Homeobox gene Cdx1 regulates Ras, Rho and PI3 kinase pathways leading to transformation and tumorigenesis of intestinal epithelial cells.

The Cdx1 homeobox gene encodes for an intestine-specific transcription factor involved in the control of proliferation and differentiation of epithelial cells. Although it has been indicated that Cdx1 may act as a proto-oncogene in cultured fibroblasts, its direct role in the regulation of intestinal tumorigenesis has not been demonstrated. Here we show that expression of Cdx1 in an intestinal epithelial cell line (IEC-6) induces anchorage-independent growth in soft agar and promotes the formation of adenocarcinoma in vivo. The phenotype of Cdx1-induced tumors was exacerbated when IEC-6/Cdx1 cells were injected together with matrigel containing mitogens and extracellular matrix components. These changes were correlated with an increase in the GTP-bound form of Ras, modulation of Cdc42 and Rho-A activities, and accumulation of phosphatidyl inositol 3 (PI3) kinase products. Moreover, combined inhibition of Ras/Rho and PI3 kinase signaling by synthethic inhibitors blocked colony formation of IEC-6/Cdx1 cells in soft agar. Taken together, these results demonstrate a direct involvement of Cdx1, and its collaboration with Ras, Rho and PI3 kinase pathways, in transformation and tumorigenesis of intestinal epithelial cells.

Pyk2 and FAK regulate neurite outgrowth induced by growth factors and integrins.

Integration of signalling pathways initiated by receptor tyrosine kinases and integrins is essential for growth-factor-mediated biological responses. Here we show that co-stimulation of growth-factor receptors and integrins activates the focal-adhesion kinase (FAK) family to promote outgrowth of neurites in PC12 and SH-SY5Y cells. Pyk2 and FAK associate with adhesion-based complexes that contain epidermal growth factor (EGF) receptors, through their carboxy- and amino-terminal domains. Expression of the C-terminal domain of Pyk2 or of FAK is sufficient to block neurite outgrowth, but not activation of extracellular-signal-regulated kinase (ERK). Moreover, activation and autophosphorylation of Pyk2/FAK, as well as of effectors of their adhesion-targeting domains, such as paxillin, are important for propagation of signals that control neurite formation. Thus, Pyk2/FAK have important functions in signal integration proximal to integrin/growth-factor receptor complexes in neurons.

Oligomerization-dependent folding of the membrane fusion protein of Semliki Forest virus.

The spikes of alphaviruses are composed of three copies of an E2-E1 heterodimer. The E1 protein possesses membrane fusion activity, and the E2 protein, or its precursor form, p62 (sometimes called PE2), controls this function. Both proteins are, together with the viral capsid protein, translated from a common C-p62-E1 coding unit. In an earlier study, we showed that the p62 protein of Semliki Forest virus (SFV) dimerizes rapidly and efficiently in the endoplasmic reticulum (ER) with the E1 protein originating from the same translation product (so-called heterodimerization in cis) (B.-U. Barth, J. M. Wahlberg, and H. Garoff, J. Cell Biol. 128:283-291, 1995). In the present work, we analyzed the ER translocation and folding efficiencies of the p62 and E1 proteins of SFV expressed from separate coding units versus a common one. We found that the separately expressed p62 protein translocated and folded almost as efficiently as when it was expressed from a common coding unit, whereas the independently expressed E1 protein was inefficient in both processes. In particular, we found that the majority of the translocated E1 chains were engaged in disulfide-linked aggregates. This result suggests that the E1 protein needs to form a complex with p62 to avoid aggregation. Further analyses of the E1 aggregation showed that it occurred very rapidly after E1 synthesis and could not be avoided significantly by the coexpression of an excess of p62 from a separate coding unit. These latter results suggest that the p62-E1 heterodimerization has to occur very soon after E1 synthesis and that this is possible only in a cis-directed reaction which follows the synthesis of p62 and E1 from a common coding unit. We propose that the p62 protein, whose synthesis precedes that of the E1 protein, remains in the translocon of the ER and awaits the completion of E1. This strategy enables the p62 protein to complex with the E1 protein immediately after the latter has been made and thereby to control (suppress) its fusion activity.

The nucleocapsid-binding spike subunit E2 of Semliki Forest virus requires complex formation with the E1 subunit for activity.

Alphaviruses, such as Semliki Forest virus (SFV), mature by budding at the plasma membrane (PM) of infected cells and enter uninfected ones by a membrane fusion process in the endosomes. Both processes are directed by the p62/E2-E1 membrane protein heterodimer of the virus. The p62 protein, or its mature form E2, provides a cytoplasmic protein domain for interaction with the nucleocapsid (NC) of the virus, and the E1 protein functions as a membrane fusogen. We have previously shown that the p62/E2 protein of SFV controls the membrane fusion activity of E1 through its complex formation with the latter (A. Salminen, J. M. Wahlberg, M. Lobigs, P. Liljeström, and H. Garoff, J. Cell Biol. 116:349-357, 1992). In the present work, we show that the E1 protein controls the NC-binding activity of p62/E2. We have studied E1 expression-deficient SFV variants and shown that although the p62/E2 proteins can be transported to the PM they cannot establish stable NC associations.

The E1 protein is mandatory for pore formation by Semliki Forest virus spikes.

Insect cells (Aedes albopictus, clone C6/36) were infected with various variants of Semliki Forest virus including the wild type using the SFV replicon system. The variants included deletion mutants lacking one of the structural proteins and a mutant with a point mutation in p62 (SQL). The latter mutation results in a failure to process p62 to E2 and E3. After infection of the cells with different variant viruses and subsequent expression of viral proteins in the host cell plasma membrane low pH-induced pore formation was detected by measuring the efflux of a radiolabeled compound. The results of these experiments clearly showed that the E1 protein is mandatory for the acid-induced pore formation. A participation of the 6K or C-protein could be excluded. Furthermore, results obtained with the SQL mutant suggest that dissociation of the E1/E2 heterodimer and subsequent homooligomerization of E1 are required for pore formation.

The oligomerization reaction of the Semliki Forest virus membrane protein subunits.

The Semliki Forest virus (SFV) spike is composed of three copies of a membrane protein heterodimer. The two subunits of this heterodimer (p62 and E1) are synthesized sequentially from a common mRNA together with the capsid (C) in the order C-p62-E1. In this work heterodimerization of the spike proteins has been studied in BHK 21 cells. The results indicate that: (a) the polyprotein is cotranslationally cleaved into individual chains; (b) the two membrane protein subunits are initially not associated with each other in the endoplasmic reticulum (ER); (c) heterodimerization occurs predominantly between subunits that originate from the same translation product (heterodimerization in cis); (d) the kinetics of subunit association are very fast (t1/2 = 4 min); and (e) this heterodimerization is highly efficient. To explain the cis-directed heterodimerization reaction we suggest that the p62 protein, which is made before E1 during 26S mRNA translation, is retained at its translocation site until also the E1 chain has been synthesized and translocated at this same site. The mechanism for p62 retention could either be that the p62 anchor sequence cannot diffuse out from an "active" translocation site or that the p62 protein is complexed with a protein folding facilitating machinery that is physically linked to the translocation apparatus.

Assembly and entry mechanisms of Semliki Forest virus.

The alphavirus Semliki Forest (SFV) is an enveloped virus with a positive single-stranded RNA genome. The genome is complexed with 240 copies of a capsid protein into a nucleocapsid structure. In the membrane the virus carries an equal number of copies of a membrane protein heterodimer. The latter oligomers are grouped into clusters of three. These structures form the spikes of the virus and carry its entry functions, that is receptor binding and membrane fusion activity. The membrane protein heterodimer is synthesized as a p62E1 precursor protein which upon transport to the cell surface is cleaved into the mature E2E1 form. Recent studies have given much new information on the assembly and entry mechanism of this simple RNA virus. Much of this work has been possible through the construction of a complete cDNA clone of the SFV genome which can be used for in vitro transcription of infectious RNA. One important finding has been to show that a spike deletion variant and a capsid protein deletion variant are budding-negative when expressed separately but can easily complement each other when transfected into the same cell. This shows clearly that enveloped viruses use different budding strategies: one which depends on a nucleocapsid-spike interaction as exemplified by SFV and another one which is based on a direct core-lipid bilayer interaction as shown before to be the case with retroviruses. Another important finding concerns the activation process of the presumed fusion protein of SFV, the E1 subunit. In the original p62E1 heterodimer E1 is completely inactive. Activation proceeds in several steps. First p62 cleavage activates the potential for low pH inducible fusion. Next the low pH which surrounds incoming virus in endosomes induces dissociation of the heterodimeric structure. This is followed by a rearrangement of E1 subunits into homotrimers which are fusion active.

Alphavirus assembly and entry: role of the cytoplasmic tail of the E1 spike subunit.

The alphavirus Semliki Forest virus (SFV) matures by budding at the cell surface. This process is driven by interactions of its membrane protein heterodimer E2-E1 and the nucleocapsid. The virus penetrates into new cells by an E1-mediated membrane fusion event. The E1 subunit has a short, strongly positively charged cytoplasmic tail peptide (Arg-Arg) which is very conserved among different alphavirus E1 proteins. In this work, we have used in vitro mutagenesis of a full-size cDNA clone of SFV to study the role of the tail peptide of the E1 subunit in virus budding and fusion processes in baby hamster kidney cell culture. Our results suggest that the E1 tail plays no major role in SFV multiplication in animal cell culture.