Abstract
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.
Highlights
Thepurification and characterization of proteins phosphorylated on tyrosine residues during mid to late GI in response to FGleFd-1to 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 facto(rPDGF)-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 fibroblast growth factor-1 (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]
To inhibit the immunoprecipitation of cytosol-associated p130 (Fig. 1B).In contrast, treatment of the NIH 3T3 cellmonolayer present in NIH 3T3 cellpopulations not exposed to FGF-1, and with FGF-1 for 2-12 h resulted in a --fold increase in the this may suggest a mobilization of the FGFR-1 protein in reintensity of the p130 band in the cytosol fraction and in the sponse to FGF-1 from this compartment to a juxtanuclear loappearance of this band in thenuclear fraction
Summary
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-1treatment. .FGF-I-induced FGFR-I tyrosine-kinase activity in nuclear fractions ofNIH 3T3ce1ls.A. quiescent NIH
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