Intact and Functional Fibroblast Growth Factor (FGF) Receptor-I. Trafficks near the Nucleus in Response to FGF-I*. (Received for publication, May 25, 1994, and ...
THEJOURNALOF BIOLQGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269, No. 50, Issue of December 16,pp. 3172031724, 1994 Printed in U.S.A.
Intact andFunctional Fibroblast GrowthFactor (FGF) Receptor-I Trafficks near theNucleus in Response to FGF-I* (Received forpublication, May 25, 1994, and in revised form, October 19, 1994)
Igor PrudovskySO, Naphtali Savionh, Xi Zhanll, Robert Friesel, Jianming Xu**, Jinzhao Hou**, Wallace L. McKeehan**, and Thomas MaciagSS From the Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855 and the Institute of Biosciences and Technology, Texas A&M University, Houston, Texas 77030
Exogenous fibroblast growth factor-1 (FGF-1) associates with the nucleus in a receptor-dependent manner during the entireGI period of the BALB/c 3T3cell cycle (Zhan,X.,Hu, X,Friesel, R., and Maciag, T.(1993)J. Biol. Chern. 268, 9611-9620). To further study the role of the FGF receptor (FGFR) during this translocation, the intracellular fate of FGFR-1 protein and enzymatic activity was examined. Immunoprecipitation using multiple FGFR-1 antibodies followed by an in vitro tyrosine kinase activity assay enabled us to identify FGFR-1 as a 130-kDaphosphotyrosine-containingprotein associated with the nuclear fraction of MH 3T3 cells exposed to FGF-1. While FGFR-1 tyrosine kinase activity could be detected as a nuclear-associated protein after a 2-h exposure of the MH 3T3 cells to FGF-1, this activity appeared to be maximalin thenuclear fraction between 4 and 12 h after FGF-1 treatment. In addition, analysis by confocal immunofluorescence microscopyof quiescent and FGF-1-stimulatedNIH 3T3 cells reveal a prominent perinuclear FGFR-1 staining pattern in the cells exposed to FGF-1 but not in thequiescent population. We also observedFGFR-1 associated with the nuclear fraction in FGFR-1-transfectedL6 rat myoblasts, whichare known to be refractive to exogenous FGF-1ahd express relatively low levels of endogenous FGFR-1. In addition, these cells alsoexhibited the presence of a 145-kDaphosphoprotein in the nuclear fraction that was recognized byFGFR-1 antibodies. These results suggest that the FGFR-1 may betranslocated near the nucleus upon interaction with its ligand during the entireG , period of the MH3T3 cell cycleas a structurally intactand functional tyrosine kinase that maybe accessible to perinuclear polypeptides as a regulatory enzyme.
The fibroblast growth factor (FGF)' gene family is presently comprised of nine members including two the prototypes FGF-1 (acidic) and FGF-2 (basic) (1).The biological activities of the FGFs are mediated through the interaction withcell surfaceassociated FGF receptors, and four members presently comprise the FGF receptor (FGFR) tyrosine kinase genefamily (2). Interestingly, the ability of FGF-1 to initiate DNA synthesis requires thelong term exposure of the cell to FGF-1 during the entire G, phase of the cell cycle, and this correlates with the tyrosine phosphorylation of novel intracellular polypeptides during mid to late G, (3). Indeed, thepurification and characterization of proteins phosphorylated on tyrosine residues during mid to late GI in response to FGF-1 led to the identification of cortactin (41, a protein independently characterized as the major substrate for v-Src (5).These studies are consistent with the identification of src family members as requisite mediators of platelet-derivedgrowth factor(PDGF)-mediated signal transduction and the demonstration that c-Src is phosphorylated during mid to late G, in response to PDGF in NIH 3T3 cells (6) and FGF-1 inBALB/c 3T3 cells (7). During the characterization of the FGF-1-mediated mid to late G, events in BALB/c 3T3 cells, we noted that the tyrosine phosphorylation events were mediated by a low steady-state level of FGF receptors present on the cell surface (3). We furto associate with the ther noted that exogenous FGF-1 was able nucleus in a receptor-dependent manner during the entireG, phase of the BALB/c 3T3 cycle (3). Indeed, sequences responsiblefor nucleartranslocationhavebeencharacterizedin FGF-1 (8),FGF-2 (9), and FGF-3 (10). However, the biological significance of the nuclear localization sequence in FGF-1has been difficult t o access sincemutagenesis of this sequence does not abolish nuclear translocation but rather results ain reduction in the efficiency of nuclear association (11).Thus, to further define the pathwayof FGF-1 nuclearassociation, we studied this receptor-dependent process by examining the intracellular fateof the FGFR-1protein. Using FGFR-1 immu* This work was supported by National Institutes of Health Grants noprecipitation methods followed by signal amplification with HL32348 and HL44336 (to T. M.) and HD29561, AmericanHeart Asso- a n in vitro tyrosine kinase assay as well as confocal immunciationGrant-In-Aid91013920 (to R. F.), and National Institutes of ofluorescence microscopy we report the association of FGFR-1 Health Grants DK40739 and DK35310 (to W. L. M.). The costs of pub- with the nuclear fraction of FGF-1-treated NIH 3T3cells as a lication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" structurally intact and functional jwtanuclear tyrosine kinase during the mid to late G, phase of the cell cycle. in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Contributed equally tothe generation of the data described within EXPERIMENTALPROCEDURES this manuscript. On sabbatical leave from the Engelhardt Institute of Molecular Cell Culture-NIH 3T3 cells (AmericanType Culture Collection) and Biology, Russian Academy of Sciences, Moscow 117984, Russia. L6 cells (a gift from Dr. Lewis T. Williams, University of California at ll On sabbatical leave from the Goldschleger Eye Research Institute, Sackler Facultyof Medicine, T e l Aviv University, TelAviv, Israel, 52621. San Francisco)weregrowninDulbecco'smediatedEagle'smedium supplemented with 10%(v/v)calf serum (Hyclone) and density arrested I( Present address: Dept. of Experimental Pathology, Holland Labora- at Go in Dulbecco's mediated Eagle's medium containing 0.5%(v/v) calf tory, American Red Cross, Rockville, MD 20855. $$ To whom correspondence should be addressed. Dept.of Molecular The abbreviations used are: FGF, fibroblast growth factor; FGFR, Laboratory, 15601 Crabbs Biology,American Red Cross,Holland Branch Way,Rockville, MD 20855. Tel.: 301-738-0653; Fax: 301-738- FGF receptor-I; NPB, nuclear preparation buf€er; PDGF, platelet-derived growth factor; PAGE, polyacrylamidegel electrophoresis. 0465.
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Nuclear Association of Fibroblast Growth Factor Receptor-1
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FIG.1.FGF-I-induced FGFR-I tyrosine-kinase activity in nuclear fractions of NIH 3T3 ce1ls.A. quiescent NIH 3T3 cells were stimulated with 25 ng/ml of FGF-1 in the presence of 10 unitdm1of heparin for the time periodsindicated. Cells were scraped and lysed; cytosol (C) and nuclear ( N ) fractions were prepared and subjected to immunoprecipitation usingrabbitanti-XenopusFGFR-1antiserum (i) or preimmune serum ( p ) followed by an in vitro kinase amplification as described under "Experimental Procedures."Aliquots of cytosol and nuclear fractions of 8 pl and 40 p1, respectively, were subjected to 7.5% (w/v) SDS-PAGE analysis followed by treatment with 1 M KOH a t 55 "C for 2 h. Molecular mass standards (Bio-Rad) are indicated on the left side. B, quiescent NIH 3T3cells were stimulated with FGF-1 for 9 h, and cytosol and nuclear fractions were prepared. Immunoprecipitationwithanti-FGFR-1 antiserumandpreimmuneserumwas performed in the presence (+) or absence (-) of the carboxyl-terminalpeptide of FGFR-1 (FR-I;20 pg/ml), which was used to raise the FGFR-1antibody. Aliquots of cytosol and nuclear fractions of 8 and 40 p1, respectively, weresubjectedto 7.5% (w/v) SDS-PAGE analysis as described for A. C, densitometry of the nuclear p130 bands observed inA.
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serum for either18 h (NIH 3T3 cells) or 30 h (L6cells). Quiescent cells demonstrated the absence of cytosol-derived membranes and organelles were stimulated with recombinant human FGF-1, which was expressed in the nuclear fractions. and purified a s described previously (8).At the end of the incubation Immunoprecipitation and inVitro Kinase Assay-Aliquots of the nuperiod, the cells were lysed in 1 ml of nuclei preparation buffer (NPB) clear andcytosol fractions (0.5 ml each) were immunoprecipitated with containing 15 mM HEPES, pH 7.5,300 mM sucrose, 60 mM KCI, 15 mM a rabbit antibody (5 pl) prepared against a synthetic peptide correNaCl, 0.5 mM EDTA, 0.5% (dv) Triton X-100, 1mM phenylmethylsulfo- sponding to residues 764-776of the deduced amino acid sequence of nyl fluoride, pg/ml 2 aprotinin, and 10 pg/ml leupeptin, and the cytosolic Xenopus FGFR-1 (3,13).The monoclonal antibody, M2F12, was generand nuclear fractions were prepared domain 50-kDa as described previously (3,11,12). ated against the bacterially expressed extracellular FGFR-1 three immunoglobulin-like disulfide loop antigen, with a speThe nuclear fraction was washed with NPB and resuspended in ml 0.25 of NPB containing0.4% (w/v) SDS, 1mM MgCI, and 10 unitsof DNase cific recognition domain onthe first immunoglobulin-likeloop (residues 1(Promega) for 3 min followed by the addition of 0.75 ml of NPB. The 50-74) as described (14). The various FGFR-1 antibody:FGFR-1 comnuclear extract was incubatedat 37 "C for 5 min and at room temper- plexes were precipitated using protein A-Sepharose beads and washed, ature for a n additional 10 min and centrifuged at 14,000 x g for 10 min. and the invitro kinase reaction initiatedby the incubation of the preof [y-32PlATPas described (3).The productsof the The purityof the nuclear fractions was assessed by the presence of acid cipitates with 10 pCi phosphatase activity in the nuclear fraction, and total cell lysate and in vitro kinase reaction wereresolved by 7.5% (w/v) SDS-polyacrylamless than2% acid phosphatase activity was associated with the nuclearide gel electrophoresis (PAGE); the gel was treated with 1 M KOH fractions. In addition,analysis by transmission electron microscopy (55 "C, 2 h) to remove non-tyrosyl phosphates(15) and thephosphoryl-
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Nuclear Association of Fibroblast Growth Factor Receptor-1
ated proteins visualized by autoradiography. Quantitation of the FGFR-1 bands was performed by densitometry using a Lynx 4000 workstation. Cell I).ansfectwn-"H 3T3 cells and L6 myoblasts were transfected with an expression construct encoding the human FGFR-1 gene (14) according to the recommended protocol from Stratagene. The FGFR-1 cDNA in a pBluescript SK plasmid was digested with BumHI, inserted into the pMEXne0 plasmid (16), and used to transfect NIH 3T3 and L6 cells. The expression of FGFR-1 in the individual transfectants was confirmed by Western blot analysis (molecular mass, -130 kDa) and the ability of the in vitro tyrosine kinase assay to detect the -130-kDa polypeptide as a phosphorylated protein (~130). Confocal ImmunofluorescenceMicroscopy-uiescent NIH 3T3 cells were stimulated with FGF-1 on coverslips, fixed for 3 min in acetone (-80 "C), air-dried, incubated for 1h in pliosphate-buffered saline containing 1% (w/v) bovine serum albumin and 0.1% (v/v) Triton X-100 (Buffer A) and incubated with 10 pg/ml FGFR-1 monoclonal antibody (Santa Cruz Biotechnology, Inc.) for 1 h in Buffer A. The coverslips were washed 3 times with phosphate-buffered saline, incubated for 1h with 10 & mlfluoresceine-conjugatedgoat anti-mouse IgM (Sigma), washed 3 times with phosphate-bufferedsaline, and mounted in 50% (v/v) glycerol on microscope slides. Confocal microscopy of the quiescent and FGF-1-stimulated NIH 3T3 cells was performed with a Odyssey laser confocal microscope (Noran Instruments) usinga 10 pn confocal slit.
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RESULTS ANJJ DISCUSSION
Quiescent NIH 3T3 cells were treated with or without FGF-1, cytosol, and nuclear fractions were prepared and immunoprecipitated with antibodies directed against a synthetic peptide whose sequence is near thecarboxyl terminus of Xenopus FGFR-1 and is conserved inthehumanand murine FGFR-1 proteins (13). The immunoprecipitated pellets were subjected to an in vitro kinaseassay inthe presence of [?PIAT€and ' analyzed by SDS-PAGE followedby KOH treatment in order to hydrolyze and remove non-tyrosyl phosphates (15).In the cytosol fraction of quiescent NIH 3T3 cell monolayers treatedwithout FGF-1, a major phosphotyrosyl-containing band with an apparent molecular mass of 130 kDa was detected (Fig. lA)representing autophosphorylated FGFR-l(l7, 18).However, nophosphorylated p130 was observed associated with the nuclear fraction (Fig. lA).The assignment of p130 as FGFR-1 was determined by the absence of this band in preimmune-treated aliquots (Fig. lA)and by the ability of the synthetic peptide that was used to generate the FGFR-1 antibody to inhibit the immunoprecipitation of cytosol-associated p130 (Fig. 1B).In contrast, treatment of the NIH 3T3 cellmonolayer increase in the with FGF-1 for 2-12 h resulted in a --fold intensity of the p130 band in the cytosol fraction and in the appearance of this band in thenuclear fraction. The autophosphorylated FGFR-1 appeared to be associated with the nuclear fraction after 2 h of treatment with FGF-1 initially as a faint band and after 4 h of exposure to FGF-1 as a prominent p130 band (Fig. LA). The p130 band remained associated with the nuclear fraction 12 h after the addition of FGF-1 (Fig. lA)and represents approximately 10% of the total autophosphorylated FGFR-1 in the FGF-1-treated cells. In addition, densitometry of the phosphorylated p130 band demonstrated that thelevel of the p130 band associated with the nuclear fraction was increased approximately %fold between 2 and 12 h after stimulation with FGF-1. In order to define the intracellular locale of the FGFR-1 protein in NIH 3T3 cells after stimulation withFGF-1, confocal immunofluorescence microscopy was performed. As shown in Fig. ?A,median optical nuclear section of FGFR-1-stained cells revealed diffuse and punctate s t a i n i n g patterns consistent with the presence of FGFR-1 as a membrane-associated protein. In contrast, NIH3T3 cells exposedto FGF-1 for 4 h demonstrated a prominent perinuclear FGFR-1 staining pattern with relatively sparce FGFR-1 staining within the nucleus (Fig. 2B). We also observed a loss of the punctate FGFR-1-staining pattern
c FIG.2. Confocal immunofluorescence microscopy of FGFRl in NIH 3T3 cells. Quiescent (A) and FGF-1-stimulated ( B ) NIH 3T3 cells (4 h with 25 ng/d FGF-1 and 10 unitdmlheparin) were examined by confocalimmunofluorescenceusing a FGFR-1 antibody as described under "Experimental Procedures." Magnificationis x1500, and median nuclear optical sections are shown.
present in NIH 3T3 cellpopulations not exposed to FGF-1, and this may suggest a mobilization of the FGFR-1 protein in response to FGF-1 from this compartment to a juxtanuclear locale. It is unlikely that thepresence of FGFR-1 as a perinuclear protein represents FGFR-1 staining associated with the endoplasmic reticulum-Golgi apparatus since FGFR-1 mRNA synthesis is not modified byFGF-1 in NIH 3T3 cells: and a prominent endoplasmic reticulum-Golgi staining patternfor FGFR-1 is not represented in thequiescent NIH 3T3 cellpopulation. In addition, staining of the FGF-1-stimulated NIH 3T3 cell population with rhodamine-labeled Lens culinaris lectin, a Golgispecific marker, did not colocalize with FGFR-1 staining (data not shown). Thus, the resultsfrom analysis by confocalimmunofluorescencemicroscopy suggest thatthe association of FGFR-1 with the nuclear fraction of FGF-1-stimulated NIH 3T3 cells does not represent intranuclear FGFR-1 but rather juxtanuclear FGFR-1. nuclear association of FGFR-1, In order to further study the NIH 3T3 cells were transfectedwith the cDNA encoding FGFR-1. As shown in Fig. 3, the presence of FGFR-1 was readily detected in thenuclear fraction of FGFR-1 NIH 3T3 cell transfectants in response to FGF-1. In addition, the specific association of the FGFR-1 translation product with the nuclear P. J. Donohue and J.A. Winkles, personal communication.
Nuclear Association Cells:
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FIG.3. Nuclear association of FGFR-1 kinase activity in NM 3T3 cells transfected with FGFR-1. NIH 3T3 cells transfected with the FGFR-1 cDNA were treated with FGF-1(25 mg/ml) and heparin (10 units/ml) for 9 h, and cytosol and nuclear fractions were prepared as described in the legend to Fig. 1. The nuclear pellet was further dissolved in 1ml of NPB (see “Experimental Procedures”) and subjected ultrasonication (10pulses of 0.5 s each) witha microtip (Sonicator,Heat Systems). This extraction method wasemployed instead of nuclei lysis with SDS followed by DNase treatment because the monoclonal antibody M2F12 did not precipitate the FGFR-1 protein in the presence of 0.1% (w/v) SDS. Both cytosol ( C )and nuclear( N )fractions were immunoprecipitatedusingrabbit anti-Xenopus FGFR-1fragment-1antiserum (i), preimmune sera( p ) ,and a monoclonal antibody M2F12 (M2) followed by an in vitro kinase assay and analysis by SDS-PAGE a s described under ”Experimental Procedures.”
fraction was also studied usinga murine monoclonal antibody that specifically recognizes the first immunoglobulin-like loop of human FGFR-1. As shown in Fig. 3, the monoclonal antiFGFR-1 antibody is able to recognize the human FGFR-1 protein as a nuclear-associated enzyme. Thus, our ability to precipitate the p130 band from the nuclear fraction of FGF-1treated NIH 3T3 cells utilizing a monoclonal antibody, which is not only specific for the humanFGFR-1 polypeptide but which is also able to discriminate FGFR-1 from the three additional members of the FGF receptor gene family, argues that this nuclear-associated phosphotyrosine-containingp130 protein is FGFR-1. Rat L6 myoblasts were also used to study the FGF-1-induced nuclear association of FGFR-1 sincethese cells, unlike theNIH 3T3 cells, have been characterized with a poor proliferative response to exogenous FGF-1, and thesecells express relatively low FGFR-1 levels (19). We anticipated that transfectionof L6 cells with the p130 FGFR-1 cDNA would enable us to determine whether the proliferative response to FGF-1 correlates with the nuclear association of the FGFR-1 protein. Thus, the presence of FGFR-1 in the cytosol and nuclear fractions of quiescent (41 h in low serum) and FGF-1-stimulated (11 h) normal and FGFR-1-transfected L6 cells was examined using the in vitro tyrosine kinase assay. While wild-type L6 cells contained p130 band in thecytosol fraction, it was not possible to detect thep130 band within the nuclear fraction (Fig. 4). In contrast with theNIH 3T3 cell data, the stimulationof the L6 cells with FGF-1 did not result in the association of the endogenous p130 band with the nuclear fraction, and the wild-type L6 cells were not able to initiateDNA synthesis inresponse to
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49.5 FIG.4. FGFR-1 tyrosine kinase activity in L6 cells. Quiescent L6 cells were stimulated with (+) or without (-) 25 ng/ml of FGF-1 and 10 unitdm1 of heparin for 11 h. The assay for FGFR-1 tyrosine kinase activity in cytosol ( C ) and nuclear ( N ) fractions of L6 cells was performed as described under “Experimental Procedures” using the rabbit anti-Xenopus FGFR-1 anti-serum (i) or preimmune serum ( p ) a s a control.
FGF-1 (data notshown). However, both FGF-1-stimulated and quiescent L6 cells exhibited a weak band associated with the nuclear fraction corresponding to a phosphoprotein with amolecular massof approximately 145 kDa (p145),which was precipitated by the anti-FGFR-1 antibodies (Fig. 4). FGFR-1-transfected L6 cells, which were able to proliferate under serum-free conditions, exhibit morphologic changes and demonstrate a moderate increase in DNA synthesis inresponse to FGF-1; these cells also presented prominentp130 and p145 bands both in immunoblots with anti-FGFR-1 antibodies and in the in vitro kinase assay. Further analysis demonstrated that both FGF-1-stimulated and unstimulated FGFR-l-transfected L6 cells exhibited p130 and p145 bands associated with the nuclearfraction (Fig.5). The p145 was also readily detected by immunoblot analysisusing FGFR-1 antibody (data not shown). Interestingly, the intensityof the p130 and p145 bands was not significantly affected by stimulation with exogenous FGF-1, and thismay be related to theinability of these cells to become quiescent under low serum or serum-free conditions (data not shown). While the significance of the p145 band is unclear, it mayrepresentanalternativeposttranslational modification of FGFR-1. Our data suggest thatFGF-1 can induce the association of the FGFR-1 with the nuclear fraction derived from FGF-1stimulated cells. It is interesting that the association of the p130 protein with the nuclear fraction of NIH 3T3 cells requires 2-4 h exposure of the cell culture system to FGF-1 because prior studies have demonstrated that FGF-1 is continuously
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FIG.5. FGFR-1 tyrosine kinase activityin L6 cells transfected with the FGFR-1 cDNA (L6-pXZ106 cells). L6-pXZ106 cells were of stimulated with (+) or without (-) 25 ng/ml of FGF-1 and 10 unitdm1 heparin for 11 h. The assay of FGFR-1-related tyrosine kinase activity in cytosol ( C ) and nuclear (N)fractions of L6-pXZ106 cells was performed a s described under "Experimental Procedures."
Factor Receptor-1
ficking of the FGFR-1 protein is regulated by FGF-1, The observation that FGFR-1 exists as a structurally intact and functional intracellular protein during the entire G, period of the NIH 3T3 cell cycle implies that it may play an intracellular role near thenucleus as an enzymatic modifier of proteins involved in the regulation ofDNA synthesis in response to FGF-1. Although c-Src and theF-actin-binding protein, cortactin arephosphorylated in a biphasic manner during theimmediate-early and mid to late G, phase of the NIH 3T3 cell cycle (3,4,7), we do not know whether these proteins are involved in the regulation of FGFR-1 traffic. However, our data are consistent with the observation that thePDGF receptor-P is able to undergo a transition to a juxtanuclear locale in response to PDGF in HepG2 cells (20). While the perinuclear trafficking of PDGF receptor+ involves the association of PDGF-p with the catalytic subunit of phosphatidylinositol 3-kinase (201, it is presently unclear whetherphosphatidylinositol-3 kinase is involved in the intracellulartrafficking of FGFR-1(3). Likewise, it has recently been reported that thecytosolic tyrosine kinase domain of the neu protooncogene, p185"'", a member of the epidermal growth factor receptor family, is able to function as a n activator of transcription in the yeast two hybrid system (21) and that ~185"'" associated is with the nuclearfraction of neutransfected NIH 3T3 cells as a phospotyrosine-containingprotein (22). Even though the ~185"'" studydid not examine the ligand dependence of ~185"'"trafficking (221, i t serves to reinforce the premise that the intracellular traffic of structurally intact and enzymatically functional receptor tyrosine kinases may be involved in cell cycle regulation. Acknowledgments-We thank K. Wawzinski for expert secretarial support andC. C. Haudenschild (Holland Laboratory)for transmission electron microscopy analysis of the nuclear fractions and use of the confocal fluorescence microscope. REFERENCES 1. Burgess, W. H., and Maciag, T. (1989) Annu. Rev. Biochem. 58,575-606 2. Jaye, M., Schlessinger, J., and Dionne, C.A. (1992) Biochim. Biophys. Acfa 1135, 185-199 3. Zhan, X., Hu, X., Friesel, R., and Maciag, T. (1993) J. Biol. Chem. 268,96119620 4. Zhan, X., Hu, X., Hampton, B., Burgess, W. H., Friesel, R., and Maciag, T. (1993) J. Biol. Chem. 268,24427-24431 5. Wong, S., Reynolds, A. B., and Rapkoff, J. (1992) Oncogene 7,2407-2415 6. Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge,S. (1993) Proc. Natl. Acad. Sci. U. S. A. 91,76967700 7. Zhan, X..Plourde, C., Hu, X., Friesel, R., and Maciag, T. (1994)J. Biol. Chem. 269,20221-20224 8. Imamura, T., Engleka, K., Zhan, X., Tokita, Y.,Forough, R., Roeder, D., Jackson, A., Maier, J. A.M., Hla, T., and Maciag, T. (1990) Science 249,
translocated from the surface of the BALBlc 3T3 cell to the nucleus during the entire G, phase of the cell cycle (3). Likewise, the efficiency of the association of p130 protein with the nuclear fraction(approximately 10%) agrees well with the amount of FGF-1 that is able to undergo receptor-dependent nuclear translocation (3). It is also likely that our failure to detect the p130 protein associated with the nuclear fraction IF. M. - IW..~. prior to 2 hafter theaddition of FGF-1 to NIH 3T3 cells may be . 9. Bouche, G., Gas, N., Prats, H., Baldin, V., Tauber, J.-P., Teissie, J., a n d h a l r i c , a consequence of the sensitivity of our detection system. I t is F. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,6770-6774 also interesting that the p130 protein is associated with the 10. Ackland, P., Dixon, M., Peters, G., and Dickson, C. (199O)Nuture343,662-665 functional tyrosine 11. Friedman, S., Zhan, X., and Maciag, T. (1992) Biochem.Biophys.Res. nuclear fraction as a structurally intact and Commum. 198,1203-1208 kinase during the mid to late G, phase of the NIH 3T3 cell 12. Baldin, V.,Roman, A.-M., Bosc-Bierne, I., Amalric, F., and Bouche, G. (1990) EMBO J. 9, 1511-1517 cycle. Although we do not know the functional consequences of Friesel, R., and Dawid, I. B. (1991) Mol. Cell. Biol. 11,2481-2488 13. the association of the p130 band with the nuclear fraction of 14. Xu, J., Nakahara, M., Crabb, J. W., Shi, E., Matuo, Y., Fraser, M.,Kan, M., FGF-1-induced cells, it is unfortunate thatwe were unable to Hou, J., andMcKeehan, W. L. (1992) J. Biol. Chem. 267,17792-17803 association 15. Cooper, J. A,, Sefton, B.M., and Hunter, T. (1983) MpthodsEnzymol. 99, utilize the rat L6 myoblast cell line to correlate the 387402 of the p130 protein with the nuclearfraction and the abilityof 16. Martin-Zanca, D., Oskam, R., Mitra, G., Copeland, T., and Barbacid,M.(1989) FGF-1 to regulateDNA synthesis in theFGFR-1 L6cell transMol. Cell. Biol. 9, 24-33 Huang, S. S., and Huang, J. S. (1986) J. Bid. Chem. 261,9568-9571 fectants. These efforts were compromised by the ability of the 17. 18. Friesel, R., Burgess, W. H., and Maciag, T. (1989)Mol. Cell. Biol. 9,1857-1865 various FGFR-1 L6 transfectants togrow not only in thepres- 19. Olwin, B. B.. and Hnuschkn, S.D.(1989) J. Cell. Biochem. 39,443454 ence of low levels of serum but also under serum-free condi- 20. Joly, M., Kazlauskas, A., Fay, F. S., and Corvera, S. (1994) Science 263,684f". iA7 tions. However, our data suggest that p130 the FGFR-1 protein 21. Fields, S., and Song, 0.(1989) Nature 340,245-246 exists as a juxtanuclear protein and that the intracellular traf-22. Xie, Y.,and Hung, M.-C. (1994)Biochem.Biophys. Res. Comm. 203,1589-1598
