Background:The von Hippel–Lindau (VHL) gene encodes two mRNA variants. Variant 1 encodes two protein isoforms, pVHL213 and pVHL160, that have been extensively documented in the literature. Variant 2 is produced by alternative splicing of exon 2 and encodes a pVHL isoform of 172 amino acids with a theoretical molecular weight of 19 kDa (pVHL172), the expression of which has never been demonstrated so far due to the absence of suitable antibodies.Methods:We have generated an anti-pVHL monoclonal antibody (JD-1956) using pVHL172 recombinant protein. We tested the antibody against exogenous or endogenous expressed proteins in different cell lines. We identified the pVHL172 using a silencing RNA strategy. The epitope of the antibody was mapped using a peptide array.Results:We efficiently detected the three different isoforms of pVHL in cell lines and tumorigenic tissues by western blotting and immunohistochemistry and confirmed for the first time the endogenous expression of pVHL172.Conclusions:The endogenous expression of the three isoforms and particularly the pVHL172 has never been shown before due to a lack of a highly specific antibody since none of the available commercial antibodies distinguish the three isoforms of pVHL in cells or in both normal and cancerous human tissues. Evidence of pVHL172 expression emphasises the need to further study its implication in renal tumorigenesis and VHL disease.

SummaryCell morphogenesis is a complex process that depends on cytoskeleton and membrane organization, intracellular signalling and vesicular trafficking. The rod shape of the fission yeast Schizosaccharomyces pombe and the availability of powerful genetic tools make this species an excellent model to study cell morphology. Here we have investigated the function of the conserved Kin1 kinase. Kin1-GFP associates dynamically with the plasma membrane at sites of active cell surface remodelling and is present in the membrane fraction. Kin1D null cells show severe defects in cell wall structure and are unable to maintain a rod shape. To explore Kin1 primary function, we constructed an ATP analoguesensitive allele kin1-as1. Kin1 inhibition primarily promotes delocalization of plasma membraneassociated markers of actively growing cell surface regions. Kin1 itself is depolarized and its mobility is strongly reduced. Subsequently, amorphous cell wall material accumulates at the cell surface, a phenotype that is dependent on vesicular trafficking, and the cell wall integrity mitogen-activated protein kinase pathway is activated. Deletion of cell wall integrity mitogen-activated protein kinase components reduces kin1D hypersensitivity to stresses such as those induced by Calcofluor white and SDS. We propose that Kin1 is required for a tight link between the plasma membrane and the cell wall.

The von Hippel-Lindau (VHL) tumor suppressor gene is often deleted or mutated in ccRCC (clear cell renal cell carcinoma) producing a non-functional protein. The gene encodes two mRNA, and three protein isoforms (pVHL213, pVHL160 and pVHL172). The pVHL protein is part of an E3 ligase complex involved in the ubiquitination and proteasomal degradation of different proteins, particularly hypoxia inducible factors (HIF) that drive the transcription of genes involved in the regulation of cell proliferation, angiogenesis or extracellular matrix remodelling. Other non-canonical (HIF-independent) pVHL functions have been described. A recent work reported the expression of the uncharacterized protein isoform pVHL172 which is translated from the variant 2 by alternative splicing of the exon 2. This splice variant is sometimes enriched in the ccRCCs and the protein has been identified in the respective samples of ccRCCs and different renal cell lines. Functional studies on pVHL have only concerned the pVHL213 and pVHL160 isoforms, but no function was assigned to pVHL172. Here we show that pVHL172 stable expression in renal cancer cells does not regulate the level of HIF, exacerbates tumorigenicity when 786-O-pVHL172 cells were xenografted in mice. The pVHL172-induced tumors developed a sarcomatoid phenotype. Moreover, pVHL172 expression was shown to up regulate a subset of pro-tumorigenic genes including TGFB1, MMP1 and MMP13. In summary we identified that pVHL172 is not a tumor suppressor. Furthermore our findings suggest an antagonistic function of this pVHL isoform in the HIF-independent aggressiveness of renal tumors compared to pVHL213.