts, although some Grb2 was present after TPA treatment in the presence of U0126 (Fig. 2A). These data suggest that serine-threonine phosphorylation of FRS2 is neither sufficient nor necessary for the recruitment of Grb2 to lipid rafts; however, enhancing tyrosine phosphorylation of FRS2 recruits more Grb2 to lipid rafts. Equal loading of the lipid raft fractions was confirmed by Western blotting for caveolin protein levels (Fig. 2A).

Protein kinase C Isoforms Show Differential Association with Lipid Rafts-- To address which PKC isoform might be involved in FRS2 signaling, we looked at the expression of PKC isoforms in fractionated LAN-1 cells to determine whether there was any localization to lipid raft microdomains. Several PKC isoforms were expressed in LAN-1 cells; however, PKCgamma was the only isoform detected that showed strong lipid raft association, although the localization of PKCgamma did not appear to be affected by FGF2 treatment (Fig. 2B). Of the other conventional PKC isoforms, PKCalpha was excluded from lipid rafts (Fig. 2B) and PKCbeta was not detected in Western blots (data not shown), suggesting that PKCgamma may be the isoform involved in signaling through FRS2 in LAN-1 cells.

Activation of MAPK by FGF2 Is Not Dependent on Serine-Threonine Phosphorylation of FRS2-- We have already shown that Grb2 is recruited to tyrosine-phosphorylated FRS2 in lipid rafts in the absence of FRS2 serine-threonine phosphorylation. We next examined whether the serine-threonine phosphorylation levels of FRS2 affected downstream MAPK activation.

After the treatment of LAN-1 cells with 20 ng/ml FGF2 for 5 min, FRS2 was immunoprecipitated and examined by SDS-PAGE for a change in electrophoretic mobility under various inhibitory conditions. Using the Src family kinase inhibitor PP2 or the PKC family inhibitor Bis I, we observed an inhibition of the change in mobility of FRS2 induced by FGF2 treatment. The inhibitory effects of PP2 and Bis I were not observed when the control compound, PP3 or Bis V, was used (Fig. 3A). As observed previously, neither PP2 nor Bis I inhibited the tyrosine phosphorylation of FRS2; however, there was an increase in the tyrosine phosphorylated FRS2 seen at 75 kDa that corresponds to the accumulation of FRS2 in the non-serine-threonine-phosphorylated form (Fig. 3A).


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Fig. 3.   FGF2-induced MAPK activation does not require serine-threonine phosphorylation of FRS2. LAN-1 cells were treated with 20 ng/ml FGF2 after a 30-min preincubation with either 10 µM PP2 or PP3 or 1 µM Bis I or Bis V and then lysed in 1% Nonidet P-40 lysis buffer and subject to immunoprecipitation with antibodies to FRS2. A, examination of FRS2 showed that FGF2-induced serine-threonine phosphorylation was inhibited by treatment with PP2 or Bis I but not control compounds PP3 or Bis V. FGF2-induced tyrosine phosphorylation of FRS2 was not affected by either inhibitor. B, whole cell lysates treated the same as above were examined for MAPK activation. FGF2 addition lead to MAPK activation, and this was not inhibited by PP2 or Bis I treatment.

Whole cell lysates from cells treated with the same conditions were used to determine the phosphorylation levels of MAPK. It was found that FGF2 activated p42ERK and that neither PP2 nor Bis I blocked MAPK activation by FGF2 (Fig. 3B). These data suggest that MAPK activation correlates with FRS2 tyrosine phosphorylation, whereas global serine-threonine phosphorylation of FRS2 is not required for the propagation of signals that lead to MAPK activation.

FGF2 Induces Cell-rounding Morphology That Is Dependent on MEK Activity but Not PKC-- LAN-1 cells that were treated with 20 ng/ml FGF2 for 18 h showed a change from a flattened dendritic morphology to a rounded refractile morphology (Fig. 4A). This change in morphology was not seen when the cells were treated with 50 ng/ml TPA or 100 nM thymeleatoxin for the same time period (Fig. 4A). When the cells were pretreated with the MEK inhibitor U0126, the FGF2-induced cell rounding was completely blocked and the cells remained in a flattened adherent morphology (Fig. 4B). In contrast, the pretreatment of the cells with the PKC inhibitor Bis I failed to inhibit the FGF2-induced cell rounding (Fig. 4B). Together, these results suggest that FGF2 acts to induce cell rounding through a MEK-dependent pathway and that this pathway does not require the activation of PKC.


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Fig. 4.   FGF2-induced cell rounding requires MAPK but not PKC. LAN-1 cells were treated with 20 ng/ml FGF2 or 50 ng/ml TPA or 100 nM thymeleatoxin for 18 h after pretreatment with either 10 µM U0126 or 1 µM Bis I. A, FGF2 treatment induced the rounding of the cells, whereas TPA and thymeleatoxin only had a slight effect on cell rounding. B, FGF2-induced cell rounding was blocked by inhibition of MEK using U0126 but not inhibition of PKC using Bis I.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In summary, we have shown that FRS2 is a lipid raft-associated protein, both in the cell culture model used here and also in vivo. We have also shown that FGF2 signaling through FRS2 involves multiple pathways that appear to regulate distinct phenotypic responses in the cell.

FRS2 was initially described as an adaptor protein involved in FGF signaling that was modified at the N terminus with the addition of a myristoylation moiety (7). The acyl modification functions to target FRS2 to the membrane, and several reports (7, 40) have shown that FRS2 is indeed localized to the membrane portion of fractionated cells and that membrane association is required for efficient signaling to occur through FRS2. Here, we have shown that FRS2 is not only targeted to the plasma membrane but that it is localized specifically to lipid rafts within the membrane, both in cultured LAN-1 cells and also in lipid rafts isolated from various stages of developing mouse brain.

Many signaling molecules have been reported to be present within lipid rafts, and membrane microdomains are thought to act as sights for regulated formation of signal transduction complexes that influence downstream signaling from a variety of membrane receptors (22). The localization of FRS2 to lipid rafts is probably important in regulating its interaction with other molecules such as bringing FRS2 into close proximity with FGFR to enable the association of the two proteins that has been demonstrated to occur between the juxtamembrane region of FGFR and the phosphotyrosine binding domain of FRS2 (42). FRS2 is known to be expressed in the developing nervous system (43). Our observation that FRS2 is also localized to lipid rafts in developing mouse brain provides evidence that this localization may be functionally important for efficient signaling through FRS2 in vivo during neural development.

We have been unable to determine whether any of the FGFRs are localized to lipid rafts (35), possibly because of the fact that the receptors are extracted from the lipid raft fraction under the conditions used as has been noted for the epidermal growth factor receptor (44).

FRS2 contains several tyrosine residues that have been shown to be phosphorylated in response to growth factor signaling (7, 8). The tyrosine phosphorylation of FRS2 is important in signal propagation and has been shown to be required for association with Grb2 and downstream activation of the MAPK pathway (40, 42) as well as in the recruitment of several other signaling molecules such as Cbl (45) and phosphatidylinositol 3-kinase (46). However, it has also been shown that the majority of phosphorylation on FRS2 is on serine and threonine rather than tyrosine (7, 10). Previous reports of the molecular weight of FRS2 have varied somewhat, possibly because of the change in electrophoretic mobility that is associated with serine-threonine kinase activity toward FRS2 (10). In this report, we have examined the tyrosine and serine-threonine levels of FRS2 under various conditions to determine the effect they have on FRS2 signaling within lipid rafts.

It has previously been shown that phospholipase Cgamma associates directly with the FGFR through its SH2 domain and is involved in FGF signaling (38). Because phospholipase C is an upstream activator of PKC signaling, we examined the possibility that PKC was involved in the serine-threonine phosphorylation of FRS2. It has been reported that FRS2 associates with the atypical isoform PKClambda , although it was not found to be a substrate of PKClambda in vitro (47). Our data suggest that a conventional PKC isoform is involved in the phosphorylation of FRS2, although in agreement with previous studies, FRS2 may not be a direct substrate of PKC because we observed an inhibition of TPA-induced serine-threonine phosphorylation of FRS2 by both PP2 and U0126 treatment, suggesting a requirement for Src family kinase and MEK1/2 activity downstream of PKC. PKC can associate with Src family kinases through adaptor proteins, and tyrosine phosphorylation of PKC has been implicated in the modulation of downstream signaling (48, 49). Of the three conventional PKC isoforms, we have shown that the PKCgamma isoform is present in lipid rafts, whereas the PKCalpha isoform is excluded from lipid rafts isolated from LAN-1 cells. The PKCbeta isoform was not detected (data not shown). Therefore, it is possible that PKCgamma is the isoform involved in either direct or indirect phosphorylation of FRS2 in LAN-1 cells.

During the preparation of this paper, it has been demonstrated that the activation of MAPK results in serine phosphorylation of FRS2, which acts to negatively regulate the tyrosine phosphorylation-dependent signaling through FRS2 (50). We also observed that the inhibition of serine-threonine phosphorylation of FRS2 leads to increased tyrosine phosphorylation levels of FRS2 as well as a correlating increase in the level of Grb2 recruited to lipid rafts that suggests a negative regulation of FRS2 phosphotyrosine by serine-threonine phosphorylation. Although Lax et al. (50) did not find PKC activity to be required for the serine-threonine phosphorylation of FRS2 to occur, we have shown that PKC is involved in serine-threonine phosphorylation of FRS2 in LAN-1 cells, possibly through MEK1/2-dependent activation of MAPK. Both FGF and TPA have been shown to activate the Ras/MAPK signaling cascade in a PKC-dependent manner (51), and it is possible that PKC-dependent activation of Ras in response to FGF2 is only utilized by certain cell types or under certain conditions.

Our data also suggest a requirement for the activation of MEK1/2 in both FGF2- and TPA-stimulated serine-threonine phosphorylation of FRS2. However, only FGF2 stimulation recruits Grb2 to lipid rafts and induces LAN-1 cell rounding in a MEK1/2-dependent manner. This would suggest that despite both FGF2 and TPA activating a MEK1/2-dependent pathway, only the FGF2-activated MEK1/2 leads to LAN-1 cell rounding. The data presented here suggest that FGF2 may activate MEK1/2 through the recruitment of Grb2 to FRS2 and also by activation of Ras by PKC. The activation of MEK1/2 through these different mechanisms may lead to different downstream responses in the cell as has been noted in other cell types where activation of different isoforms of PKC lead to differential localization of MAPK within the cell, leading to different cellular responses (37). A model showing FRS2 signaling pathways is shown in Fig. 5.


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Fig. 5.   A putative model for FGF-induced phosphorylation of FRS2 and downstream signaling. Tyrosine phosphorylation of FRS2 (Y), possibly through the action of the tyrosine kinase receptor itself, leads to activation of MAPK through multiple pathways. Serine-threonine phosphorylation of FRS2 (S and T) involving PKC and Src family kinases leads to the activation of pathways distinct from those downstream of FRS2 tyrosine phosphorylation but still involve MEK. Whether MAPK is the direct mediator of the PKC-dependent pathway remains to be determined.

In addition to FGF, a diverse array of signaling molecules has been shown to utilize lipid rafts for various aspects of their signaling mechanisms (36). We have previously shown that the glycosylphosphatidylinositol-anchored ephrins are also able to induce a compartmentalized signaling response within the lipid rafts of cells including neural cells. As is the case for FGF, ephrin signaling induces the phosphorylation of a p75-80 phosphoprotein (35); however, this protein is not FRS2.2 Therefore, there is specificity of signaling within lipid rafts through the recruitment of different signaling molecules to distinct signaling pathways

In summary, we have shown that the FGF-responsive adaptor protein FRS2 is localized exclusively to lipid rafts within the cell membrane and that this localization is probably an important factor in the regulation of signaling via FRS2 through the regulation of interacting signaling molecules that either regulate the phosphorylation of FRS2 or are responsive to FRS2 phosphorylation levels. We have also shown that MEK1/2 is involved in multiple signaling pathways within lipid rafts and that the activation of MEK1/2 through different mechanisms may lead to different cellular responses.

    ACKNOWLEDGEMENTS

We thank the members of the laboratory for their constructive comments and discussions during the course of this work. We also thank Dr. Alice Davy for valuable input during the initial stages of this work.

    FOOTNOTES

* This work was supported by a grant from the Canadian Institutes of Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by an Alberta Cancer Board fellowship.

§ Senior Scholar of the Alberta Heritage Foundation for Medical Research and holds a Canada Research Chair in Cancer Biology. To whom correspondence should be addressed: Depts. of Oncology and Biochemistry & Molecular Biology, 3330 Hospital Dr., N. W., University of Calgary, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-4054; Fax: 403-283-2787; E-mail: srobbins@ucalgary.ca.

Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M210245200

2 A. Davy, M. S. Ridyard, and S. M. Robbins, unpublished data.

    ABBREVIATIONS

The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; SH, Src homology; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; Bis, bisindolylmaleimide; FRS2, fibroblast growth factor receptor substrate 2; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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