Requirement of Phosphatidylinositol 3-Kinase Activity for Translocation of Exogenous aFGF to the Cytosol and Nucleus

Abstract

Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution. Acidic fibroblast growth factor (aFGF)1 is considered to be involved in several important physiological and pathological processes, such as angiogenesis, wound healing (1), and atheromatosis (2-6). In cell cultures it can stimulate growth, cell migration, and differentiation. As the growth factor lacks a classical signal sequence, its mechanism of secretion is still unclear, but it appears to involve a pathway different from the classical secretion pathway through the endoplasmic reticulum and Golgi apparatus (7-9). At the cell surface, aFGF binds with high affinity to transmembrane FGF-receptors (FGFR) containing a cytoplasmic split tyrosine kinase domain. There are four different genes encoding FGFR (FGFR1-4) with several different splicing variants. aFGF also binds to heparan sulfate proteoglycans at the cell surface, although with lower affinity. Upon activation, the FGFR is phosphorylated on tyrosine residues, and it activates downstream effectors such as phosholipase C␥ and the mitogen-activated protein kinase pathway. There are some conflicting data as to whether phosphatidylinositol (PI) 3-kinase is also activated (10 -13). After binding to high affinity cell surface receptors, aFGF is translocated across cellular membranes and transported to the nucleus (14 -22). The mechanism of this process is not well defined. It depends on FGFR and, at least in the case of FGFR4, also on the intactness of the C terminus of its cytosolic part. 2Human PI 3-kinases phosphorylate PI in the 3Ј position of the inositol ring and can be divided into three main classes on the basis of in vitro substrate specificity (23). Class I phosphorylates PI, PI 4-phosphate and PI 4,5-diphosphate and include...

Phosphorylation of Fibroblast Growth Factor (FGF) Receptor 1 at Ser777 by p38 Mitogen-Activated Protein Kinase Regulates Translocation of Exogenous FGF1 to the Cytosol and Nucleus

“…To test for the transport of FGF1 from the cell surface and into the cytosol and nucleus, we have developed an in vivo FGF1 phosphorylation assay which has been extensively described and validated in earlier studies (23,25, 31, 32, 36, 47, 52, 57). In this assay the translocation of exogenously added FGF1 into the cytosol and nucleus of the cells is monitored by phosphorylation of the growth factor.…”

“…The translocation of exogenous FGF1 or FGF2 into the cytosol and nucleus is a highly regulated process that requires phosphatidylinositol 3-kinase (PI3K) activity (23) and active hsp90 (52) and is strictly dependent on binding of FGF to either FGFR1 or FGFR4 (47). Furthermore, translocation was found to be cell cycle dependent (3,31,63), it can be stimulated by serum deprivation of cells (1,3,18,25,31,32,55,63), and it occurs after a several-hour delay compared to the endocytic uptake of FGF (31,47).…”

Fibroblast Growth Factor-12 (FGF12) Translocation into Intestinal Epithelial Cells Is Dependent on a Novel Cell-penetrating Peptide Domain

“…Generally, FGFs can function through the activation of surface FGFRs, while receptor-bound FGF1 can be endocytosed to reach the nucleus via the presence of a NLS, leading to DNA synthesis and cell proliferation (9,10). Its translocation depends on binding with FGFR1 and FGFR4 (11) and requires phosphatidylinositol 3-kinase (PI3K) activity (12) and Hsp90 (13). Moreover, FGF1 can interact with intracellular proteins such as FIBP (14), p34 (15), casein kinase 2 (CK2) (16), and mortalin (17), suggesting that endocytosed FGF1 plays multiple roles inside cells.…”

Different abilities of the four FGFRs to mediate FGF-1 translocation are linked to differences in the receptor C-terminal tail

“…cDNA encoding IIIc splice variants of fulllength human FGFR1, FGFR2, FGFR3 and FGFR4, as well as the shorter splice variants FGFR1␤ and FGFR2␤ (lacking Iglike domain I) were cloned in the pcDNA3 expression vector and expressed by transient transfection in COS-1 cells. We have previously shown that exogenous FGF-1 can be translocated to the cytosol in COS-1 cells transfected with FGFR4 (Klingenberg et al, 2000a). To maintain the cells in a state that allows a reproducible response to FGF stimulation, they were propagated in low serum conditions (defined medium for fibroblasts supplemented with 2% serum) before the experiment.…”

“…Translocation of exogenous FGF-1 or FGF-2 into cytosol and nucleus is a regulated process, which has been found to occur in the G1 phase of the cell cycle (Bouche et al, 1987;Baldin et al, 1990;Zhan et al, 1993;Imamura et al, 1994;Malecki et al, 2004) and it requires PI3K activity (Klingenberg et al, 2000a;Malecki et al, 2004). It has been found that translocation of both FGF-1 and FGF-2 occurs from the lumen of intracellular vesicles possessing vacuolar proton pumps, most likely early endosomes and that it depends on the electrical potential across the vesicular membrane (Malecki et al, 2002;Malecki et al, 2004).…”