The Ubiquitin Ligase COP1 Promotes Glioma Cell Proliferation by Preferentially Downregulating Tumor Suppressor p53

Human glioma causes substantial morbidity and mortality worldwide. However, the molecular mechanisms underlying glioma progression are still largely unknown. COP1 (constitutively photomorphogenic 1), an E3 ubiquitin ligase, is important in cell survival, development, cell growth, and cancer biology by regulating different substrates. As is well known, both tumor suppressor p53 and oncogenic protein c-JUN could be ubiquitinated and degraded by ubiquitin ligase COP1, which may be the reason that COP1 serves as an oncogene or a tumor suppressor in different cancer types. Up to now, the possible role of COP1 in human glioma is still unclear. In the present study, we found that the expression of COP1 was upregulated in human glioma tissues. The role of COP1 in glioma cell proliferation was investigated using COP1 loss- and gain-of-function. The results showed that downregulation of COP1 by short hairpin RNA (shRNA) inhibited glioma cell proliferation, while overexpression of COP1 significantly promoted it. Furthermore, we demonstrated that COP1 only interacted with and regulated p53, but not c-JUN. Taken together, these results indicate that COP1 may play a role in promoting glioma cell proliferation by interacting with and downregulating tumor suppressor p53 rather than oncogenic protein c-JUN.     

Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27(Kip1) at G1 phase

The cyclin-dependent kinase inhibitor p27(Kip1) is degraded at the G0-G1 transition of the cell cycle by the ubiquitin-proteasome pathway. Although the nuclear ubiquitin ligase (E3) SCF(Skp2) is implicated in p27(Kip1) degradation, proteolysis of p27(Kip1) at the G0-G1 transition proceeds normally in Skp2(-/-) cells. Moreover, p27(Kip1) is exported from the nucleus to the cytoplasm at G0-G1 (refs 9-11). These data suggest the existence of a Skp2-independent pathway for the degradation of p27(Kip1) at G1 phase. We now describe a previously unidentified E3 complex: KPC (Kip1 ubiquitination-promoting complex), consisting of KPC1 and KPC2. KPC1 contains a RING-finger domain, and KPC2 contains a ubiquitin-like domain and two ubiquitin-associated domains. KPC interacts with and ubiquitinates p27(Kip1) and is localized to the cytoplasm. Overexpression of KPC promoted the degradation of p27(Kip1), whereas a dominant-negative mutant of KPC1 delayed p27(Kip1) degradation. The nuclear export of p27(Kip1) by CRM1 seems to be necessary for KPC-mediated proteolysis. Depletion of KPC1 by RNA interference also inhibited p27(Kip1) degradation. KPC thus probably controls degradation of p27(Kip1) in G1 phase after export of the latter from the nucleus.     

Regulation of p27Kip1 by mitogen-induced tyrosine phosphorylation

Extracellular mitogen signal transduction is initiated by ligand binding to specific receptors of target cells. This causes a cellular response that frequently triggers the activation of tyrosine kinases. Non-receptor kinases like Src and Lyn can directly phosphorylate the Cdk inhibitor protein p27^Kip1. Tyrosine phosphorylation can cause impaired Cdk-inhibitory activity and decreased stability of p27. In addition to these non-receptor tyrosine kinases, the receptor-associated tyrosine kinase Janus kinase 2 (JAK2) was recently identified to phosphorylate p27. JAK2 becomes activated through binding of various cytokines and growth factors to their corresponding receptors and can directly bind and selectively phosphorylate tyrosine residue 88 (Y88) of the Cdk inhibitor p27. This impairs Cdk inhibition by p27 and promotes its ubiquitin-dependent proteasomal degradation. Via this mechanism, JAK2 can link cytokine and growth factor initiated signal transduction to p27 regulation, whereas oncogenes like JAK2V617F or BCR-Abl can use this mechanism to inactivate the Cdk inhibitor.     

Regulation of p27Kip1 by intracellular iron levels

Enhanced intracellular iron levels are essential for proliferation of mammalian cells. If cells have entered S phase when iron is limiting, an adequate supply of deoxynucleotides cannot be maintained and the cells arrest with incompletely replicated DNA. In contrast, proliferating cells that are not in S phase, but have low iron pools, arrest in late G1. In this report the mechanism of iron-dependent G1 arrest in normal fibroblasts was investigated. Cells were synchronized in G0 by contact inhibition and serum deprivation. Addition of serum caused the cells to re-enter the cell cycle and enter S phase. However, if the cells were also treated with the iron chelator deferoxamine, S phase entry was blocked. This corresponded to elevated levels of the cyclin dependent kinase inhibitor p27^Kip1 and inhibition of CDK2 activity. Expression of other cell cycle regulatory proteins was not affected, including the induction of cyclins D1 and E. When the quiescent serum starved cells were supplemented with a readily usable form of iron in the absence of serum or any other growth factors, a significant population of the cells entered S phase. This was associated with downregulation of p27^Kip1 and increased CDK2 activity. Using an IPTG-responsive construct to artificially raise p27^Kip1 levels blocked the ability of iron supplementation to promote S phase entry. Thus it appears that p27^Kip1 is a mediator of G1 arrest in iron depleted Swiss 3T3 fibroblasts. We propose that this is part of an iron-sensitive checkpoint that functions to ensure that cells have sufficient iron pools to support DNA synthesis prior to entry into S phase.     

MAP kinase-dependent degradation of p27Kip1 by calpains in choroidal melanoma cells. Requirement of p27Kip1 nuclear export

We investigated the status and the regulation of the cyclin-dependent kinases (CDK) inhibitor p27(Kip1) in a choroidal melanoma tumor-derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27(Kip1) was low in these cells and the mitogen-activated protein (MAP) kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27(Kip1) accumulation, whereas MAP kinase reactivation triggered a down-regulation of p27(Kip1) that could be partially reversed by calpain inhibitors. In good accordance, ectopic expression of the cellular calpain inhibitor calpastatin led to an increase of endogenous p27(Kip1) expression. In vitro, p27(Kip1) was degraded by calpains, and OCM-1 cell extracts contained a calcium-dependent p27(Kip1) degradation activity. MAP kinase inhibition partially inhibited both calpain activity and calcium-dependent p27(Kip1) degradation by cellular extracts. Immunofluorescence labeling and subcellular fractionation revealed that p27(Kip1) was in part localized in the cytoplasmic compartment of OCM-1 cells but not of melanocytes, and accumulated into the nucleus upon MAP kinase inhibition. MAP kinase activation triggered a cytoplasmic translocation of the protein, as well as a change in its phosphorylation status. This CRM-1-dependent cytoplasmic translocation was necessary for MAP kinase- and calpain-dependent degradation. Taken together, these data suggest that in tumor-derived cells, p27(Kip1) could be degraded by calpains through a MAP kinase-dependent process, and that abnormal cytoplasmic localization of the protein, probably linked to modifications of its phosphorylation state, could be involved in this alternative mechanism of degradation.     

Multiple Mechanisms for p27Kip1 Translocation and Degradation

The nuclear export and rapid degradation of p27^Kip1 at the G₀–G₁ transition are critical events for effective progression of the cell cycle. Several pathways have been proposed at the molecular level for the export of this cyclin-dependent kinase inhibitor from the nucleus. However, the addition of each new pathway renders the situation more complicated. We recently showed that cyclin D2 links growth signals to the cytoplasmic translocation and degradation of p27 at the G₀–G₁ transition. Here we describe our findings and discuss how the multiple potential mechanisms for p27 translocation that precedes its degradation might be integrated in the context of growth stimulation and G1 progression.     

Cytokine-stimulated T lymphocyte proliferation is regulated by p27Kip1

T lymphocyte growth is regulated by the cyclin-dependent kinase inhibitor p27(Kip1). Mice deficient in p27(Kip1) have increased proliferative responses to multiple cytokines, including IL-2, IL-4, and IL-12, but not to anti-CD3. In the absence of p27(Kip1), T cells proliferate faster than control cells, as evidenced by increased [(3)H]thymidine uptake, increased cell growth and division, and an increased number of cells in S phase. Importantly, this regulation is specific for p27(Kip1) in T cells, because hyperproliferation of T cells from mice deficient in p21(Cip1/Waf1) was not observed. In vivo, there is an expansion of activated/memory CD4(+) cells in p27(Kip1)-deficient mice before and after immunization. Furthermore, Ag-stimulated spleen cells from immunized p27(Kip1)-deficient mice demonstrated increased proliferative responses to IL-2 and increased secretion of IFN-gamma. Although IL-4 stimulated proliferative responses are diminished in Stat6-deficient T cells, activated T cells from mice doubly deficient in both p27(Kip1) and Stat6 recover normal proliferative responses to IL-4. Together, these data firmly support a role for p27(Kip1) as a negative regulator of cytokine-stimulated T cell growth.     

Regulation of SCFSKP2 Ubiquitin E3 Ligase Assembly and p27KIP1 Proteolysis by the PTEN Pathway and Cyclin D1

The PTEN tumor suppressor functions as a phosphatase of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and negatively regulates the PI 3-kinase signaling pathway. Our previous studies showed that PTEN expression causes accumulation of cyclin-dependent kinase inhibitor p27Kip1 and G1 cell cycle arrest. Here, we show that PTEN negatively regulates expression of cyclin D1 and that cyclin D1 plays a unique role in p27 proteolysis. Co-expression of cyclin D1, but not cyclin E, is sufficient to restore p27 levels in PTEN-expressing cells. Conversely, loss of cyclin D1 by siRNA causes p27 accumulation. Silencing of the cyclin D1 gene or inhibition of the PI 3-kinase pathway prevents formation of the SCFSKP2 complex, with a simultaneous increase in CUL1 binding to CAND1. CAND1-CUL1 binding is known to block the accessibility of CUL1 to SKP1 and SKP2. We have found that CUL1 is less neddylated in cells that have lost cyclin D1 expression. Using an in vitro extract system, we found that the extracts prepared from cells lacking cyclin D1 have reduced activity to neddylate CUL1, in a manner similar to extracts from cells treated with a PI 3-kinase inhibitor or in G0 resting cells. Consistenly, the steady state levels of CUL1 neddylation were found lower under these conditions. Our studies reveal that PTEN/PI 3-kinase signaling and cyclin D1 control a novel pathway that regulates assembly of the SCFSKP2 complex by modulating cullin neddylation and CAND1 binding at the G1/S cell cycle transition.     

Inhibition of PDGF-B Induction and Cell Growth by Syndecan-1 Involves the Ubiquitin and SUMO-1 Ligase, Topors

Syndecans are receptors for soluble ligands, including heparin-binding growth factors, and matrix proteins. However, intracellular targets of syndecan-1 (Sdc-1)-mediated signaling are not fully understood. A yeast two-hybrid protein interaction screening of a mouse embryo library identified the ubiquitin and SUMO-1 E3 ligase, Topors, as a novel ligand of the Sdc-1 cytoplasmic domain (S1CD), a finding confirmed by ligand blotting and co-precipitation with Sdc-1 from cell lysates. Deletion mutagenesis identified an 18-amino acid sequence of Topors required for the interaction with the S1CD. By immunohistochemistry, Topors and Sdc-1 co-localized near the cell periphery in normal murine mammary gland (NMuMG) cells in vitro and in mouse embryonic epithelia in vivo. Finally, siRNA-mediated knockdown of Topors demonstrated that Topors is a growth promoter for murine arterial smooth muscle cells and is required for the inhibitory effect of Sdc-1 on cell growth and platelet-derived growth factor-B induction. These data suggest a novel mechanism for the inhibitory effects of Sdc-1 on cell growth that involves the interaction between the cytoplasmic domain of Sdc-1 and the SUMO-1 E3 ligase, Topors.     

Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension

The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27^Kip1, and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.     

Direct and Indirect Control of Mitogen-activated Protein Kinase Pathway-associated Components,BRAP/IMP E3 Ubiquitin Ligase and CRAF/RAF1 Kinase,by the Deubiquitylating Enzyme USP15

The opposing regulators of ubiquitylation status, E3 ligases and deubiquitylases, are often found to be associated in complexes. Here we report on a novel interaction between the E3 ligase BRAP (also referred to as IMP), a negative regulator of the MAPK scaffold protein KSR, and two closely related deubiquitylases, USP15 and USP4. We map the interaction to the N-terminal DUSP-UBL domain of USP15 and the coiled coil region of BRAP. USP15 as well as USP4 oppose the autoubiquitylation of BRAP, whereas BRAP promotes the ubiquitylation of USP15. Importantly, USP15 but not USP4 depletion destabilizes BRAP by promoting its proteasomal degradation, and BRAP-protein levels can be rescued by reintroducing catalytically active but not inactive mutant USP15. Unexpectedly, USP15 depletion results in a decrease in amplitude of MAPK signaling in response to EGF and PDGF. We provide evidence for a model in which the dominant effect of prolonged USP15 depletion upon signal amplitude is due to a decrease in CRAF levels while allowing for the possibility that USP15 may also function to dampen MAPK signaling through direct stabilization of a negative regulator, the E3 ligase BRAP.     

JosD1, a Membrane-targeted Deubiquitinating Enzyme,Is Activated by Ubiquitination and Regulates Membrane Dynamics,Cell Motility,and Endocytosis

The functional diversity of deubiquitinating enzymes (DUBs) is not well understood. The MJD family of DUBs consists of four cysteine proteases that share a catalytic “Josephin” domain. The family is named after the DUB ATXN3, which causes the neurodegenerative disease Machado-Joseph disease. The two closely related Josephin domain-containing (JosD) proteins 1 and 2 consist of little more than the Josephin domain. To gain insight into the properties of Josephin domains, we investigated JosD1 and JosD2. JosD1 and JosD2 were found to differ fundamentally in many respects. In vitro, only JosD2 can cleave ubiquitin chains. In contrast, JosD1 cleaves ubiquitin chains only after it is monoubiquitinated, a form of posttranslational-dependent regulation shared with ATXN3. A significant fraction of JosD1 is monoubiquitinated in diverse mouse tissues. In cell-based studies, JosD2 localizes to the cytoplasm whereas JosD1 preferentially localizes to the plasma membrane, particularly when ubiquitinated. The membrane occupancy by JosD1 suggests that it could participate in membrane-dependent events such as cell motility and endocytosis. Indeed, time-lapse imaging revealed that JosD1 enhances membrane dynamics and cell motility. JosD1 also influences endocytosis in cultured cells by increasing the uptake of endocytic markers of macropinocytosis while decreasing those for clathrin- and caveolae-mediated endocytosis. Our results establish that two closely related DUBs differ markedly in activity and function and that JosD1, a membrane-associated DUB whose activity is regulated by ubiquitination, helps regulate membrane dynamics, cell motility, and endocytosis.     

Hsp70 Targets a Cytoplasmic Quality Control Substrate to the San1p Ubiquitin Ligase

Accumulation of misfolded proteins in cellular compartments can result in stress-induced cell death. In the endoplasmic reticulum (ER), ER-associated degradation clears aberrant proteins from the secretory pathway. In the cytoplasm and nucleus, this job is left to the cytoplasmic quality control (CytoQC) machinery. Both processes utilize chaperones and the ubiquitin-proteasome system to aid in protein elimination. Previous studies in yeast have drawn comparisons between these processes using data from structurally and topologically different substrates. We sought to draw a direct comparison between ERAD and CytoQC by studying the elimination of a single misfolded domain that, depending on its residence, is disposed by either of these pathways. The truncated, second nucleotide binding domain (NBD2*) from a yeast ERAD substrate, Ste6p*, resides at the cytoplasmic face of the ER. We show that a soluble form of NBD2* is cytoplasmic and unlike wild-type NBD2 is targeted for proteasome-mediated degradation. In contrast to Ste6p*, which employs the ER-localized Doa10p ubiquitin ligase, NBD2* is ubiquitinated by a nuclear E3 ligase San1p, a factor that is also required for its degradation. Although the yeast cytoplasmic Hsp70 chaperone, Ssa1p, has been thought to facilitate the nuclear import or to maintain the solubility of most CytoQC substrates, we discovered that Ssa1p facilitates the interaction between San1p and NBD2*, demonstrating that chaperones can aid in substrate recognition and San1p-dependent protein degradation. These results emphasize the diverse action of molecular chaperones during CytoQC.     

p27Kip1, a double-edged sword in Shh-mediated medulloblastoma

Medulloblastoma, a brain tumor arising in the cerebellum, is the most common solid childhood malignancy. The current standard of care for medulloblastoma leaves survivors with life-long side effects. Gaining insight into mechanisms regulating transformation of medulloblastoma cells-of-origin may lead to development of better treatments for these tumors. Cerebellar granule neuron precursors (CGNPs) are proposed cells-of-origin for certain classes of medulloblastoma, specifically those marked by aberrant Sonic hedgehog (Shh) signaling pathway activation. CGNPs require signaling by Shh for proliferation during brain development. In mitogen-stimulated cells, nuclear localized cyclin dependent kinase (cdk) inhibitor p27 (Kip1) functions as a checkpoint control at the G1- to S-phase transition by inhibiting cdk2. Recent studies have suggested cytoplasmically localized p27^kip1 acquires oncogenic functions. Here, we show that p27^Kip1 is cytoplasmically localized in CGNPs and mouse Shh-mediated medulloblastomas. Tranasgenic mice bearing an activating mutation in the Shh pathway and lacking one or both p27^Kip1 alleles have accelerated tumor incidence compared to mice bearing both p27^Kip1 alleles. Interestingly, mice heterozygous for p27^Kip1 have decreased survival latency compared to p27^Kip1-null animals. Our data indicate that this may reflect the requirement for at least one copy of p27^Kip1 for recruiting cyclin D/cdk4/6 to promote cell cycle progression yet insufficient expression in the heterozygous or null state to inhibit cyclin E/cdk2. Finally, we find that mis-localized p27^Kip1 may play a positive role in motility in medulloblastoma cells. Together, our data indicate that the dosage of p27^Kip1 plays a role in cell cycle progression and tumor suppression in Shh-mediated medulloblastoma expansion.     

Phosphorylation of p27Kip1 at Thr187 by Cyclin-dependent Kinase 5 Modulates Neural Stem Cell Differentiation

Cyclin-dependent kinase 5 (Cdk5) plays a key role in the development of the mammalian nervous system; it phosphorylates a number of targeted proteins involved in neuronal migration during development to synaptic activity in the mature nervous system. Its role in the initial stages of neuronal commitment and differentiation of neural stem cells (NSCs), however, is poorly understood. In this study, we show that Cdk5 phosphorylation of p27^Kip1 at Thr187 is crucial to neural differentiation because 1) neurogenesis is specifically suppressed by transfection of p27^Kip1 siRNA into Cdk5^+/+ NSCs; 2) reduced neuronal differentiation in Cdk5^−/− compared with Cdk5^+/+ NSCs; 3) Cdk5^+/+ NSCs, whose differentiation is inhibited by a nonphosphorylatable mutant, p27/Thr187A, are rescued by cotransfection of a phosphorylation-mimicking mutant, p27/Thr187D; and 4) transfection of mutant p27^Kip1 (p27/187A) into Cdk5^+/+ NSCs inhibits differentiation. These data suggest that Cdk5 regulates the neural differentiation of NSCs by phosphorylation of p27^Kip1 at theThr187 site. Additional experiments exploring the role of Ser10 phosphorylation by Cdk5 suggest that together with Thr187 phosphorylation, Ser10 phosphorylation by Cdk5 promotes neurite outgrowth as neurons differentiate. Cdk5 phosphorylation of p27^Kip1, a modular molecule, may regulate the progress of neuronal differentiation from cell cycle arrest through differentiation, neurite outgrowth, and migration.     

The N Terminus of Cbl-c Regulates Ubiquitin Ligase Activity by Modulating Affinity for the Ubiquitin-conjugating Enzyme

Cbl proteins are ubiquitin ligases (E3s) that play a significant role in regulating tyrosine kinase signaling. There are three mammalian family members: Cbl, Cbl-b, and Cbl-c. All have a highly conserved N-terminal tyrosine kinase binding domain, a catalytic RING finger domain, and a C-terminal proline-rich domain that mediates interactions with Src homology 3 (SH3) containing proteins. Although both Cbl and Cbl-b have been studied widely, little is known about Cbl-c. Published reports have demonstrated that the N terminus of Cbl and Cbl-b have an inhibitory effect on their respective E3 activity. However, the mechanism for this inhibition is still unknown. In this study we demonstrate that the N terminus of Cbl-c, like that of Cbl and Cbl-b, inhibits the E3 activity of Cbl-c. Furthermore, we map the region responsible for the inhibition to the EF-hand and SH2 domains. Phosphorylation of a critical tyrosine (Tyr-341) in the linker region of Cbl-c by Src or a phosphomimetic mutation of this tyrosine (Y341E) is sufficient to increase the E3 activity of Cbl-c. We also demonstrate for the first time that phosphorylation of Tyr-341 or the Y341E mutation leads to a decrease in affinity for the ubiquitin-conjugating enzyme (E2), UbcH5b. The decreased affinity of the Y341E mutant Cbl-c for UbcH5b results in a more rapid turnover of bound UbcH5b coincident with the increased E3 activity. These data suggest that the N terminus of Cbl-c contributes to the binding to the E2 and that phosphorylation of Tyr-341 leads to a decrease in affinity and an increase in the E3 activity of Cbl-c.