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.

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.

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.

Unconventional Functions of Mitotic Kinases in Kidney Tumorigenesis.

Human tumors exhibit a variety of genetic alterations, including point mutations, translocations, gene amplifications and deletions, as well as aneuploid chromosome numbers. For carcinomas, aneuploidy is associated with poor patient outcome for a large variety of tumor types, including breast, colon, and renal cell carcinoma. The Renal cell carcinoma (RCC) is a heterogeneous carcinoma consisting of different histologic types. The clear renal cell carcinoma (ccRCC) is the most common subtype and represents 85% of the RCC. Central to the biology of the ccRCC is the loss of function of the Von Hippel-Lindau gene, but is also associated with genetic instability that could be caused by abrogation of the cell cycle mitotic spindle checkpoint and may involve the Aurora kinases, which regulate centrosome maturation. Aneuploidy can also result from the loss of cell-cell adhesion and apical-basal cell polarity that also may be regulated by the mitotic kinases (polo-like kinase 1, casein kinase 2, doublecortin-like kinase 1, and Aurora kinases). In this review, we describe the "non-mitotic" unconventional functions of these kinases in renal tumorigenesis.

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.