Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases

The ubiquitin-mediated proteolysis of cyclin E plays a central role in cell-cycle progression, and cyclin E accumulation is a common event in cancer. Cyclin E degradation is triggered by multisite phosphorylation, which induces binding to the SCF(Fbw7) ubiquitin ligase complex. Structures of the Skp1-Fbw7 complex bound to cyclin E peptides identify a doubly phosphorylated pThr380/pSer384 cyclin E motif as an optimal, high-affinity degron and a singly phosphorylated pThr62 motif as a low-affinity one. Biochemical data indicate that the closely related yeast SCF(Cdc4) complex recognizes the multisite phosphorylated Sic1 substrate similarly and identify three doubly phosphorylated Sic1 degrons, each capable of high-affinity interactions with two Cdc4 phosphate binding sites. A model that explains the role of multiple cyclin E/Sic1 degrons is provided by the findings that Fbw7 and Cdc4 dimerize, that Fbw7 dimerization enhances the turnover of a weakly associated cyclin E in vivo, and that Cdc4 dimerization increases the rate and processivity of Sic1 ubiquitination in vitro.     

Emi1 proteolysis: how SCF(beta-Trcp1) helps to activate the anaphase-promoting complex

Emi1 inhibits the anaphase-promoting complex (APC) during S and G2 phase. Two papers by Guardavaccaro et al. and Margottin-Goguet et al. in the June issue of Developmental Cell now show that Emi1 degradation in early mitosis is mediated by beta-Trcp1, an adaptor protein that recruits proteins to the SCF ubiquitin ligase.     

Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex

Growth factor-dependent accumulation of the cyclin D1 proto-oncogene is balanced by its rapid phosphorylation-dependent proteolysis. Degradation is triggered by threonine 286 phosphorylation, which promotes its ubiquitination by an unknown E3 ligase. We demonstrate that Thr286-phosphorylated cyclin D1 is recognized by a Skp1-Cul1-F box (SCF) ubiquitin ligase where FBX4 and alphaB crystallin govern substrate specificity. Overexpression of FBX4 and alphaB crystallin triggered cyclin D1 ubiquitination and increased cyclin D1 turnover. Impairment of SCF(FBX4-alphaB crystallin) function attenuated cyclin D1 ubiquitination, promoting cyclin D1 overexpression and accelerated cell-cycle progression. Purified SCF(FBX4-alphaB crystallin) catalyzed polyubiquitination of cyclin D1 in vitro. Consistent with a putative role for a cyclin D1 E3 ligase in tumorigenesis, FBX4 and alphaB crystallin expression was reduced in tumor-derived cell lines and a subset of primary human cancers that overexpress cyclin D1. We conclude that SCF(FBX4-alphaB crystallin) is an E3 ubiquitin ligase that promotes ubiquitin-dependent degradation of Thr286-phosphorylated cyclin D1.     

Mitosis persists in the absence of Cdk1 activity when proteolysis or protein phosphatase activity is suppressed

Cellular transition to anaphase and mitotic exit has been linked to the loss of cyclin-dependent kinase 1 (Cdk1) kinase activity as a result of anaphase-promoting complex/cyclosome (APC/C)–dependent specific degradation of its cyclin B1 subunit. Cdk1 inhibition by roscovitine is known to induce premature mitotic exit, whereas inhibition of the APC/C-dependent degradation of cyclin B1 by MG132 induces mitotic arrest. In this study, we find that combining both drugs causes prolonged mitotic arrest in the absence of Cdk1 activity. Different Cdk1 and proteasome inhibitors produce similar results, indicating that the effect is not drug specific. We verify mitotic status by the retention of mitosis-specific markers and Cdk1 phosphorylation substrates, although cells can undergo late mitotic furrowing while still in mitosis. Overall, we conclude that continuous Cdk1 activity is not essential to maintain the mitotic state and that phosphatase activity directed at Cdk1 substrates is largely quiescent during mitosis. Furthermore, the degradation of a protein other than cyclin B1 is essential to activate a phosphatase that, in turn, enables mitotic exit.     

Potential relation of aberrant proteolysis of human protein tyrosine kinase 7 (PTK7) chuzhoi by membrane type 1 matrix metalloproteinase (MT1-MMP) to congenital defects

Membrane PTK7 pseudo-kinase plays an essential role in planar cell polarity and the non-canonical Wnt pathway in vertebrates. Recently, a new N-ethyl-N-nitrosourea-induced mutant named chuzhoi (chz) was isolated in mice. chz embryos have severe birth defects, including a defective neural tube, defective heart and lung development, and a shortened anterior-posterior body axis. The chz mutation was mapped to the Ala-Asn-Pro tripeptide insertion into the junction region between the fifth and the sixth Ig-like domains of PTK7. Unexpectedly, chz reduced membrane localization of the PTK7 protein. We hypothesized and then proved that the chz mutation caused an insertion of an additional membrane type 1 matrix metalloproteinase cleavage site in PTK7 and that the resulting aberrant proteolysis of chz affected the migratory parameters of the cells. It is likely that aberrations in the membrane type 1 matrix metalloproteinase/PTK7 axis are detrimental to cell movements that shape the body plan and that chz represents a novel model system for increasing our understanding of the role of proteolysis in developmental pathologies, including congenital defects.     

Identification of a common protein association region in the neuronal Cdk5 activator

Cyclin-dependent protein kinase 5 (Cdk5) depends on the association with neuronal Cdk5 activator (Nck5a) for kinase activity. A variety of cellular proteins have been shown to undergo high affinity association with Nck5a, including three novel proteins, C42, C48, and C53 found by a yeast two-hybrid screen (Ching, Y. P., Qi, Z., and Wang, J. H. (2000) Gene 242, 285-294). The three proteins show competitive binding to Nck5a suggesting that they bind at a common site. The binding site has been mapped to a region of 26 amino acid residues (residues 145 to 170) at the N-terminal boundary of the kinase activation domain of Nck5a. This region of Nck5a contains an amphipathic alpha-helix whose hydrophobic face is involved in Cdk5 activation (Chin, K. T., Ohki, S, Tang, D., Cheng, H. C., Wang, J. H. , and Zhang, M. (1999) J. Biol. Chem. 274, 7120-7127). Several lines of evidence suggest that Nck5a interacts with the binding proteins at the hydrophilic face of the amphipathic alpha-helix. First, the Nck5a-(145-170) peptide can bind Cdk5 and Nck5a-binding proteins simultaneously. Second, the association of Nck5a-(145-170) to C48 can be markedly reduced by high ionic strength whereas the interaction between Nck5a and Cdk5 is not affected. Third, substitution of Glu(157) by glutamine in Nck5a-(145-170) abolishes the peptide's ability to bind to the three Nck5a-binding proteins without diminishing its Cdk5 binding activity.     

Selective chemical inhibition as a tool to study Cdk1 and Cdk2 functions in the cell cycle

Cyclin-dependent kinases are highly conserved among all eukaryotes, and have essential roles in the cell cycle. However, these roles are still only poorly understood at a molecular level, partly due to the functional redundance of different Cdk complexes. Indeed, mice knockouts have even thrown into some doubt the assumed essential roles for Cdk2-cyclin E in triggering S-phase, but this is almost certainly due to compensation by Cdk1 complexes. By combining both knockout approaches and chemical Cdk inhibition in Xenopus egg extracts, we have shown that one reason for functional redundancy of Cdk control of S-phase is that Cdk activity required to trigger S-phase is very low. Cdk1 contributes to this activity even in the presence of Cdk2, and Cdk activity at this stage does not show "switch-like" regulation, as at the onset of mitosis. It is important to try to confirm and extend these findings to other cell-types, and to explain why different cells might have evolved different requirements for Cdk activity. In this paper, we present data that suggest that selective chemical Cdk inhibition will be a useful tool towards achieving this goal.     

Interaction between the SH3 domains and C-terminal proline-rich region in NADPH oxidase organizer 1 (Noxo1)

NADPH oxidase organizer 1 (Noxo1), harboring a PX domain, two SH3 domains, and a proline-rich region (PRR), participates in activation of superoxide-producing Nox-family NADPH oxidases. Here, we show that Noxo1 supports superoxide production in a cell-free system for gp91(phox)/Nox2 activation by arachidonic acid. This lipid enhances an SH3-mediated binding of Noxo1 to p22(phox), a protein complexed with Nox oxidases; the binding is known to be required for Nox activation. We also demonstrate that the bis-SH3 domain directly interacts with the Noxo1 PRR. The interaction appears to prevent the bis-SH3 domain and PRR from binding to their target proteins; disruption of the intramolecular interaction facilitates Noxo1 binding to p22(phox) and also allows the PRR to associate with the Nox activator Noxa1, which association is crucial for Nox activation as well. These findings suggest that Nox activation involves a conformational change leading to disruption of the bis-SH3-PRR interaction in Noxo1.     

Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly

Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV. The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as beta-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity. In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature. The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis. We have identified a P. carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function. The P. carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Delta yeast. Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Delta yeast to maintain expression of the temperature-inducible beta-1-3-glucan synthetase gene, FKS2. PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation. Since glucan machinery is not present in mammals, a better understanding of this pathway in P. carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall.     

A genetic interaction between a ubiquitin-like protein and ubiquitin-mediated proteolysis in Dictyostelium discoideum(1)

A ubiquitination factor, NosA, is essential for cellular differentiation in Dictyostelium discoideum. In the absence of nosA, development is blocked, resulting in a developmental arrest at the tight-aggregate stage, when cells differentiate into two precursor cell types, prespore and prestalk cells. Development is restored when a second gene, encoding the ubiquitin-like protein SonA, is inactivated in nosA-mutant cells. SonA has homology over its entire length to Dsk2 from Saccharomyces cerevisiae, a ubiquitin-like protein that is involved in the assembly of the spindle pole body. Dsk2 and SonA are both stable proteins that do not seem to be subjected to degradation via the ubiquitin pathway. SonA does not become ubiquitinated and the intracellular levels of SonA are not affected by the absence of NosA. The high degree of suppression suggests that SonA rescues most or all of the defects caused by the absence of nosA. We propose that NosA and SonA act in concert to control the activity of a developmental regulator that must be deactivated for cells to cross a developmental boundary.     

Translation of the alzheimer amyloid precursor protein mRNA is up-regulated by interleukin-1 through 5'-untranslated region sequences

The amyloid precursor protein (APP) has been associated with Alzheimer's disease (AD) because APP is processed into the beta-peptide that accumulates in amyloid plaques, and APP gene mutations can cause early onset AD. Inflammation is also associated with AD as exemplified by increased expression of interleukin-1 (IL-1) in microglia in affected areas of the AD brain. Here we demonstrate that IL-1alpha and IL-1beta increase APP synthesis by up to 6-fold in primary human astrocytes and by 15-fold in human astrocytoma cells without changing the steady-state levels of APP mRNA. A 90-nucleotide sequence in the APP gene 5'-untranslated region (5'-UTR) conferred translational regulation by IL-1alpha and IL-1beta to a chloramphenicol acetyltransferase (CAT) reporter gene. Steady-state levels of transfected APP(5'-UTR)/CAT mRNAs were unchanged, whereas both base-line and IL-1-dependent CAT protein synthesis were increased. This APP mRNA translational enhancer maps from +55 to +144 nucleotides from the 5'-cap site and is homologous to related translational control elements in the 5'-UTR of the light and and heavy ferritin genes. Enhanced translation of APP mRNA provides a mechanism by which IL-1 influences the pathogenesis of AD.     

Budding yeast Cdc6p induces re-replication in fission yeast by inhibition of SCF(Pop)-mediated proteolysis

In fission yeast, overexpression of the replication initiator protein Cdc18p induces re-replication, a phenotype characterized by continuous DNA synthesis in the absence of cell division. In contrast, overexpression of Cdc6p, the budding yeast homolog of Cdc18p, does not cause re-replication in S. cerevisiae. However, we have found that Cdc6p has the ability to induce re-replication in fission yeast. Cdc6p cannot functionally replace Cdc18p, but instead interferes with the proteolysis of both Cdc18p and Rum1p, the inhibitor of the protein kinase Cdc2p. This activity of Cdc6p is entirely contained within a short N-terminal peptide, which forms a tight complex with Cdc2p and the F-box/WD-repeat protein Sud1p/Pop2p, a component of the SCF^Pop ubiquitin ligase in fission yeast. These interactions are mediated by two distinct regions within the N-terminal region of Cdc6p and depend on the integrity of its Cdc2p phosphorylation sites. The data suggest that disruption of re-replication control by overexpression of Cdc6p in fission yeast is a consequence of sequestration of Cdc2p and Pop2p, two factors involved in the negative regulation of Rum1p, Cdc18p and potentially other replication proteins. Received: 29 April 1999 / Accepted: 27 June 1999     

Role of hepatitis B virus X protein in regulating LIM and SH3 protein 1 (LASP-1) expression to mediate proliferation and migration of hepatoma cells

ABSTRACT: BACKGROUND: Hepatitis B virus X protein (HBx) has been shown to be responsible for the development of hepatocellular carcinoma (HCC) caused by Hepatitis B virus infection. However, its potential effect on the progression of hepatocellular carcinoma remains yet unclear. LIM and SH3 protein 1 (LASP-1), a focal adhesion protein, is expressed in an up-regulation manner in the HCC tissues. LASP-1 plays an important role in the regulation of proliferation and migration of HCC. In this study, we investigated the effect of LASP-1 involved in HBx-related tumor progression. METHODS: LASP-1 levels in the HBx stable transfected HepG2 and Huh-7 cells were detected by RT-PCR and western blot analysis. The cellular localization of LASP-1 was assessed by immunofluorescence analysis. The activity of phosphatidylinositol 3-kinase (PI3-K) pathway was demonstrated by western blot assay. The HBx-expressing cells were transfected with specific small interference RNA (siRNA) against LASP-1. The proliferation and migration ability of cells were evaluated by cell viability assay and plate clone formation assay. The migration ability of cells was detected by transwell assay and wound healing assay. RESULTS: RT-PCR and western blot analysis indicated the expression of LASP-1 was increased in the stable HBx-expressing cells compared with the control cells. Immunofluorescence study revealed that the distributions of LASP-1 in HepG2-HBx cells were mainly in pseudopods and the cytoplasm while they were mainly localized in the cytoplasm of HepG2-mock cells. The cellular localizations of LASP-1 in Huh-7-HBx cells were in the perinuclear fractions while they were mainly localized in the cytoplasm of Huh-7-Mock cells. The upregulation of LASP-1 was inhibited after treatment with LY294002, PI3-K pathway inhibitor. Overexpression of LASP-1 in the stable HBx-expressing cells enhanced the proliferation and migration ability of hepatocellular cells. siRNA-mediated LASP-1 knowdown in the stable HBx-expressing cells significantly suppressed hepatocellular cells proliferation and migration. CONCLUSIONS: These results demonstrated that HBx could upregulate LASP-1 through PI3-K pathway to promote the proliferation and migration of hepatoma cells.     

Elevation of highly up-regulated in liver cancer (HULC) by hepatitis B virus X protein promotes hepatoma cell proliferation via down-regulating p18

Long noncoding RNAs (lncRNAs) play crucial roles in human cancers. It has been reported that lncRNA highly up-regulated in liver cancer (HULC) is dramatically up-regulated in hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) contributes importantly to the development of HCC. However, the function of HULC in HCC mediated by HBx remains unclear. Here, we report that HULC is involved in HBx-mediated hepatocarcinogenesis. We found that the expression levels of HULC were positively correlated with those of HBx in clinical HCC tissues. Moreover, we revealed that HBx up-regulated HULC in human immortalized normal liver L-O2 cells and hepatoma HepG2 cells. Luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay showed that HBx activated the HULC promoter via cAMP-responsive element-binding protein. We further demonstrated that HULC promoted cell proliferation by methyl thiazolyl tetrazolium, 5-ethynyl-2'-deoxyuridine, colony formation assay, and tumorigenicity assay. Next, we hypothesized that HULC might function through regulating a tumor suppressor gene p18 located near HULC in the same chromosome. We found that the mRNA levels of p18 were inversely correlated with those of HULC in the above clinical HCC specimens. Then, we validated that HULC down-regulated p18, which was involved in the HULC-enhanced cell proliferation in vitro and in vivo. Furthermore, we observed that knockdown of HULC could abolish the HBx-enhanced cell proliferation through up-regulating p18. Thus, we conclude that the up-regulated HULC by HBx promotes proliferation of hepatoma cells through suppressing p18. This finding provides new insight into the roles of lncRNAs in HBx-related hepatocarcinogenesis.     

miR-204 functions as a tumor suppressor by regulating SIX1 in NSCLC

The involvement of miR-204 in lung cancer development is unclear. In our study, we analyzed the expression of miR-204 in tumor- and adjacent-tissue samples from 141 patients with non-small cell lung cancer (NSCLC). MiR-204 expression was decreased in tumor samples compared with non-cancerous tissue-derived controls. Moreover, miR-204 expression negatively correlated with homeobox protein SIX1 expression, tumor size and metastasis. MiR-204 silencing in miR-204-positive NSCLC cell lines promoted cell invasion and proliferation. Concomitantly, MiR-204 overexpression resulted in reduced cell proliferation and invasion, upregulated E-cadherin and downregulated N-cadherin and Vimentin expression. SIX1 was identified as a potential target of miR-204, and SIX1 silencing partially compromised the invasive and proliferative capacity of miR-204-deficient cells. Thus, miR-204 may be involved in the NSCLC development.