The tumour suppressor VHL is known to stabilize the ubiquitin ligase Jade-1. Jade-1 targets both phosphorylated and unphosphorylated -catenin, thereby linking VHL function directly to inhibition of Wnt signalling. The von Hippel–Lindau protein pVHL suppresses renal tumorigenesis in part by promoting the degradation of hypoxia-inducible HIF-α transcription factors1; additional mechanisms have been proposed2. pVHL also stabilizes the plant homeodomain protein Jade-1, which is a candidate renal tumour suppressor that may correlate with renal cancer risk3,4,5. Here we show that Jade-1 binds the oncoprotein β-catenin in Wnt-responsive fashion. Moreover, Jade-1 destabilizes wild-type β-catenin but not a cancer-causing form of β-catenin. Whereas the well-established β-catenin E3 ubiquitin ligase component β-TrCP ubiquitylates only phosphorylated β-catenin6, Jade-1 ubiquitylates both phosphorylated and non-phosphorylated β-catenin and therefore regulates canonical Wnt signalling in both Wnt-off and Wnt-on phases. Thus, the different characteristics of β-TrCP and Jade-1 may ensure optimal Wnt pathway regulation. Furthermore, pVHL downregulates β-catenin in a Jade-1-dependent manner and inhibits Wnt signalling, supporting a role for Jade-1 and Wnt signalling in renal tumorigenesis. The pVHL tumour suppressor and the Wnt tumorigenesis pathway are therefore directly linked through Jade-1.

DNA topoisomerase I (topo I) is involved in the regulation of DNA supercoiling, gene transcription, and rDNA recombination. However, little is known about interactions between topo I and other nuclear proteins. We used affinity chromatography with a topo I fusion protein to screen U-937 leukemic cell extracts and have identified nucleolin as a topo I-binding protein. Coimmunoprecipitation and other studies demonstrate that the interaction between topo I and nucleolin is direct. Furthermore, deletion analyses have identified the 166-210-amino acid region of topo I as sufficient for the interaction with nucleolin. Since nucleolin has been implicated in nuclear transport and in a variety of transcriptional processes, the interaction with topo I may relate to the cellular localization of topo I or to the known role of this topoisomerase in transcription.

Protein kinase C (PKC) δ is cleaved by caspase-3 to a kinase-active catalytic fragment (PKCδCF) in the apoptotic response of cells to DNA damage. Expression of PKCδCF contributes to the induction of apoptosis by mechanisms that are presently unknown. Here we demonstrate that PKCδCF associates with p73β, a structural and functional homologue of the p53 tumor suppressor. The results show that PKCδCF phosphorylates the p73β transactivation and DNA-binding domains. One PKCδCF-phosphorylation site has been mapped to Ser-289 in the p73β DNA-binding domain. PKCδCF-mediated phosphorylation of p73β is associated with accumulation of p73β and induction of p73β-mediated transactivation. By contrast, PKCδCF-induced activation of p73β is attenuated by mutating Ser-289 to Ala (S289A). The results also demonstrate that PKCδCF stimulates p73β-mediated apoptosis and that this response is attenuated with the p73β(S289A) mutant. These findings demonstrate that cleavage of PKCδ to PKCδCF induces apoptosis by a mechanism in part dependent on PKCδCF-mediated phosphorylation of the p73β Ser-289 site.

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF–SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.