Wilhelm Krek

Chemokine receptor CXCR4 downregulated by von Hippel–Lindau tumour suppressor pVHL

Organ-specific metastasis is governed, in part, by interactions between chemokine receptors on cancer cells and matching chemokines in target organs. For example, malignant breast cancer cells express the chemokine receptor CXCR4 and commonly metastasize to organs that are an abundant source of the CXCR4-specific ligand stromal cell-derived factor-1α (ref. 1). It is still uncertain how an evolving tumour cell is reprogrammed to express CXCR4, thus implementing the tendency to metastasize to specific organs. Here we show that the von Hippel–Lindau tumour suppressor protein pVHL negatively regulates CXCR4 expression owing to its capacity to target hypoxia-inducible factor (HIF) for degradation under normoxic conditions. This process is suppressed under hypoxic conditions, resulting in HIF-dependent CXCR4 activation. An analysis of clear cell renal carcinoma that manifests mutation of the VHL gene in most cases revealed an association of strong CXCR4 expression with poor tumour-specific survival. These results suggest a mechanism for CXCR4 activation during tumour cell evolution and imply that VHL inactivation acquired by incipient tumour cells early in tumorigenesis confers not only a selective survival advantage but also the tendency to home to selected organs.

p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells.

The F-box protein p45SKP2 is the substrate-targeting subunit of the ubiquitin–protein ligase SCFSKP2 and is frequently overexpressed in transformed cells. Here we report that expression of p45SKP2 in untransformed fibroblasts activates DNA synthesis in cells that would otherwise growth-arrest. Expression of p45SKP2 in quiescent fibroblasts promotes p27Kip1 degradation, allows the generation of cyclin-A-dependent kinase activity and induces S phase. Coexpression of a degradation-resistant p27Kip1 mutant suppresses p45SKP2-induced cyclin-A-kinase activation and S-phase entry. We propose that p45SKP2 is important in the progression from quiescence to S phase and that the ability of p45SKP2 to promote p27Kip1 degradation is a key aspect of its S-phase-inducing function. In transformed cells, p45SKP2 may contribute to deregulated initiation of DNA replication by interfering with p27Kip1 function.

A CDK-Independent Function of Mammalian Cks1: Targeting of SCFSkp2 to the CDK Inhibitor p27Kip1

The Cks/Suc1 proteins associate with CDK/cyclin complexes, but their precise function(s) is not well defined. Here we demonstrate that Cks1 directs the ubiquitin-mediated proteolysis of the CDK-bound substrate p27Kip1 by the protein ubiquitin ligase (E3) SCF(Skp2). Cks1 associates with the F box protein Skp2 and is essential for recognition of the p27Kip1 substrate for ubiquitination in vivo and in vitro. Using purified recombinant proteins, we reconstituted p27Kip1 ubiquitination activity and show that it is dependent on Cks1. CKS1-/- mice are abnormally small, and cells derived from them proliferate poorly, particularly under limiting mitogen conditions, possibly due to elevated levels of p27Kip1.

Interaction between ubiquitin–protein ligase SCF SKP2 and E2F-1 underlies the regulation of E2F-1 degradation

The transcription factor E2F-1 is important in the control of cell proliferation. Its activity must be tightly regulated in a cell-cycle-dependent manner to enable programs of gene expression to be coupled closely with cell-cycle position. Here we show that, following its accumulation in the late G1 phase of the cell cycle, E2F-1 is rapidly degraded in S/G2 phase. This event is linked to a specific interaction of E2F-1 with the F-box-containing protein p45SKP2, which is the cell-cycle-regulated component of the ubiquitin–protein ligase SCFSKP2 that recognizes substrates for this ligase. Disruption of the interaction between E2F-1 and p45SKP2 results in a reduction in ubiquitination of E2F-1 and the stabilization and accumulation of transcriptionally active E2F-1 protein. These results indicate that an SCFSKP2-dependent ubiquitination pathway may be involved in the downregulation of E2F-1 activity in the S/G2 phase of the cell cycle, and suggest a link between SCFSKP2 and cell-cycle-dependent gene control.

Activation of a HIF1alpha-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

Association of human CUL‐1 and ubiquitin‐conjugating enzyme CDC34 with the F‐box protein p45SKP2: evidence for evolutionary conservation in the subunit composition of the CDC34–SCF pathway

In normal and transformed cells, the F‐box protein p45SKP2 is required for S phase and forms stable complexes with p19SKP1 and cyclin A–cyclin‐dependent kinase (CDK)2. Here we identify human CUL‐1, a member of the cullin family, and the ubiquitin‐conjugating enzyme CDC34 as additional partners of p45SKP2 in vivo. CUL‐1 also associates with cyclin A and p19SKP1 in vivo and, with p45SKP2, they assemble into a large multiprotein complex. In Saccharomyces cerevisiae, a complex of similar molecular composition (an F‐box protein, a member of the cullin family and a homolog of p19SKP1) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45SKP2–CUL‐1–p19SKP1 complex may be a human representative of an SCF‐type E3 ubiquitin protein ligase. We propose that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase. Finally, we show that multiprotein complex formation involving p45SKP2–CUL‐1 and p19SKP1 is governed, in part, by periodic, S phase‐specific accumulation of the p45SKP2 subunit and by the p45SKP2‐bound cyclin A–CDK2. The dependency of p45SKP2–p19SKP1 complex formation on cyclin A–CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway.

PKA phosphorylates and inactivates AMPKα to promote efficient lipolysis

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by β‐adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone‐sensitive lipase (HSL). TAG resynthesis is associated with high‐energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA‐mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKα1 at Ser‐173 to impede threonine (Thr‐172) phosphorylation and thus activation of AMPKα1 by LKB1 in response to lipolytic signals. Activation of AMPKα1 by LKB1 is also blocked by PKA‐mediated phosphorylation of AMPKα1 in vitro. Functional analysis of an AMPKα1 species carrying a non‐phosphorylatable mutation at Ser‐173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA‐activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.

pVHL and GSK3|[beta]| are components of a primary cilium-maintenance signalling network

Defects in the structure or function of the primary cilium, an antennae-like structure whose functional integrity has been linked to the suppression of uncontrolled kidney epithelial cell proliferation, are a common feature of genetic disorders characterized by kidney cysts. However, the mechanisms by which primary cilia are maintained remain poorly defined. von Hippel-Lindau (VHL) disease is characterized by the development of premalignant renal cysts and arises because of functional inactivation of the VHL tumour suppressor gene product, pVHL. Here, we show that pVHL and glycogen synthase kinase (GSK)3β are key components of an interlinked signalling pathway that maintains the primary cilium. Although inactivation of either pVHL or GSK3β alone did not affect cilia maintenance, their combined inactivation leads to loss of cilia. In VHL patients, GSK3β is subjected to inhibitory phosphorylation in renal cysts, but not in early VHL mutant lesions, and these cysts exhibit reduced frequencies of primary cilia. We propose that pVHL and GSK3β function together in a ciliary-maintenance signalling network, disruption of which enhances the vulnerability of cells to lose their cilia, thereby promoting cyst formation.

3D cell culture systems modeling tumor growth determinants in cancer target discovery

Phenotypic heterogeneity of cancer cells, cell biological context, heterotypic crosstalk and the microenvironment are key determinants of the multistep process of tumor development. They sign responsible, to a significant extent, for the limited response and resistance of cancer cells to molecular-targeted therapies. Better functional knowledge of the complex intra- and intercellular signaling circuits underlying communication between the different cell types populating a tumor tissue and of the systemic and local factors that shape the tumor microenvironment is therefore imperative. Sophisticated 3D multicellular tumor spheroid (MCTS) systems provide an emerging tool to model the phenotypic and cellular heterogeneity as well as microenvironmental aspects of in vivo tumor growth. In this review we discuss the cellular, chemical and physical factors contributing to zonation and cellular crosstalk within tumor masses. On this basis, we further describe 3D cell culture technologies for growth of MCTS as advanced tools for exploring molecular tumor growth determinants and facilitating drug discovery efforts. We conclude with a synopsis on technological aspects for on-line analysis and post-processing of 3D MCTS models.

The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts

The ubiquitin protein ligase SCFSkp2 is composed of Skp1, Cul1, Roc1/Rbx1 and the F‐box protein Skp2, the substrate‐recognition subunit. Levels of Skp2 decrease as cells exit the cell cycle and increase as cells re‐enter the cycle. Ectopic expression of Skp2 in quiescent fibroblasts causes mitogen‐independent S‐phase entry. Hence, mechanisms must exist for limiting Skp2 protein expression during the G0/G1 phases. Here we show that Skp2 is degraded by the proteasome in G0/G1 and is stabilized when cells re‐enter the cell cycle. Rapid degradation of Skp2 in quiescent cells depends on Skp2 sequences that contribute to Cul1 binding and interference with endogenous Cul1 function in serum‐deprived cells induces Skp2 expression. Furthermore, recombinant Cul1–Roc1/Rbx1–Skp1 complexes can catalyse Skp2 ubiquitylation in vitro. These results suggest that degradation of Skp2 in G0/G1 is mediated, at least in part, by an autocatalytic mechanism involving a Skp2‐bound Cul1‐based core ubiquitin ligase and imply a role for this mechanism in the suppression of SCFSkp2 ubiquitin protein ligase function during the G0/G1 phases of the cell cycle.