Faculty Bibliography

The activity of the cyclin-dependent kinase inhibitor p27 is controlled by its concentration and subcellular localization. However, the mechanisms that regulate its intracellular transport are poorly understood. Here we show that p27 is phosphorylated on Ser10 in vivo and that mutation of Ser10 to Ala inhibits p27 cytoplasmic relocalization in response to mitogenic stimulation. In contrast, a fraction of wild-type p27 and a p27(S10D)-phospho-mimetic mutant translocates to the cytoplasm in the presence of mitogens. G1 nuclear export of p27 and its Ser10 phosphorylation precede cyclin-dependent kinase 2 (Cdk2) activation and degradation of the bulk of p27. Interestingly, leptomycin B-mediated nuclear accumulation accelerates the turnover of endogenous p27; the p27(S10A) mutant, which is trapped in the nucleus, has a shorter half-life than wild-type p27 and the p27(S10D) mutant. In summary, p27 is efficiently degraded in the nucleus and phosphorylation of Ser10 is necessary for the nuclear to cytoplasmic redistribution of a fraction of p27 in response to mitogenic stimulation. This cytoplasmic localization may serve to decrease the abundance of p27 in the nucleus below a certain threshold required for activation of cyclin-Cdk2 complexes.

Tax, an oncogenic viral protein encoded by human T cell leukemia virus type 1 (HTLV-1), induces cellular transformation of T lymphocytes by modulating a variety of cellular gene expressions [1]. Identifying cellular partners that interact with Tax constitutes the first step toward elucidating the molecular basis of Tax-induced transformation. Here, we report a novel Tax-interacting protein, hTid-1. hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, was initially characterized based on its interaction with the HPV-16 E7 oncoprotein [2]. hTid-1 and Tid56 are members of the DnaJ family [2,3], which contains a highly conserved signature J domain that regulates the activities of heat shock protein 70 (Hsp70) by serving as cochaperone [4-6]. In this context, the molecular chaperone complex is involved in cellular signaling pathways linked to apoptosis, protein folding, and membrane translocation and in modulation of the activities of tumor suppressor proteins, including retinoblastoma, p53, and WT1[7-12]. We find that expression of hTid-1 inhibits the transformation phenotype of two human lung adenocarcinoma cell lines. We show that Tax interacts with hTid-1 via a central cysteine-rich domain of hTid-1 while a signature J domain of hTid-1 mediates its binding to Hsp70 in HEK cells. Importantly, Tax associates with the molecular chaperone complex containing both hTid-1 and Hsp70 and alters the cellular localization of hTid-1 and Hsp70. In the absence of Tax, expression of the hTid-1/Hsp70 molecular complex is targeted to perinuclear mitochondrial clusters. In the presence of Tax, hTid-1 and its associated Hsp70 are sequestered within a cytoplasmic 'hot spot' structure, a subcellular distribution that is characteristic of Tax in HEK cells

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