Acland, Piers Dyke; (1991) Synthesis and subcellular fate of proteins encoded by the mouse int-2 (FGF3) gene. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The int-2 gene encodes a member of the FGF family and was discovered as a proto-oncogene transcriptionally activated in tumours induced by Mouse Mammary Tumour Virus. Int-2 transcription is rarely detected in adult mouse tissues apart from low levels in brain and testis, but in situ hybridisation has revealed widespread expression during embryogenesis. The predicted int-2 gene product has a molecular mass of 27 kD and a hydrophobic sequence at the N-terminus which acts as a signal peptide for vectorial synthesis into the endoplasmic reticulum. The products have proved difficult to detect in natural sources known to contain int-2 RNA such as mammary tumours and embryo-derived cell lines. Consequently the protein has been characterised by expression of cloned cDNAs in COS-1 cells using an SV40-based plasmid vector, in insect cells infected with recombinant baculoviruses and by translation of synthetic sense RNA in cell-free systems supplemented with canine pancreatic microsomes. These studies identified four major int-2 products ranging in size from 27.5 kD to 31.5 kD which arise by post-translational modification of a 28.5 kD primary translation product. Although int-2 proteins are targeted to the secretory pathway, they have only been detected at very low levels in the medium and the associated extracellular matrix of transfected COS-1 cells. Cell-free translation systems programmed with synthetic int-2 RNA identified an additional N-terminally extended product which initiates from an in-frame CUG codon located upstream of the first AUG. Immunofluorescent staining of transfected COS-1 cells showed that a substantial proportion of this extended product localised in the cell nucleus, while a truncated int-2 protein lacking both the N-terminal extension and the signal peptide was exclusively nuclear. These observations suggested that the signals required for nuclear localisation of int-2 were encoded in the body of the molecule, but only functioned when entry into the secretory pathway was compromised. Two nuclear targeting sequences were identified by mutagenesis. Fusion of these motifs to the cytosolic protein pyruvate kinase did not result in effective nuclear localisation. However chimaeras containing int-2 and hst, an exclusively secreted member of the FGF-family, identified a targeting signal sequence with superficial resemblance to the bipartite nuclear localisation sequence of Xenopus nucleoplasmin. Initiation at alternative codons changes the sub-cellular fate of the protein and in principle provides a means imparting distinct functions upon the different int-2 products. Although a biological activity has yet to be assigned to the nuclear forms, the secreted protein is transforming in a NIH3T3 focus assay. The efficiency of focus formation was augmented by mutations which prevented initiation at the CUG codon or at an upstream AUG in the +1 reading frame, resulting in elevated levels of secreted int-2. Thus transformation was shown to require a high level of int-2 synthesis and secretion. In agreement with these findings, mutations which reduce the efficiency of secretion reduce the focus forming ability of NIH3T3 cells transfected with int-2 cDNAs, substantiating the notion that transformation is effected through an autocrine loop mechanism that involves a cell surface receptor.

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