S100A13, a new marker of angiogenesis in human astrocytic gliomas.

S100 proteins are Ca(2+)-binding polypeptides involved in the tumourigenesis of several human neoplasms. S100A13 is a key regulator of the stress-dependent release of FGF1, the prototype of the FGF protein family involved in angiogenesis. Indeed, S100A13 is a copper binding protein able to enhance the export of FGF1 in response to stress in vitro and to induce the formation of a multiprotein aggregate responsible for FGF1 release. We investigated the expression of S100A13 in human astrocytic gliomas in relation to tumour grading and vascularization. A series of 26 astrocytic gliomas was studied to evaluate microvessel density and to assess FGF1, S100A13 and VEGF-A expression. FGF1 was equally expressed in the vast majority of tumours, whereas S100A13 and VEGF-A were significantly up-regulated in high-grade vascularized gliomas. Moreover, both S100A13 and VEGF-A expression significantly correlated with microvessel density and tumour grading. These data suggest that the up-regulation of S100A13 and VEGF-A expression correlates with the activation of angiogenesis in high-grade human astrocytic gliomas.

The alternative translation of synaptotagmin 1 mediates the non-classical release of FGF1.

Although the extravesicular p40 domain of the transmembrane protein, p65 synaptotagmin (Syt) 1, is essential for the non-classical export of the signal peptide-less structure, FGF1, it was not possible to identify a specific intracellular protease responsible for the processing of p65 Syt1. Surprisingly, analysis of the p65 Syt1 coding sequence revealed the presence of two potential alternative ATG codons corresponding to Met103 and Met113 both of which were flanked by Kozak sequences. Indeed, in vitro translation of a Met103Ile but not a Met113Ile p65 Syt1 point mutant exhibited reduced expression of p40 Syt1 and the double p65 Syt1 Met103Ile and Met113Ile point mutant was unable to translate the p40 Syt1 isoform. Since the expression of the p65 Syt1 double point mutant inhibited the stress-induced release of FGF1, it is likely that the alternative translation of the p65 Syt1 transcript at Met103 may be involved in the generation of intracellular p40 Syt1, a critical component of the FGF1 release pathway.

Members of the Jagged/Notch gene families are expressed in injured arteries and regulate cell phenotype via alterations in cell matrix and cell-cell interaction.

The Jagged/Notch signaling pathways control cell fate determination and differentiation, and their dysfunction is associated with human pathologies involving cardiovascular abnormalities. To determine the presence of these genes during vascular response to injury, we analyzed expression of Jagged1, Jagged2, and Notch1 through 4 after balloon catheter denudation of the rat carotid artery. Although low levels of Jagged1, Jagged2, and constitutive expression of Notch1 were seen in uninjured endothelium, expression of all was significantly increased in injured vascular cells. High Jagged1 expression was restricted to the regenerating endothelial wound edge, whereas Notch transcripts were abundant in endothelial and smooth muscle cells. To understand the basis for Jagged/Notch control of cellular phenotype, we studied an in vitro model of NIH3T3 cells transfected with a secreted form of the extracellular domain of Jagged1. We report that the soluble Jagged1 protein caused decreased cell-matrix adhesion and cell migration defects. Cadherin-mediated intercellular junctions as well as focal adhesions were modified in soluble Jagged1 transfectants, demonstrating that cell-cell contacts and adhesion plaques may be targets of Jagged/Notch activity. We suggest that Jagged regulation of cell-cell and cell-matrix interactions may contribute to the control of cell migration in situations of tissue remodeling in vivo.

The comparative release of FGF1 by hypoxia and temperature stress.

The signal peptide-less FGF gene family prototype, FGF1 is released in response to temperature stress in vitro as a latent reducing agent-sensitive homodimer non-covalently complexed with the extravesicular p40 domain of p65 synaptotagmin (Syt)1. Because FGF1 is well recognized as an angiogenesis factor in vivo and angiogenesis is known to be induced by hypoxia, we examined the release of FGF1 and p40 Syt1 under conditions of hypoxia and temperature stress using a chemostatic microcarrier cell culture system. We report that like the pathway used by FGF1 and p40 Syt1 release under temperature stress, hypoxia also induces the release of FGF1 and p40 Syt1 with similar kinetic and pharmacologic properties including the requirement for functional cysteine residues. Lastly, FGF1 and p40 Syt1 release in response to hypoxia and temperature stress is sensitive to lipoxygenase and cyclooxygenase inhibitors suggesting that arachidonic acid metabolism may play an important role in the mechanism of FGF1 release in vitro.

Copper induces the assembly of a multiprotein aggregate implicated in the release of fibroblast growth factor 1 in response to stress.

Fibroblast growth factor (FGF) 1 is known to be released in response to stress conditions as a component of a multiprotein aggregate containing the p40 extravescicular domain of p65 synaptotagmin (Syt) 1 and S100A13. Since FGF1 is a Cu2+-binding protein and Cu2+ is known to induce its dimerization, we evaluated the capacity of recombinant FGF1, p40 Syt1, and S100A13 to interact in a cell-free system and the role of Cu2+ in this interaction. We report that FGF1, p40 Syt1, and S100A13 are able to bind Cu2+ with similar affinity and to interact in the presence of Cu2+ to form a multiprotein aggregate which is resistant to low concentrations of SDS and sensitive to reducing conditions and ultracentrifugation. The formation of this aggregate in the presence of Cu2+ is dependent on the presence of S100A13 and is mediated by cysteine-independent interactions between S100A13 and either FGF1 or p40 Syt1. Interestingly, S100A13 is also able to interact in the presence of Cu2+ with Cys-free FGF1 and this observation may account for the ability of S100A13 to export Cys-free FGF1 in response to stress. Lastly, tetrathiomolybdate, a Cu2+ chelator, significantly represses in a dose-dependent manner the heat shock-induced release of FGF1 and S100A13. These data suggest that S100A13 may be involved in the assembly of the multiprotein aggregate required for the release of FGF1 and that Cu2+ oxidation may be an essential post-translational intracellular modifier of this process.

Soluble Jagged 1 represses the function of its transmembrane form to induce the formation of the Src-dependent chord-like phenotype.

We have previously demonstrated that the expression of the soluble extracellular domain of the transmembrane ligand for Notch receptors, Jagged 1 (sJ1), in NIH 3T3 cells results in the formation of a matrix-dependent chord-like phenotype, the loss of contact inhibition of growth, and an inhibition of pro-alpha 1(I) collagen expression. In an effort to define the mechanism by which sJ1 induces this phenotype, we report that sJ1 transfectants display biochemical and cytoskeletal alterations consistent with the activation of Src. Indeed, cotransfection of sJ1 transfectants with a dominant-negative mutant of Src resulted in the loss of matrix-dependent chord formation and correlated with the restoration of type I collagen expression and contact inhibition of growth. We also report that the sJ1-mediated induction of Src activity and related phenotypes, including chord formation, may result from the inhibition of endogenous Jagged 1-mediated Notch signaling since it was not possible to detect an sJ1-dependent induction of CSL-dependent transcription in these cells. Interestingly, NIH 3T3 cells transfected with dominant-negative (but not constitutively active) mutants of either Notch 1 or Notch 2 displayed a similar Src-related phenotype as the sJ1 transfectants. These data suggest that the ability of sJ1 to mediate chord formation is Src-dependent and requires the repression of endogenous Jagged 1-mediated Notch signaling, which is tolerant to the destabilization of the actin cytoskeleton, a mediator of cell migration.

S100A13 participates in the release of fibroblast growth factor 1 in response to heat shock in vitro.

S100A13, a member of the S100 gene family of Ca(2+)-binding proteins has been previously characterized as a component of a brain-derived heparin-binding multiprotein aggregate/complex containing fibroblast growth factor 1 (FGF1). We report that while expression of S100A13 in NIH 3T3 cells results in the constitutive release of S100A13 into the extracellular compartment at 37 degrees C, co-expression of S100A13 with FGF1 represses the constitutive release of S100A13 and enables NIH 3T3 cells to release S100A13 in response to temperature stress. S100A13 release in response to stress occurs with kinetics similar to that observed for the stress-induced release of FGF1, but S100A13 expression is able to reverse the sensitivity of FGF1 release to inhibitors of transcription and translation. The release of FGF1 and S100A13 in response to heat shock results in the solubility of FGF1 at 100% (w/v) ammonium sulfate saturation, and the expression of a S100A13 deletion mutant lacking its novel basic residue-rich domain acts as a dominant negative effector of FGF1 release in vitro. Surprisingly, the expression of S100A13 also results in the stress-induced release of a Cys-free FGF1 mutant, which is normally not released from NIH 3T3 cells in response to heat shock. These data suggest that S100A13 may be a component of the pathway for the release of the signal peptide-less polypeptide, FGF1, and may involve a role for S100A13 in the formation of a noncovalent FGF1 homodimer.

The precursor but not the mature form of IL1alpha blocks the release of FGF1 in response to heat shock.

Interleukin (IL)1alpha mediates proinflammatory events through its extracellular interaction with the IL1 type I receptor. However, IL1alpha does not contain a conventional signal peptide sequence that provides access to the endoplasmic reticulum-Golgi apparatus for secretion. Thus, we have studied the release of the precursor (p) and mature (m) forms of IL1alpha from NIH 3T3 cells. We have demonstrated that mIL1alpha but not pIL1alpha was released in response to heat shock with biochemical and pharmacological properties similar to those reported for the stress-mediated release pathway utilized by fibroblast growth factor (FGF)1. However, unlike the FGF1 release pathway, the IL1alpha release pathway appears to function independently of synaptotagmin (Syt)1 because the expression of a dominant-negative form of Syt1, which represses the release of FGF1, did not inhibit the release of mIL1alpha in response to temperature stress. Interestingly, whereas the expression of both mIL1alpha and FGF1 in NIH 3T3 cells did not impair the stress-induced release of either polypeptide, the expression of both pIL1alpha and FGF1 repressed the release of FGF1 in response to temperature stress. These data suggest that the release of mIL1alpha requires proteolytic processing of its precursor form and that mIL1alpha and FGF1 may utilize similar but distinct mechanisms for export.

Amlexanox reversibly inhibits cell migration and proliferation and induces the Src-dependent disassembly of actin stress fibers in vitro.

Amlexanox binds S100A13 and inhibits the release of fibroblast growth factor 1 (FGF1). Because members of the S100 gene family are known to be involved with the function of the cytoskeleton, we examined the ability of amlexanox to modify the cytoskeleton and report that amlexanox induces a dramatic reduction in the presence of actin stress fibers and the appearance of a random, non-oriented distribution of focal adhesion sites. Correspondingly, amlexanox induces the complete and reversible non-apoptotic inhibition of cell migration and proliferation, and although amlexanox does not induce either the down-regulation of F-actin levels or the depolymerization of actin filaments, it does induce the tyrosine phosphorylation of cortactin, a Src substrate known to regulate actin bundling. In addition, a dominant negative form of Src is able to partially rescue cells from the effect of amlexanox on both the actin cytoskeleton and cell migration. In contrast, the inhibition of cell proliferation by amlexanox correlates with the inhibition of cyclin D1 expression without interference of the receptor tyrosine kinase/mitogen-activated protein kinase signaling pathway. Last, the ability of amlexanox to inhibit FGF1 release is reversible and correlates with the restoration of the actin cytoskeleton, suggesting a role for the actin cytoskeleton in the FGF1 release pathway.

A non-transmembrane form of Jagged-1 regulates the formation of matrix-dependent chord-like structures.

Jagged-Notch interactions regulate a transmembrane ligand-receptor signaling pathway involved in the regulation of cell fate determination as well as myoblast and endothelial cell differentiation. To further examine the role of the transmembrane ligand, Jagged-1, in the regulation of cell differentiation, we stably transfected NIH 3T3 cells with a truncated form of Jagged(J)-1, which results in the secretion of a soluble(s) form of the protein. Comparison of gene expression by serial analysis demonstrated that among the 227 transcripts differentially regulated in the sJ-1 transfectants, the expression of the pro-alpha-2(I) collagen transcript and pro-alpha-1(I) collagen translation product was predominantly repressed in sJ-1 transfectants. When plated on extracellular matrices, sJ-1 transfectants formed prominent chord-like structures on type I collagen but not on fibrin, fibronectin, or vitronectin. While the sJ-1 transfectants exhibited growth kinetics similar to control cells and were unable to grow in soft agar, the cells were less sensitive to contact inhibition of growth in vitro and sJ-1 allografts formed tissue masses in nude mice after a prolonged latency period and exhibited an abundance of host-derived microvascular endothelial cells. These data suggest that J-1 may be able to modulate, in a matrix-dependent manner, the organization of cell to cell interactions including its ability to promote the development of chord-like structures.

Telomerase activity of cells during alterations of their proliferative states.

We showed that the telomerase activity (TAC) of human promyelocytic leukemic cells U-937 and HL-60 sharply decreased after induction of macrophagal differentiation. Dedifferentiation which occurred several days after removing the inductor was accompanied by the resumption of proliferation and increase of TAC. Telomerase activity significantly decreased also when U-937 cells ceased to proliferate in response to long-term inhibition of the telomerase function by azidothymidine. TAC was observed to decrease slowly for 6-8 days in the course of transition of mouse fibroblasts 3T3 Swiss to the quiescent state. TAC decreased both in serum-deprived cells and in slowly proliferating high-density inhibited cells. During the exit from quiescence and in dedifferentiation, the increase in TAC preceded the resumption of proliferation. In all the cases described the alterations of TAC correlated with alterations in the nonspecific polymerase activity which we had found earlier (D. N. Chernov, Y. E. Yegorov, and S. S. Akimov, DokL Biochemistry 349:55-58 (1996)). The problems of TAC regulation are discussed.

Endogenous beta-galactosidase activity in continuously nonproliferating cells.

Cytochemically detectable activity of endogenous beta-galactosidase was found at pH 6.0 in Swiss 3T3 cells after long-term incubation in low serum or in the presence of heparin concentrations known to reversibly inhibit cell proliferation. A high percentage of beta-galactosidase-positive cells were detected in U937 and HL60 cultures at the late stage of macrophage-like differentiation induced by TPA. Interestingly, a small number of beta-galactosidase-positive cells were found even in the growing Swiss 3T3 cultures. These positive cells expressed morphological features similar to those of senescent cells. Thus, the activity of beta-galactosidase at pH 6.0 cannot be considered an exclusive marker of senescent cells since it is expressed in other types of nonproliferating cells.

Activation of the MAP kinase pathway by FGF-1 correlates with cell proliferation induction while activation of the Src pathway correlates with migration.

FGF regulates both cell migration and proliferation by receptor-dependent induction of immediate-early gene expression and tyrosine phosphorylation of intracellular polypeptides. Because little is known about the disparate nature of intracellular signaling pathways, which are able to discriminate between cell migration and proliferation, we used a washout strategy to examine the relationship between immediate-early gene expression and tyrosine phosphorylation with respect to the potential of cells either to migrate or to initiate DNA synthesis in response to FGF-1. We demonstrate that transient exposure to FGF-1 results in a significant decrease in Fos transcript expression and a decrease in tyrosine phosphorylation of the FGFR-1, p42(mapk), and p44(mapk). Consistent with these biochemical effects, we demonstrate that attenuation in the level of DNA synthesis such that a 1.5-h withdrawal is sufficient to return the population to a state similar to quiescence. In contrast, the level of Myc mRNA, the activity of Src, the tyrosine phosphorylation of cortactin, and the FGF-1-induced redistribution of cortactin and F-actin were unaffected by transient FGF-1 stimulation. These biochemical responses are consistent with an implied uncompromised migratory potential of the cells in response to growth factor withdrawal. These results suggest a correlation between Fos expression and the mitogen-activated protein kinase pathway with initiation of DNA synthesis and a correlation between high levels of Myc mRNA and Src kinase activity with the regulation of cell migration.

Intracellular precursor interleukin (IL)-1alpha, but not mature IL-1alpha, is able to regulate human endothelial cell migration in vitro.

The human umbilical vein endothelial cell (HUVEC) has a finite lifespan in vitro, and senescent HUVEC contain elevated levels of the negative growth regulator interleukin (IL)-1alpha. IL-1alpha is translated as a signal peptide sequence-less cytosolic 31-kDa precursor (IL-1alpha p), which undergoes proteolytic activation to release the mature carboxyl terminus 17-kDa protein (IL-1alpha m). Both the IL-1alpha p and IL-1alpha m proteins are biologically active as exogenous cytokines. Interestingly, only IL-1alpha p contains a nuclear localization sequence between residues 79 and 85. To further study the role of intracellular IL-1alpha in the regulation of human endothelial cell function, a spontaneous HUVEC transformant was stably transfected with IL-1alpha p, IL-1alpha m, and the IL-1alpha p K82N mutant, which attenuates the nuclear traffic of IL-1alpha p. Interestingly, the IL-1alpha p transfectants were found to have a lower migratory potential than either IL-1alpha m or IL-1alpha p K82N transfectants, and the addition of the IL-1 receptor antagonist did not alter the migration of these cells. Immunofluorescence microscopy demonstrated that only the IL-1alpha p transfectants exhibited prominent staining for beta-catenin-associated cell-to-cell contacts, as well as pronounced vimentin intermediate filaments and actin cytoskeleton staining. These data suggest that IL-1alpha p, and not IL-1alpha m, may function as an intracellular regulator of the migratory capacity of the human endothelial cell and that the nuclear localization sequence present within IL-1alpha p may be involved in regulating this function.

Opposite effects of cell differentiation and apoptosis on Ap3A/Ap4A ratio in human cell cultures.

The biological role of diadenosine oligophosphates (DAOP) remains obscure in spite of numerous attempts to solve this enigma. It is known that Ap3A contrary to Ap4A accumulates in human cultured cells treated with interferons (IFNs) alpha or gamma. Since IFNs are considered as antiproliferative regulators, we assumed that different cell status may be associated with varying intracellular levels of DAOP. Promyelocytic human cell line HL60 induced by phorbol ester (TPA) to differentiate to macrophage-like cells in culture exhibits a profound loss of proliferative potential. Here we have shown a 4-5-fold increase in Ap3A concentration in HL60 cells induced by TPA, similar to the effect of IFN, while the Ap4A concentration remained unchanged. On the contrary, in cells undergoing apoptosis induced by VP16, a topoisomerase II inhibitor, the Ap3A concentration considerably decreased, while the Ap4A concentration increased. These findings combined with earlier results suggest an involvement of the Ap3A/Ap4A ratio in signal transduction pathways controlling the cell status.

Differential effects of phorbol ester on signaling and gene expression in human leukemia cells.

Human U937 myeloid leukemia cells were treated with different concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) to determine signals that contribute to growth arrest and differentiation. While 0.5 nM TPA had little if any effect, exposure of U937 cells to higher TPA concentrations (5-500 nM) revealed a complete growth arrest after 48 h. Cytosolic PKC activity decreased by 50% after exposure to 0.5 nM TPA and by 80 and 95% after stimulation with 5 nM and 50 nM TPA, respectively. Simultaneously, the PKC activity in the particulate fraction of U937 cells increased accordingly. These events were associated with induction of a differentiated monocytic phenotype. Expression of the c-myc gene was down-regulated and c-jun and c-fms transcripts increased following exposure to 5-500 nM TPA. In contrast, exposure to 0.5 nM TPA decreased c-myc expression and increased c-jun transcripts only transiently between 4 and 8 h while little if any effect was detectable on c-fms mRNA expression and subsequent differentiation. Taken together, these data suggest that a certain initial threshold of PKC activation is required for induction of a differentiated monocytic phenotype while beyond this threshold, a growth-arrested and differentiated state in these human leukemic cells can be maintained regardless of TPA concentrations.

FGF-1-dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothelial cells and correlate with the inability to signal tyrosine phosphorylation of fibroblast growth factor receptor-1 substrates.

Senescent cells do not proliferate in response to exogenous growth factors, yet the number and affinity of growth factor receptors on the cell surface appear to be similar to presenescent cell populations. To determine whether a defect in receptor signaling exists, we analyzed human umbilical vein endothelial cells (HUVEC) since HUVEC growth is absolutely dependent upon the presence of FGF. We report that in both presenescent and senescent HUVEC populations, FGF-1 induces the expression of cell cycle-specific genes, suggesting that functional FGF receptor (FGFR) may exist on the surface of these cells. However, the tyrosine phosphorylation of FGFR-1 substrates, Src and cortactin, is impaired in senescent HUVEC, and only the presenescent cell populations exhibit a FGF-1-dependent Src tyrosine kinase activity. Moreover, we demonstrate that senescent HUVEC are unable to migrate in response to FGF-1, and these data correlate with an altered organization of focal adhesion sites. These data suggest that the induction of gene expression is insufficient to promote a proliferative or migratory phenotype in senescent HUVEC and that the attenuation of the FGFR-1 signal transduction pathway may be involved in the inability of senescent HUVEC to proliferate and/or migrate.

The nuclear trafficking of extracellular fibroblast growth factor (FGF)-1 correlates with the perinuclear association of the FGF receptor-1alpha isoforms but not the FGF receptor-1beta isoforms.

The alternatively spliced fibroblast growth factor receptor (FGFR)-1 isoforms, FGFR-1alpha and FGFR-1beta, are characterized by the presence of either three or two Ig-like loops in the extracellular domain and are differentially expressed during embryonic development and tumor progression. We have previously shown that in cells irreversibly committed to DNA synthesis by FGF-1, approximately 15% of cell surface FGFR-1 traffics to a perinuclear locale as a structurally intact and functional tyrosine kinase (Prudovsky, I., Savion, N., Zhan, X., Friesel, R., Xu, J., Hou, J., McKeehan, W. L., and Maciag, T. (1994) J. Biol. Chem. 269, 31720-31724). In order to define the structural requirement for association of FGFR-1 with the nucleus, the expression and trafficking of FGFR-1 in FGFR-1alpha and FGFR-1beta L6 myoblast transfectants was studied. Although FGFR-1alpha was expressed as p145 and p125 forms, FGFR-1beta was expressed as p120 and p100 forms in the L6 myoblast transfectants. Tunicamycin and N-glyconase experiments suggest that these forms of FGFR-1alpha and FGFR-1beta are the result of differential glycosylation. However, only the p145 form of FGFR-1alpha and the p120 form of FGFR-1beta were able to bind FGF-1 and activate tyrosine phosphorylation. Pulse-chase analysis of FGFR-1 biosynthesis suggests that the p125 and p100 proteins are the precursor forms of p145 FGFR-1alpha and p120 FGFR-1beta, respectively. Because ligand-chase analysis demonstrated that FGFR-1beta L6 myoblast transfectants exhibited a reduced efficiency of nuclear translocation of exogenous FGF-1 when compared with FGFR-1alpha transfectants, the intracellular trafficking of the FGFR-1alpha and FGFR-1beta isoforms was studied using an in vitro kinase assay to amplify immunoprecipitated FGFR-1. Indeed, the appearance of the FGFR-1alpha but not FGFR-1beta isoform in the nuclear fraction of L6 myoblast transfectants suggests that the distal Ig-like loop in FGFR-1alpha mediates the differential nuclear association of FGFR-1alpha as a structurally intact and functional tyrosine kinase. Further, the FGFR-1beta L6 myoblast transfectants but not the FGFR-1alpha myoblast transfectants exhibited a pronounced morphologic change in response to exogenous FGF-1. Because this phenotype change involves the induction of a rounded cellular shape, it is possible that the FGFR-1alpha and FGFR-1beta may ultimately exhibit differential trafficking to adhesion sites.

Intact and functional fibroblast growth factor (FGF) receptor-1 trafficks near the nucleus in response to FGF-1.

Exogenous fibroblast growth factor-1 (FGF-1) associates with the nucleus in a receptor-dependent manner during the entire G1 period of the BALB/c 3T3 cell cycle (Zhan, X., Hu, X., Friesel, R., and Maciag, T. (1993) J. Biol. Chem. 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-kDa phosphotyrosine-containing protein associated with the nuclear fraction of NIH 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 NIH 3T3 cells to FGF-1, this activity appeared to be maximal in the nuclear fraction between 4 and 12 h after FGF-1 treatment. In addition, analysis by confocal immunofluorescence microscopy of quiescent and FGF-1-stimulated NIH 3T3 cells reveal a prominent perinuclear FGFR-1 staining pattern in the cells exposed to FGF-1 but not in the quiescent population. We also observed FGFR-1 associated with the nuclear fraction in FGFR-1-transfected L6 rat myoblasts, which are known to be refractive to exogenous FGF-1 and express relatively low levels of endogenous FGFR-1. In addition, these cells also exhibited the presence of a 145-kDa phosphoprotein in the nuclear fraction that was recognized by FGFR-1 antibodies. These results suggest that the FGFR-1 may be translocated near the nucleus upon interaction with its ligand during the entire G1 period of the NIH 3T3 cell cycle as a structurally intact and functional tyrosine kinase that may be accessible to perinuclear polypeptides as a regulatory enzyme.