Projects

Current research for the Antoni Wiedlocha group

Transport and translocation into cytosol and nucleus of fibroblast growth factors
This group has for many years been working on the mechanism of uptake of protein toxins into the cytosol. Many protein toxins are able to cross cellular membranes and get access to components in the cytosol and nucleus. Certain growth factors are also able to be translocated into the cell which could be an efficient signal transduction principle. There are similarities and differences in the uptake and translocation of the two groups of proteins.

Translocation of exogenous growth factors into the cytosol and nucleus could be an efficient mode of signal transduction. Such direct interaction of growth factors with intracellular key proteins, e.g. transcription factors, could overcome the logistic problem involved in simultaneous encoding of many signals at the level of the plasma membrane and decoding them in the cytosol. The efficiency of the translocation (of the order 100 000 molecules translocated per cell) makes such a possibility realistic.
The group has presented evidence that FGF-1 has a dual mode of signal transduction involving both stimulation of the surface receptor and translocation of the growth factor to the nucleus. Binding of the growth factors to specific receptors (FGFRs) is a requirement for transport to the cytosol and to the nucleus. Binding to cell surface heparans does not induce translocation. Thee group has therefore focussed on the role of the receptor in the translocation process. One approach is to make various deletions and mutations in the receptor and study to what extent they interfere with the translocation. The full-length receptor is not required for translocation. Surprisingly, the kinase domain of the receptor is not required. Using systematic deletion and mutagenesis of the receptor they have identified a serine residue, Ser777, which must be phosphorylated by MAP-kinase p38 for translocation to occur.
The translocation occurs from electrically polarised endosomes (positive inside the vesicles). The potential is normally generated by the proton pump present in the endosome membrane. After FGF-1 is translocated to the cytosol, it is transported to the nucleus. The growth factor has two nuclear localisation sequences, one at the N-terminus and a bipartite one close to the C-terminus.
Once in the nucleus, the growth factor is phosphorylated by protein kinase C delta. This modification generates a nuclear export signal and the growth factor is subsequently exported to the cytosol where it is dephosphorylated and degraded. It is not clear what function the growth factor carries out in the nucleus, but whatever it is, it has a moderately stimulatory effect on the DNA synthesis. In attempts to elucidate the function of the growth factor in the nucleus and they are now trying to identify nuclear proteins that interact with the mutant FGF-1-Ser123Ala which is not recognised by exportin and accumulates in the nucleus.

Research projects

Characterisation of the mechanism of translocation of FGF1 into cells

The translocation of FGF1 to the cytosol appears to occur from intracellular acidic vesicular compartments and requires the transmembrane potential. Brefeldin A which blocks retrograde transport through the Golgi apparatus to the endoplasmic reticulum did not prevent translocation. Introduction of artificial disulfide bonds in the growth factor did not prevent translocation to the cytosol. In the case of diphtheria toxin and ricin a disulfide in the A-chain blocks translocation, indicating that different translocation mechanisms are operating. In addition, the translocation of FGF1 to the nucleus is highly sensitive to inhibitors of PI 3-kinase, indicating that a 3-phosphoinositide regulates this process.

A major effort will be made to identify membrane proteins involved in the translocation. An attractive possibility is that the growth factor may be translocated by an ABC transporter present in the membrane of an intracellular organelle. Mammalian cells contain a large number of different ABC transporters which fulfil a number of different functions. We plan to test the possibility that such transporters participate in the translocation. One approach is to label ATP-binding proteins in the cell (among them the ABC transporters) to see if some of the labelled proteins bind to FGF1 or to its receptor. If so, we will try to identify the protein

Characterisation of proteins that bind to FGF1 and to the cytoplasmic part of FGF-receptor 4

If FGF1 plays an important role inside the cells, it must interact with components in the cytosol and nucleus. Using the yeast two-hybrid system we have earlier cloned a nuclear protein, FIBP, which binds to FGF1 (but not FGF2). We have not been able to assign any other function to this protein. After the sequencing of the Drosophila gene was complete, we have found a homologous protein (43 % homology) in the fly. Hopefully this can lead to some understanding of its function.

Using a fusion protein of FGF1 and maltose-binding protein as a bait, we have identified several proteins from a cell lysate that bind to the growth factor. We have purified these proteins by microsequencing and electrospray mass spectrometry. We have in this way identified two proteins; protein kinase CK2 and ribosom-binding protein p34. These two proteins may be involved in FGF1 intracelluar signalling.

In a similar way we have used the minimal part of FGF receptor 4 that is sufficient to facilitate translocation of the growth factor into cells to identify proteins that bind to it. We will use the same approach to identify these proteins which may be involved in the translocation mechanism.

Characterisation of the intracellular routing of endocytosed FGF1.

After FGF1 is bound to its specific receptor, it is endocytosed and transported to the juxtanuclear recycling compartment. FGF1 bound to surface heparans is also endocytosed, but it is not transported to the recycling compartment, but appears to be routed to the lysosomes instead. We have made a number of mutations and deletions in the cytoplasmic part of the receptor in attempts to identify the signal determining the routing to the recycling compartment. We have found that mutations of a putative caveolin binding domain in the receptor prevents the transport to the recycling compartment. On the other hand, a receptor lacking the whole kinase domain including the putative caveolin binding domain is able to direct the growth factor to the recycling compartment provided that the remaining part of the receptor is retained. This part of the receptor is also sufficient to facilitate translocation of the growth factor to the cytosol.

We are now studying a series of mutations in the cytoplasmic part of the receptor for these two abilities. We hope the results will tell us if transport to the recycling compartment is a prerequisite for translocation of the growth factor to the cytosol and nucleus.

Endocytosis of aFGF bound to its specific receptors occurs only partly from clathrin coated pits. Thus inhibition of clathrin dependent endocytosis by acidification of the cytosol reduces the uptake only and to different extents in different cell lines. In COS cells it is almost not reduced at all, whereas in HeLa cells it is reduced to the half. In HeLa cells similar results were obtained in cells expressing inactive dynamin which also inhibits endocytosis from coated pits. We are now studying if FGF receptors can be used to characterise further the mechanism of uncoated endocytosis.

It has been claimed in the literature that FGF receptors are present in the nucleus. The mechanism for transport of the receptor to this location (if it occurs) is unclear. By immunostaining with anti-receptor 4 we have not been able to demonstrate convincingly that the receptor is in the nucleus of transfected cells. However, we have found accumulation of receptor in the perinuclear region, some of which could be present in the inner nuclear membrane. Thus it is not removed by treatment of the nuclei with Triton X-100.