Inability of the Acidic Fibroblast Growth Factor Mutant K132E to Stimulate DNA Synthesis after Translocation into Cells

Klingenberg, Olav; Wiedlocha, Antoni G; Rapak, Andrzej; Muñoz, Raquel; Falnes, Pål Ø.; Olsnes, Sjur

doi:10.1074/jbc.273.18.11164

Cited by 42 publications

(91 citation statements)

References 40 publications

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“…U2OSDr1-R4 cells, a human osteosarcoma cell line stably transfected with FGFR4, bind aFGF and transport the growth factor to the nuclear fraction (14,18,63). The results in Fig.…”

Section: Fig 1 Effect Of Pi-3 Kinase Inhibitors On Binding (A and Bmentioning

confidence: 68%

“…We recently demonstrated that aFGF contains a functional phosphorylation site for protein kinase C (18,73) and that the growth factor can be phosphorylated at this site upon incubation with COS cells transfected with functional FGFR4. 2 In this paper we use phosphorylation of aFGF as a measure of translocation to the cytosol and nucleus, since these are the only places in the cell where protein kinase C is known to exist.…”

Section: S]methionine-labeled Afgf (Upper Panel) As Well As Farnesylamentioning

confidence: 99%

“…We therefore modified the growth factor to contain the C-terminal extension CVIM (Cys-Val-Ile-Met). When unlabeled, modified aFGF (aFGF-CAAX) is incubated with FGFRpositive cells in the presence of radiolabeled mevalonic acid, a precursor of the farnesyl group, and lovastatin, an inhibitor of mevalonic acid biosynthesis included to increase labeling efficiency, the growth factor becomes labeled (14,17,18).…”

Section: Fig 1 Effect Of Pi-3 Kinase Inhibitors On Binding (A and Bmentioning

confidence: 99%

“…The reconstituted fusion proteins were incubated with cells for 1 h at 4°C. The cells were washed and treated with buffer, pH 4.5, for 5 min at 37°C, treated for 10 min at 37°C with 5 mg/ml Pronase, washed in the presence of protease inhibitors, and either lysed or further incubated for 9 -22 h at 37°C in the presence or absence of different inhibitors before lysis and fractionation into cytosol/membrane and nuclear fractions (18,61,62). In experiments with wortmannin, the medium was replaced every third hour.…”

Section: Translocation Of Afgf-dt-a Fusion Proteins Into Cells-[mentioning

confidence: 99%

“…Several proteins, including aFGF, are capable of being translocated into cells as fusion proteins with DT-A (60 -62). We have previously used this as a tool to study the intracellular role of aFGF (16,18,63).…”

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

Klingenberg¹,

dłocha

Citores³

et al. 2000

Journal of Biological Chemistry

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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...

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“…U2OSDr1-R4 cells, a human osteosarcoma cell line stably transfected with FGFR4, bind aFGF and transport the growth factor to the nuclear fraction (14,18,63). The results in Fig.…”

Section: Fig 1 Effect Of Pi-3 Kinase Inhibitors On Binding (A and Bmentioning

confidence: 68%

Section: S]methionine-labeled Afgf (Upper Panel) As Well As Farnesylamentioning

confidence: 99%

Section: Fig 1 Effect Of Pi-3 Kinase Inhibitors On Binding (A and Bmentioning

confidence: 99%

Section: Translocation Of Afgf-dt-a Fusion Proteins Into Cells-[mentioning

confidence: 99%

mentioning

confidence: 99%

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

Klingenberg¹,

dłocha

Citores³

et al. 2000

Journal of Biological Chemistry

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The mitogenic activity of fibroblast growth factor-1 correlates with its internalization and limited proteolytic processing

Grieb

Burgess

2000

J. Cell. Physiol.

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The fibroblast growth factor-1 (FGF-1) mitogenic signal transduction pathway is not well characterized, and evidence indicates that FGF-1 binding to and activation of cell-surface receptors is not solely sufficient for a full mitogenic response. Although initiation of the phosphorylation signaling cascades are likely important in FGF-1-induced mitogenic signaling, there appear to be additional signaling requirements. In this study, we demonstrate that FGF-1 internalization and subsequent processing correlates with the mitogenic potential of the growth factor on NIH 3T3 cells. Using site-directed mutants of FGF-1 and inhibitors of the endocytic and degradative pathways, we provide evidence for growth factor internalization and exposure to an acidic environment as necessary components of FGF-1-induced mitogenesis. In addition, a protease-sensitive event(s) appears critical for a complete mitogenic response to FGF-1, whereas, this protease sensitivity was not detected under the same conditions for serum-stimulated mitogenesis. Therefore, proteolytic modification of internalized FGF-1 may result in the activation of additional, intracellular signaling events.

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A recessive syndrome of intellectual disability, moderate overgrowth, and renal dysplasia predisposing to Wilms tumor is caused by a mutation inFIBPgene

Akawi

Ben-Salem

Lahti

et al. 2016

American J of Med Genetics Pt A

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Clinical classification of overgrowth syndromes represents a challenge since a wide spectrum of disorders result in marked overgrowth. Therefore, there is a continuous effort to identify the genetic basis of these disorders that will eventually facilitate their molecular classification. Here, we have identified the genetic etiology and the pathogenetic mechanism underlying a rare autosomal recessive overgrowth syndrome in three affected siblings. The overgrowth phenotype in the patients was accompanied by developmental delay, learning disabilities, and variable congenital abnormalities. To elucidate the genetic etiology of the disorder, whole-genome genotyping and whole-exome sequencing were used. The disease was mapped to 3p21.1-p14.2 and 11q13.1-q13.4, where an in-frame insertion (c.175_176insTAA) in FIBP gene was revealed. The resulting indel (p.H59LN) was predicted to change the protein conformation with likely deleterious effect on its function as one of the fibroblast growth factor signaling mediators. In vitro cellular proliferation assay and in situ hypridization in vivo were then performed to understand the pathophysiology of the disease. The patients' skin fibroblasts showed an increased proliferation capacity compared to the controls' explaining the observed overgrowth phenotype. In addition, we detected Fibp expression most notably in the brains of mice embryos suggesting a possible effect on cognitive functions early in development. To date, only one patient has been reported with a homozygous nonsense mutation in FIBP exhibiting an overgrowth syndrome with multiple congenital abnormalities. Taken all together, these findings provide convincing evidence implicating FIBP aberrations in the newly recognized overgrowth syndrome and expand the associated phenotypes to include possible Wilms tumor predisposition. © 2016 Wiley Periodicals, Inc.

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Inability of the Acidic Fibroblast Growth Factor Mutant K132E to Stimulate DNA Synthesis after Translocation into Cells

Cited by 42 publications

References 40 publications

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

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

The mitogenic activity of fibroblast growth factor-1 correlates with its internalization and limited proteolytic processing

A recessive syndrome of intellectual disability, moderate overgrowth, and renal dysplasia predisposing to Wilms tumor is caused by a mutation inFIBPgene

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