Mutation of the proline P81 into a serine modifies the tumour suppressor function of the von Hippel-Lindau gene in the ccRCC.

BACKGROUND: The von Hippel-Lindau disease is an autosomal dominant syndrome associated with tumour formation in various tissues, such as retina, central nervous system, kidney, and adrenal glands. VHL gene deletion or mutations support the development of various cancers. Unclassified VHL variants also referred as "of unknown significance" result from gene mutations that have an unknown or unclear effect on protein functions. The P81S mutation has been linked to low penetrance Type 1 disease but its pathogenic function was not clearly determined. METHODS: We established a stable cell line expressing the pVHL213 (c.241C>T, P81S) mutant. Using biochemical and physiological approaches, we herein analysed pVHL folding, stability and function in the context of this VHL single missense mutation. RESULTS: The P81S mutation mostly affects the non-canonical function of the pVHL protein. The cells expressing the pVHL213P81S acquire invasive properties in relation with modified architecture network. CONCLUSION: We demonstrated the pathogenic role of this mutation in tumour development in vhl patients and confirm a medical follow up of family carrying the c.241C>T, P81S.

Pre-return to work consultation and therapeutic part-time work: Cross-sectional study on level of knowledge and use by general practitioners in France.

BACKGROUND: In France, general practitioners (GPs) may use two tools specifically designed to help employees who experience difficulties in returning to work after sick leave: the pre-return-to-work (PRW) medical consultation and therapeutic part-time (TPT) work. OBJECTIVES: The objective was to investigate the level of knowledge and use of these two tools by GPs in Maine-et-Loire, France. METHODS: This cross-sectional study was performed using a telephone questionnaire to evaluate the level of knowledge of GPs and the use of these two tools in patients having difficulties returning to work. RESULTS: Among the 200 randomly selected GPs, 122 responded (response rate: 61%). More than half of the interviewed GPs declared they 'often' (46%) or 'always' (14%) contacted the occupational physician in these situations. Moreover, 62.2% and 32.7% believed that they had a 'vague' or 'very good' level of knowledge, and 41% and 51% declared either 'frequent' or 'regular' level of use of the PRW medical consultation, respectively. Regarding TPT work, 47% and 53% reported a 'very good' or 'vague' level of knowledge, and 41% and 51% a 'frequent' or 'regular' level of use, respectively. GPs who had a better level of knowledge of this tool reported a higher level of use (p < 0.001). CONCLUSION: This study shows that while the level of knowledge and use of the PRW medical consultation and TPT work is good, it is not optimal. This could be improved by organising training courses for GPs. Obstacles to their wider use could be investigated further in a qualitative study.

The prefoldin complex stabilizes the von Hippel-Lindau protein against aggregation and degradation.

Loss of von Hippel-Lindau protein pVHL function promotes VHL diseases, including sporadic and inherited clear cell Renal Cell Carcinoma (ccRCC). Mechanisms controlling pVHL function and regulation, including folding and stability, remain elusive. Here, we have identified the conserved cochaperone prefoldin complex in a screen for pVHL interactors. The prefoldin complex delivers non-native proteins to the chaperonin T-complex-protein-1-ring (TRiC) or Cytosolic Chaperonin containing TCP-1 (CCT) to assist folding of newly synthesized polypeptides. The pVHL-prefoldin interaction was confirmed in human cells and prefoldin knock-down reduced pVHL expression levels. Furthermore, when pVHL was expressed in Schizosaccharomyces pombe, all prefoldin mutants promoted its aggregation. We mapped the interaction of prefoldin with pVHL at the exon2-exon3 junction encoded region. Low levels of the PFDN3 prefoldin subunit were associated with poor survival in ccRCC patients harboring VHL mutations. Our results link the prefoldin complex with pVHL folding and this may impact VHL diseases progression.

Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease.

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.

The pVHL172 isoform is not a tumor suppressor and up-regulates a subset of pro-tumorigenic genes including TGFB1 and MMP13.

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.

Aggregation dynamics and identification of aggregation-prone mutants of the von Hippel-Lindau tumor suppressor protein.

Quality control mechanisms promote aggregation and degradation of misfolded proteins. In budding yeast, the human von Hippel-Lindau protein (pVHL, officially known as VHL) is misfolded and forms aggregates. Here, we investigated the aggregation of three pVHL isoforms (pVHL213, pVHL160, pVHL172) in fission yeast. The full-length pVHL213 isoform aggregates in highly dynamic small puncta and in large spherical inclusions, either close to the nucleus or to the cell ends. The large inclusions contain the yeast Hsp104 chaperone. Aggregate clearance is regulated by proteasomal degradation. The pVHL160 isoform forms dense foci and large irregularly shaped aggregates. In silico, prediction of pVHL aggregation propensity identified a key aggregation-promoting region within exon 2. Consistently, the pVHL172 isoform, which lacks exon 2, formed rare reduced inclusions. We studied the aggregation propensity of pVHL variants harbouring missense mutations found in kidney carcinomas. We show that the P86L mutation stimulated small aggregate formation, the P146A mutation increased large inclusion formation, whereas the I151S mutant destabilized pVHL. The prefoldin subunit Pac10 (the human homolog VBP-1 binds to pVHL) is required for pVHL stability. Reduction of soluble functional pVHL might be crucial in VHL-related diseases.

Investigation of the functional properties and subcellular localization of alpha human and rainbow trout estrogen receptors within a unique yeast cellular context.

Estrogens are steroid hormones that play a pivotal role in growth, differentiation and function of reproductive and non-reproductive tissues, mediated through estrogen receptors (ERs). Estrogens are involved in different genomic and non-genomic cell signaling pathways which involve well-defined subcellular ER localizations. Thus, ER activity results from complex interplays between intrinsic binding properties and specific subcellular localization. Since these two factors are deeply intricate, we carried out, in a unique yeast cell context, a comparative study to better understand structure/function/subcellular distribution relationships. This was carried out by comparing two ERs: the human ER α subtype (hERα) and the short form of the α isoform of the rainbow trout ER (rtERαS). Their distinct binding properties to agonist and antagonist ligands and subcellular localizations were characterized in Saccharomyces cerevisiae yeast cells. An unexpected partial agonistic effect of ICI 182-780 was observed for rtERαS. Concomitant to distinct binding properties, distinct subcellular localizations were observed before and after ligand stimulation. Due to the unique cell context, the link between ERs intrinsic binding properties and subcellular localizations is partly unveiled and issues are hypothesized based on the role of cytoplasmic transient complexes which play a role in the ER cytoplasmic/nuclear partition, which in turn is critical for the recruitment of co-regulators in the nucleus.

Identification of pVHL as a novel substrate for Aurora-A in clear cell renal cell carcinoma (ccRCC).

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of kidney cancer and is often characterized by mutations or deletions of the Von Hippel Lindau (VHL) tumour suppressor gene. Aurora gene family members are implicated in proper mitotic progression and spindle checkpoint function and play a crucial role in cancer progression. In the present study, we assessed the expression of Aurora-A in a cohort of 30 ccRCC with fully characterized VHL status (wt/wt or mut/del) and Fuhrman grade. Aurora-A transcript and protein levels were significantly increased in high Fuhrman grade tumours and in VHLwt/wt tumours. These results suggest that Aurora-A and VHL interact in the ccRCC. We demonstrated that the two proteins interact in vivo and identified the Ser72 on the sequence of VHL as the unique site phosphorylated by Aurora-A.

The Kin1 kinase and the calcineurin phosphatase cooperate to link actin ring assembly and septum synthesis in fission yeast.

BACKGROUND INFORMATION: The Kin1 protein kinase of fission yeast, which regulates cell surface cohesiveness during interphase cell growth, is also present at the cell division site during mitosis; however, its function in cell division has remained elusive. RESULTS: In FK506-mediated calcineurin deficient cells, mitosis is extended and ring formation is transiently compromised but septation remains normal. Here, we show that Kin1 inhibition in these cells leads to polyseptation and defects in membrane closure. Actomyosin ring disassembly is prevented and ultimately the daughter cells fail to separate. We show that the Pmk1 MAP kinase pathway and the type V myosin Myo4 act downstream of the cytokinetic function of Kin1. Kin1 inhibition also promotes polyseptation in myo3Δ, a type II myosin heavy-chain mutant defective in ring assembly. In contrast, Kin1 inactivation rescues septation in a myosin light-chain cdc4-8 thermosensitive mutant. A structure/function analysis of the Kin1 protein sequence identified a novel motif outside the kinase domain that is important for its polarised localisation and its catalytic activity. This motif is remarkably conserved in all fungal Kin1 homologues but is absent in related kinases of metazoans. CONCLUSIONS: We conclude that calcineurin and Kin1 activities must be tightly coordinated to link actomyosin ring assembly with septum synthesis and membrane closure and to ensure separation of the daughter cells.

Kin1 is a plasma membrane-associated kinase that regulates the cell surface in fission yeast.

Cell 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. Kin1Δ 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 analogue-sensitive allele kin1-as1. Kin1 inhibition primarily promotes delocalization of plasma membrane-associated 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 kin1Δ 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.

Role of the protein kinase Kin1 and nuclear centering in actomyosin ring formation in fission yeast.

Cytokinesis is the last step of the cell cycle, producing two daughter cells inheriting equal genetic information. This process involves the assembly of an actomyosin ring during mitosis. In the fission yeast Schizosaccharomyces pombe, cytokinesis occurs at the geometric cell centre, a position which is defined by the interphase nucleus and the anilin-related Mid1 protein. The pom1Delta, tea1Delta and tea4Delta mutants are defective in restricting Mid1 as a band around the nucleus and misplace the division site. We previously reported that inhibition of the protein kinase Kin1 promoted failure of cytokinesis in pom1Delta and tea1Delta cells but the mechanism involving Kin1 remained elusive. Here we investigated the contribution of Kin1 in cytokinesis. We show that Kin1-GFP has a dynamic cell cycle regulated distribution. Like pom1Delta and tea1Delta, tea4Delta exhibits a strong genetic interaction with kin1Delta. Using a conditional repressible kin1 allele that only alters interphase nuclear centering, we observed that Kin1 downregulation severely compromised actomyosin ring formation and septum synthesis in tea4Delta cells. In addition, nuclear displacement induced either by overexpression of a putative catalytically inactive Kin1 mutant, by chemically mediated microtubule depolymerization or by mutation in the par1Delta gene impaired cytokinesis in tea4Delta but not tea4(+) cells. We propose that nuclear mispositioning exacerbates the tea4Delta, pom1Delta and tea1Delta cell division phenotype. Our work reveal that nuclear centering becomes essential when Pom1/Tea1/Tea4 function is compromised and that Kin1 expression level is a key regulatory element in this situation. Our results suggest the existence of distinct overlapping control mechanisms to ensure efficient cell division.

pEg6, a spire family member, is a maternal gene encoding a vegetally localized mRNA in Xenopus embryos.

BACKGROUND INFORMATION: In Xenopus, during oocyte maturation and the segmentation period, cell cycle progression is independent of new transcription, but requires de novo translation. This suggests that the completion of oocyte maturation and then the rapid cell division period is controlled exclusively at a post-transcriptional level by specific gene products. To isolate these maternal genes, a differential screening of a Xenopus egg cDNA library was performed. Several cDNAs were isolated which correspond to mRNA polyadenylated in eggs and deadenylated in embryos, and these constitute the founders members of the Eg family of mRNAs. RESULTS: We report here the characterization of Eg6 mRNA as a novel maternal gene expressed in Xenopus egg until gastrula stage. The Eg6 transcript is initially concentrated in the vegetal cytoplasm of the egg, and later the distribution of the transcript marks the posterior vegetal end of developing embryos. pEg6 is a multidomain protein with a kinase non-catalytic C-lobe domain of unknown function, a cluster of four WH2 (Wiskott-Aldrich syndrome protein homology 2) domains and a modified FYVE zinc-finger motif. The amino acid sequence of pEg6 is related to PEM-5 (posterior end mark-5), from an ascidian maternal mRNA, and spire, a Drosophila protein required to establish dorsal-ventral and anterior-posterior axes of polarity and recently described as an actin nucleation factor. In Xenopus and Schizosaccharomyces pombe cells pEg6 expression induces filamentous actin clusters and is associated with vesicular structure. CONCLUSION: These data suggest that pEg6 acts as a vegetally localized factor contributing to the actin nucleation process during Xenopus early development.

M-phase MELK activity is regulated by MPF and MAPK.

The protein kinase MELK is implicated in the control of cell proliferation, cell cycle and mRNA splicing. We previously showed that MELK activity is correlated with its phosphorylation level, is cell cycle dependent, and maximal during mitosis. Here we report on the identification of T414, T449, T451, T481 and S498 as residues phosphorylated in Xenopus MELK (xMELK) in M-phase egg extract. Phosphorylations of T449, T451, T481 are specifically detected during mitosis. Results obtained in vivo showed that MPF and MAPK pathways are involved in xMELK phosphorylation. In vitro, MPF and MAPK directly phosphorylate xMELK and MPF phosphorylates xMELK on T481. In addition, phosphorylation by MPF and MAPK enhances MELK activity in vitro. Taken together our results indicate that MELK phosphorylation by MPF and MAPK enhance its activity during M-phase.

Cell-cycle-dependent cortical localization of pEg3 protein kinase in Xenopus and human cells.

BACKGROUND INFORMATION: Protein kinase pEg3 belongs to the evolutionarily conserved KIN1/PAR-1/MARK family, whose members are involved in a variety of functions, including cell polarity, microtubule stability, intracellular signalling and the cell cycle. Activity and phosphorylation of pEg3 are cell-cycle dependent and rise to maximum levels during mitosis. pEg3 was shown to interact with and phosphorylate phosphatase CDC25B, and to potentially control cell-cycle progression. Subcellular localization of pEg3 was investigated in Xenopus and human cultured cells. RESULTS: By expression of GFP (green fluorescent protein)-tagged pEg3 and indirect immunofluorescence with specific antibodies, pEg3 was found to be localized in the cytoplasm and the nucleus in interphase cells. During mitosis pEg3 was also found in the cytoplasm. From anaphase to telophase, a proportion of the protein was detected at the cell cortex. The cortical distribution in mitotic cells was dependent on F-actin, because the actin-depolymerization-inducing drugs cytochalasin D or latrunculin A prevented pEg3 cortical localization. The protein lacking the conserved C-terminal domain was not detected at the cell cortex, whereas the C-terminal domain was targeted to the cell periphery. In contrast with full-length pEg3, the cortical localization of the C-terminal domain and construct lacking the N-terminal domain was cell-cycle independent, and these constructs were found at the cell periphery in interphase cells. CONCLUSIONS: pEg3 is localized at the cell periphery specifically during mitosis. The C-terminal domain is the only pEg3 domain found to be necessary and sufficient for cortical targeting. Cortical distribution of pEg3 also requires the F-actin cytoskeleton. The cell-cycle-independent cortical localization of the pEg3 C-terminal domain and a construct lacking the N-terminal domain indicates that a negative control mechanism involving the pEg3 catalytic N-terminal domain probably acts to prevent pEg3 cortical distribution during interphase. These results suggest that pEg3 might play a role at the cell cortex during mitosis.

Poly(A)-binding protein and eRF3 are associated in vivo in human and Xenopus cells.

An interaction between human poly(A)-binding protein (PABP) et human eRF3 has been demonstrated using a double-hybrid approach and in vitro assays. Here, we show that the binding of both proteins is conserved through evolution. We also demonstrate that the last 39 C-terminal amino acids of PABP contain the interface that interacts with eRF3. This region includes helix 5, identified by RMN, which is conserved in all known PABPs. Lastly, we demonstrate that eRF3 et PABP molecules interact in vivo.

Characterization of the poly(A) binding proteins expressed during oogenesis and early development of Xenopus laevis.

During vertebrate oogenesis and early embryogenesis, gene expression is governed mainly by translational control. The recruitment of Poly(A) Binding Protein (PABP) during poly(A) tail lengthening appears to be the key to translational activation during this period of development in Xenopus laevis. We showed that PABP1 and ePABP proteins are both present during oogenesis and early development. We selected ePABP as an eRF3 binding protein in a two-hybrid screening of a X. laevis cDNA library and demonstrated that this protein is associated with translational complexes. It can complement essential functions of the yeast homologue Pab1p. We discuss specific expression patterns of the finely tuned PABP1 and ePABP proteins.

Poly(A)-binding protein acts in translation termination via eukaryotic release factor 3 interaction and does not influence [PSI(+)] propagation.

Recent studies of translational control suggest that translation termination may not be simply the end of synthesizing a protein but rather be involved in modulating both the translation efficiency and stability of a given transcript. Using recombinant eukaryotic release factor 3 (eRF3) and cellular extracts, we have shown for Saccharomyces cerevisiae that yeast eRF3 and Pab1p can interact. This interaction, mediated by the N+M domain of eRF3 and amino acids 473 to 577 of Pab1p, was demonstrated to be direct by the two-hybrid approach. We confirmed that a genetic interaction exists between eRF3 and Pab1p and showed that Pab1p overexpression enhances the efficiency of termination in SUP35 (eRF3) mutant and [PSI(+)] cells. This effect requires the interaction of Pab1p with eRF3. These data further strengthen the possibility that Pab1p has a role in coupling translation termination events with initiation of translation. Several lines of evidence indicate that Pab1p does not influence [PSI(+)] propagation. First, "[PSI(+)]-no-more" mutations do not affect eRF3-Pab1p two-hybrid interaction. Second, overexpression of PAB1 does not cure the [PSI(+)] phenotype or solubilize detectable amounts of eRF3. Third, prion-curing properties of overexpressed HSP104p, which is required for formation and maintenance of [PSI(+)], were not modified by excess Pab1p.