Fibroblast growth factor receptor 1 (FGFR1) is a transmembrane protein capable of transducing stimulation by secreted FGFs. In addition, newly synthesized FGFR1 enters the nucleus in response to cellular stimulation and during development. Nuclear FGFR1 can transactivate CRE (cAMP responsive element), activate CRE-binding protein (CREB)-binding protein (CBP) and gene activities causing cellular growth and differentiation. Here, a yeast two-hybrid assay was performed to identify FGFR1-binding proteins and the mechanism of nuclear FGFR1 action. Ten FGFR1-binding proteins were identified. Among the proteins detected with the intracellular FGFR1 domain was a 90-kDa ribosomal S6 kinase (RSK1), a regulator of CREB, CBP, and histone phosphorylation. FGFR1 bound to the N-terminal region of RSK1. The FGFR1-RSK1 interaction was confirmed by co-immunoprecipitation and colocalization in the nucleus and cytoplasm of mammalian cells. Predominantly nuclear FGFR1-RSK1 interaction was observed in the rat brain during neurogenesis and in cAMP-stimulated cultured neural cells. In TE671 cells, transfected FGFR1 colocalized and coimmunoprecipitated, almost exclusively, with nuclear RSK1. Nuclear RSK1 kinase activity and RSK1 activation of CREB were enhanced by transfected FGFR1. In contrast, kinase-deleted FGFR1 (TK؊), which did not bind to RSK1 failed to stimulate nuclear RSK1 activity or RSK1 activation of CREB. Kinase inactive FGFR1 (K514A) bound effectively to nuclear RSK1, but it failed to stimulate RSK1. Thus, active FGFR1 kinase regulates the functions of nuclear RSK1. The interaction of nuclear FGFR1 with pluripotent RSK1 offers a new mechanism through which FGFR1 may control fundamental cellular processes.Fibroblast growth factor receptor 1 (FGFR1), 1 like other single transmembrane growth factor receptors, transduces signals across the cell membrane generated by extracellular ligands (1). Binding of secreted FGFs causes FGFR1 molecules to dimerize and undergo cross-phosphorylation (2, 3). The phosphotyrosine residues serve as docking sites for the SH2 (Src homology 2) or phosphotyrosine-binding domains of cytoplasmic proteins, which initiate downstream signaling cascades. A number of proteins that interact with the cytosolic, C-terminal portion of FGFR1 have been identified, including phospholipase C␥ (3, 4), adaptor proteins Shc (5), Grb14 (6), the regulatory subunit (p85) of the phosphatidylinositol 3Ј-kinase (PI3K), and NCK (7). However, binding of the FGF receptor substrate 2 to FGFR1 occurs independent of ligand stimulation and tyrosine phosphorylation (8, 9), suggesting multiple mechanisms of FGFR1 action.Studies in this and other laboratories have shown that upon cell stimulation, FGFR1, a typically plasma membrane-associated protein, translocates to the cell nucleus along with nonsecreted, intracellular forms of FGF-2, which lack a signal peptide but contain a functional nuclear localization signal (NLS) (10). Nuclear FGFR1 is full-length, binds FGF-2, and has an active TK domain (11,12). Nuclear FGFR1 is not deriv...

Studies of telomerase-deficient mice and human cell lines have demonstrated that telomere shortening enhances sensitivity to ionizing radiation (IR). The molecular basis for this observation remains unclear. To better understand the connection between telomere shortening and radiation sensitivity, we evaluated components of the DNA damage response pathway in normal human fibroblasts with short and long telomeres. Late-passage cells with short telomeres showed enhanced sensitivity to IR compared to early-passage cells with longer telomeres. Compared to early-passage cells, late-passage cells had a higher baseline level of phosphorylated H2AX protein (γH2AX) before IR, but diminished peak levels of H2AX phosphorylation after IR. Both the appearance and disappearance of γH2AX foci were delayed in late-passage cells, indicative of delayed DNA repair. In contrast to the situation with H2AX, ATM and p53 phosphorylation kinetics were similar in early and late-passage cells, but phosphorylation of the chromatin-bound ATM targets SMC1 and NBS1 was delayed in late-passage cells. Because impaired phosphorylation associated with short telomeres was restricted to chromatin-bound ATM targets, chromatin structure was assessed. DNA from cells with short telomeres was more resistant to digestion with micrococcal nuclease, indicative of compacted chromatin. Moreover, cells with short telomeres showed histone acetylation and methylation profiles consistent with heterochromatin. Together our data suggest a model in which short telomeres induce chromatin structure changes that limit access of activated ATM to its downstream targets on the chromatin, thereby providing a potential explanation for the increased radiation sensitivity seen with telomere shortening.

, phosphorylation of H2AX, TERC, the RNA component of telomerase; TERT, the catalytic component of telomerase; TRAP, telomeric repeat amplification protocol.The unsatisfactory outcomes for osteosarcoma necessitate novel therapeutic strategies. This study evaluated the effect of the telomerase inhibitor imetelstat in pre-clinical models of human osteosarcoma. Because the chaperone molecule HSP90 facilitates the assembly of telomerase protein, the ability of the HSP90 inhibitor alvespimycin to potentiate the effect of the telomerase inhibitor was assessed. The effect of single or combined treatment with imetelstat and alvespimycin on long-term growth was assessed in osteosarcoma cell lines (143B, HOS and MG-63) and xenografts derived from 143B cells. Results indicated that imetelstat as a single agent inhibited telomerase activity, induced telomere shortening, and inhibited growth in all 3 osteosarcoma cell lines, though the bulk cell cultures did not undergo growth arrest. Combined treatment with imetelstat and alvespimycin resulted in diminished telomerase activity and shorter telomeres compared to either agent alone as well as higher levels of gH2AX and cleaved caspase-3, indicative of increased DNA damage and apoptosis. With dual telomerase and HSP90 inhibition, complete growth arrest of bulk cell cultures was achieved. In xenograft models, all 3 treatment groups significantly inhibited tumor growth compared with the placebo-treated control group, with the greatest effect seen in the combined treatment group (imetelstat, p D 0.045, alvespimycin, p D 0.034; combined treatment, p D 0.004). In conclusion, HSP90 inhibition enhanced the effect of telomerase inhibition in pre-clinical models of osteosarcoma. Dual targeting of telomerase and HSP90 warrants further investigation as a therapeutic strategy.

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