Faculty Bibliography

The ubiquitin pathway is involved in the proteolytic turnover of many short-lived cellular regulatory proteins. Since selective degradation of substrates of this system requires the covalent attachment of a polyubiquitin chain to the substrates, degradation could be counteracted by de-ubiquitinating enzymes (or isopeptidases) which selectively remove the polyubiquitin chain. Unp is a human isopeptidase with still poorly understood biological functions. Here, we show that cellular Unp specifically interacts with the retinoblastoma gene product (pRb)

Activation of Jun N-kinase (JNK) and NF-kappaB transcription factor are the hallmarks of cellular response to stress. Phosphorylation of NF-kappaB inhibitor (IkappaB) by respective stress-inducible kinases (IKK) is a key event in NF-kappaB activation. beta-TrCP F-box protein mediates ubiquitination of phosphorylated IkappaB via recruitment of SCF(beta-TrCP)-Roc1 E3 ubiquitin ligase complex. Subsequent proteasome-dependent degradation of IkappaB results in activation of the NF-kappaB pathway. We found that a variety of cellular stress stimuli induce an increase in the steady state levels of beta-TrCP mRNA and protein levels in human cells. Activation of stress-activated protein kinases JNK (and, to a lesser extent, p38) by forced expression of constitutively active mutants of JNKK2 and MKK6 (but not MEK1 or IKKbeta) also leads to accumulation of beta-TrCP. Transcription of the beta-TrCP gene is not required for JNK-mediated induction of beta-TrCP. A synergistic effect of stimulation of IKK and JNK on the transcriptional activity of NF-kappaB was observed. The mechanisms of beta-TrCP induction via stress and its role in NF-kappaB activation are discussed

Cell adhesion to the extracellular matrix (ECM) is a requirement for proliferation that is typically lost in malignant cells. In the absence of adhesion, nontransformed cells arrest in G1 with increased levels of the cyclin-dependent kinase inhibitor p27. We have reported previously that the degradation of p27 requires its phosphorylation on Thr-187 and is mediated by Skp2, an F-box protein that associates with Skp1, Cul1, and Roc1/Rbx1 to form the SCF(Skp2) ubiquitin ligase complex. Here, we show that the accumulation of Skp2 protein is dependent on both cell adhesion and growth factors but that the induction of Skp2 mRNA is exclusively dependent on cell adhesion to the ECM. Conversely, the expression of the other three subunits of the SCF(Skp2) complex is independent of cell anchorage. Phosphorylation of p27 on Thr-187 is also not affected significantly by the loss of cell adhesion, demonstrating that increased p27 stability is not dependent on p27 dephosphorylation. Significantly, ectopic expression of Skp2 in nonadherent G1 cells resulted in p27 downregulation, entry into S phase, and cell division. The ability to induce adhesion-independent cell cycle progression was potentiated by coexpressing Skp2 with cyclin D1 but not with cyclin E, indicating that Skp2 and cyclin D1 cooperate to rescue proliferation in suspension cells. Our study shows that Skp2 is a key target of ECM signaling that controls cell proliferation

BACKGROUND: Previous studies have shown that low levels of p27(Kip1), an inhibitor of G1 cyclin-dependent kinases, are associated with high aggressiveness and poor prognosis in a variety of cancers. Decreased levels of p27 are caused, at least in part, by acceleration of the rate of its ubiquitin-mediated degradation. In cultured cells and cell-free biochemical systems, it has been shown that p27 is targeted for degradation by a ubiquitin ligase complex that contains Skp2 (S-phase kinase-associated protein 2) as the specific substrate-recognizing and rate-limiting subunit. This investigation was undertaken to examine the possible relation between levels of p27 and of its specific ubiquitin ligase subunit Skp2 in human cancers. METHODS: Quick-frozen colorectal tumor samples from 20 patients were homogenized at 0 degrees C in buffer containing a mixture of protease inhibitors. Samples were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose, and probed with highly specific monoclonal antibodies directed against Skp2 and p27. The expression of Skp2 also was examined by immunohistochemistry using formalin fixed, paraffin embedded tissue sections from the same cases. RESULTS: A strongly significant inverse correlation was found between levels of Skp2 and p27 (r = -0.812; P < 0.0001). Thus, decreased levels of p27 were associated with strongly increased levels of Skp2, whereas high levels of p27 coincided with low levels of Skp2. Immunohistochemical examination of Skp2 expression agreed with immunoblot analysis in 89% of cases. CONCLUSIONS: The results are compatible with the notion that increased expression of Skp2 may have a causative role in decreasing the levels of p27 in aggressive colorectal carcinomas.

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded in late G1 phase by the ubiquitin pathway, allowing CDK activity to drive cells into S phase. Ubiquitinylation of p27 requires its phosphorylation at Thr 187 (refs 3, 4) and subsequent recognition by S-phase kinase associated protein 2 (Skp2; refs 5-8), a member of the F-box family of proteins that associates with Skp1, Cul-1 and ROC1/Rbx1 to form an SCF ubiquitin ligase complex. However, in vitro ligation of p27 to ubiquitin could not be reconstituted by known purified components of the SCFSkp2 complex. Here we show that the missing factor is CDK subunit 1 (Cks1), which belongs to the highly conserved Suc1/Cks family of proteins that bind to some CDKs and phosphorylated proteins and are essential for cell-cycle progression. Human Cks1, but not other members of the family, reconstitutes ubiquitin ligation of p27 in a completely purified system, binds to Skp2 and greatly increases binding of T187-phosphorylated p27 to Skp2. Our results represent the first evidence that an SCF complex requires an accessory protein for activity as well as for binding to its phosphorylated substrate

The F-box protein Skp2 (S-phase kinase-associated protein 2) positively regulates the G(1)-S transition by controlling the stability of several G(1) regulators, such as the cell cycle inhibitor p27. We show here that Skp2 expression correlates directly with grade of malignancy and inversely with p27 levels in human lymphomas. To directly evaluate the potential of Skp2 to deregulate growth in vivo, we generated transgenic mice expressing Skp2 targeted to the T-lymphoid lineage as well as double transgenic mice coexpressing Skp2 and activated N-Ras. A strong cooperative effect between these two transgenes induced T cell lymphomas with shorter latency and higher penetrance, leading to significantly decreased survival when compared with control and single transgenic animals. Furthermore, lymphomas of Nras single transgenic animals often expressed higher levels of endogenous Skp2 than tumors of double transgenic mice. This study provides evidence of a role for an F-box protein in oncogenesis and establishes SKP2 as a protooncogene causally involved in the pathogenesis of lymphomas

This year the most prestigious prize in medical sciences, the Lasker Award, has been presented to the three scientists who discovered the ubiquitin pathway: Aaron Ciechanover, Avram Hershko, and Alexander Varshavsky [Nature Med. 6 (2000) 1073-1081]. During a time when the scientific community was focused on understanding how proteins were synthesized, they intently pursued the novel idea that cells were programmed to selectively destroy proteins. Their work led to the identification of an elaborate system of protein degradation targeting a myriad of cellular substrates. A small protein called ubiquitin is at the center of this process. Although the ubiquitin pathway was first described in the early 1980s, it has only more recently advanced to the forefront of basic research as a significant regulatory network within the cell. The field continues to grow as new ubiquitination enzymes and novel functions of this system are identified. Scientists are focused on elucidating the mechanisms by which cells deploy the ubiquitin pathway to control levels of selected proteins, such as cell cycle regulatory proteins, transcription factors and signaling molecules. Accelerated or decelerated rates of degradation of particular substrates participate in the genesis of many human diseases. Thus, understanding the mechanisms that confer specificity to the ubiquitin system will allow the development of novel therapeutic approaches to target aberrations in this pathway underlying tumorigenesis and other human pathologies

p27 is an inhibitor of cyclin dependent kinase involved in the regulation of the cell cycle. In this commentary we discuss the current knowledge on p27 in breast cancer and its significance in predicting the outcome. p27 protein levels are high in most cases of breast carcinomas, are correlated with the levels of cyclin D1 and estrogen receptor, and could be a useful predictor of survival, because they are low in aggressive carcinomas. Immunodetection of p27 in breast tumors could be useful in the assessment of prognosis, especially in those cases in which the commonly used parameters are insufficient, and might ultimately influence the therapy of this disease

F-box proteins are members of a large family that regulates the cell cycle, the immune response, signalling cascades and developmental programmes by targeting proteins, such as cyclins, cyclin-dependent kinase inhibitors, IkappaBalpha and beta-catenin, for ubiquitination (reviewed in refs 1-3). F-box proteins are the substrate-recognition components of SCF (Skp1-Cullin-F-box protein) ubiquitin-protein ligases. They bind the SCF constant catalytic core by means of the F-box motif interacting with Skp1, and they bind substrates through their variable protein-protein interaction domains. The large number of F-box proteins is thought to allow ubiquitination of numerous, diverse substrates. Most organisms have several Skp1 family members, but the function of these Skp1 homologues and the rules of recognition between different F-box and Skp1 proteins remain unknown. Here we describe the crystal structure of the human F-box protein Skp2 bound to Skp1. Skp1 recruits the F-box protein through a bipartite interface involving both the F-box and the substrate-recognition domain. The structure raises the possibility that different Skp1 family members evolved to function with different subsets of F-box proteins, and suggests that the F-box protein may not only recruit substrate, but may also position it optimally for the ubiquitination reaction