Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBC^VHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBC^VHL complex, and is probably one of the new substrates of the CBC^VHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBC^VHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBC^VHL complex, and is probably one of the new substrates of the CBC^VHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.     

Identification of Elongin C and Skp1 sequences that determine Cullin selection

The multiprotein von Hippel-Lindau (VHL) tumor suppressor and Skp1-Cul1-F-box protein (SCF) complexes belong to families of structurally related E3 ubiquitin ligases. In the VHL ubiquitin ligase, the VHL protein serves as the substrate recognition subunit, which is linked by the adaptor protein Elongin C to a heterodimeric Cul2/Rbx1 module that activates ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzyme Ubc5. In SCF ubiquitin ligases, F-box proteins serve as substrate recognition subunits, which are linked by the Elongin C-like adaptor protein Skp1 to a Cul1/Rbx1 module that activates ubiquitylation of target proteins, in most cases by the E2 Cdc34. In this report, we investigate the functions of the Elongin C and Skp1 proteins in reconstitution of VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 structural elements responsible for selective interaction with their cognate Cullin/Rbx1 modules. In addition, using altered specificity Elongin C and F-box protein mutants, we investigate models for the mechanism underlying E2 selection by VHL and SCF ubiquitin ligases. Our findings provide evidence that E2 selection by VHL and SCF ubiquitin ligases is determined not solely by the Cullin/Rbx1 module, the target protein, or the integrity of the substrate recognition subunit but by yet to be elucidated features of these macromolecular complexes.     

Characterization of the Cullin7 E3 ubiquitin ligase--heterodimerization of cullin substrate receptors as a novel mechanism to regulate cullin E3 ligase activity

Cul1 and Cul7 are cullin E3 ubiquitin ligase scaffold proteins. Cul1 is known to form a complex with the RING domain protein Rbx1 and one of approximately 70 different F-box proteins. F-box proteins function as substrate receptor subunits and recruit numerous substrates for poly-ubiquitination. Similarly to Cul1, Cul7 interacts with Rbx1, however, only one F-box protein, Fbxw8, has been shown to bind to Cul7. To date only few Cul7 E3 ubiquitin ligase substrates, including cyclin D1, IRS-1 and GRASP65, have been reported, and using Fbxw8 affinity purification, we were unable to identify additional substrate proteins. Here we provide evidence for a model in which Cul7-Rbx1 can promote the ubiquitination of Cul1 substrates by forming high order complexes with Cul1-Rbx1. Binding of Cul1-Rbx1 to Cul7-Rbx1 is mediated via heterodimerization of Fbxw8 with other F-box proteins which function to recruit substrates into the E3 ligase complex. The formation of this high order complex is likely to increase polyubiquitination efficiency.     

Deletion of the Cul1 gene in mice causes arrest in early embryogenesis and accumulation of cyclin E.

The stability of many proteins is controlled by the ubiquitin proteolytic system, which recognizes specific substrates through the action of E3 ubiquitin ligases [1]. The SCFs are a recently described class of ubiquitin ligase that target a number of cell cycle regulators and other proteins for degradation in both yeast and mammalian cells [2] [3] [4] [5] [6]. Each SCF complex is composed of the core protein subunits Skp1, Rbx1 and Cul1 (known as Cdc53 in yeast), and substrate-specific adaptor subunits called F-box proteins [2] [3] [4]. To understand the physiological role of SCF complexes in mammalian cells, we generated mice carrying a deletion in the Cul1 gene. Cul1(-/-) embryos arrested around embryonic day 6.5 (E6.5) before the onset of gastrulation. In all cells of the mutant embryos, cyclin E protein, but not mRNA, was highly elevated. Outgrowths of Cul1(-/-) blastocysts had limited proliferative capacity in vitro and accumulated cyclin E in all cells. Within Cul1(-/-) blastocyst cultures, trophoblast giant cells continued to endocycle despite the elevated cyclin E levels. These results suggest that cyclin E abundance is controlled by SCF activity, possibly through SCF-dependent degradation of cyclin E.     

Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.

Mitogen-activated protein (MAP) kinases bind tightly to many of their physiologically relevant substrates. We have identified a new subfamily of murine serine/threonine kinases, whose members, MAP kinase-interacting kinase 1 (Mnk1) and Mnk2, bind tightly to the growth factor-regulated MAP kinases, Erk1 and Erk2. MNK1, but not Mnk2, also binds strongly to the stress-activated kinase, p38. MNK1 complexes more strongly with inactive than active Erk, implying that Mnk and Erk may dissociate after mitogen stimulation. Erk and p38 phosphorylate MNK1 and Mnk2, which stimulates their in vitro kinase activity toward a substrate, eukaryotic initiation factor-4E (eIF-4E). Initiation factor eIF-4E is a regulatory phosphoprotein whose phosphorylation is increased by insulin in an Erk-dependent manner. In vitro, MNK1 rapidly phosphorylates eIF-4E at the physiologically relevant site, Ser209. In cells, Mnk1 is post-translationally modified and enzymatically activated in response to treatment with either peptide growth factors, phorbol esters, anisomycin or UV. Mitogen- and stress-mediated MNK1 activation is blocked by inhibitors of MAP kinase kinase 1 (Mkk1) and p38, demonstrating that Mnk1 is downstream of multiple MAP kinases. MNK1 may define a convergence point between the growth factor-activated and one of the stress-activated protein kinase cascades and is a candidate to phosphorylate eIF-4E in cells.     

Mnk kinase pathway: Cellular functions and biological outcomes

The mitogen-activated protein kinase(MAPK) interacting protein kinases 1 and 2(Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs(p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E(eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4 E. The role of Mnk kinases in inflammation and inflammationinduced malignancies is also discussed.     

ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins from ASB1 to ASB18 and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. ASB2 was recently shown to interact with a certain Cul-Rbx module to form an E3 ubiquitin (Ub) ligase complex, but the functional composition of the ASB-containing E3 Ub ligase complexes remains to be characterized. Here, we show that ASB proteins interact with Cul5-Rbx2 but neither Cul2 nor Rbx1 in cells. Mutational analysis revealed that the highly conserved amino acid sequences of the BC box and Cul5 box in the SOCS box of ASB proteins were essential for the interaction with Cul5-Rbx2. Although ASB proteins show slight divergences from the consensus sequences of the BC box and Cul5 box, all five tested ASB proteins bound to Cul5-Rbx2. Furthermore, all three tested ASB complexes containing Cul5-Rbx2 were found to have E3 Ub ligase activity. These findings suggest that the ASB family proteins interact with Cul5-Rbx2 to form E3 Ub ligases and play significant roles via a ubiquitination-mediated pathway.     

Schistosoma mansoni: Heterologous complementation of a yeast null mutant by SmRbx, a protein similar to a RING box protein involved in ubiquitination

The SCF (Skp1-Cul1-F-box) complex is one of the several E3 ligase enzymes and it catalyzes protein ubiquitination and degradation by the 26S proteasome. Rbx1 is a member of the SCF complex in humans and HRT1 is its yeast orthologue. A cDNA encoding a Schistosoma mansoni Rbx1 homolog was cloned and functionally characterized. Heterologous functional complementation in yeast showed that the worm SmRbx gene was able to complement the HRT1yeast null mutation. Gene deletion constructs for N- and C-termini truncated proteins were used to transform hrt1(-) yeast mutant strains, allowing us to observe that regions reported to be involved in the interaction with cullin1 (Cul1) were essential for SmRbx function. Yeast two-hybrid assays using SmRbx and yeast Cul1 confirmed that SmRbx, but not the mutant SmRbxDelta24N, lacking the N-terminus of the protein, was capable of interacting with Cul1. These results suggest that SmRbx protein is involved in the SCF complex formation.     

The von Hippel-Lindau tumour-suppressor protein interaction with protein kinase Cdelta

The VHL (von Hippel-Lindau) tumour-suppressor protein forms a multi-protein complex [VCB (pVHL-elongin C-elongin B)-Cul-2 (Cullin-2)] with elongin C, elongin B, Cul-2 and Rbx1, acting as a ubiquitin-ligase (E3) and directing proteasome-dependent degradation of targeted proteins. The alpha-subunit of Hif1alpha (hypoxia-inducible factor 1alpha) is the principal substrate for the VCB-Cul-2 complex; however, other substrates such as aPKC (atypical protein kinase C) have been reported. In the present study, we show with FRET (fluorescence resonance energy transfer) analysis measured by FLIM (fluorescence lifetime imaging microscopy) that PKCdelta and pVHL (VHL protein) interact directly in cells. This occurs through the catalytic domain of PKCdelta (residues 432-508), which appears to interact with two regions of pVHL, residues 113-122 and 130-154. Despite this robust interaction, analysis of the PMA-induced proteasome-dependent degradation of PKCdelta in different RCC (renal cell carcinoma) lines (RCC4, UMRC2 and 786 O) shows that there is no correlation between the degradation of PKCdelta and the presence of active pVHL. Thus, in contrast with aPKC, PKCdelta is not a conventional substrate of the ubiquitin-ligase complex, VCB-Cul-2, and the observed interaction between these two proteins must underlie a distinct signalling output.     

Mnk2 and Mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4E but not for cell growth or development

Mnk1 and Mnk2 are protein kinases that are directly phosphorylated and activated by extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein (MAP) kinases and implicated in the regulation of protein synthesis through their phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) at Ser209. To investigate their physiological functions, we generated mice lacking the Mnk1 or Mnk2 gene or both; the resulting KO mice were viable, fertile, and developed normally. In embryonic fibroblasts prepared from Mnk1-Mnk2 DKO mice, eIF4E was not detectably phosphorylated at Ser209, even when the ERK and/or p38 MAP kinases were activated. Analysis of embryonic fibroblasts from single KO mice revealed that Mnk1 is responsible for the inducible phosphorylation of eIF4E in response to MAP kinase activation, whereas Mnk2 mainly contributes to eIF4E's basal, constitutive phosphorylation. Lipopolysaccharide (LPS)- or insulin-induced upregulation of eIF4E phosphorylation in the spleen, liver, or skeletal muscle was abolished in Mnk1(-/-) mice, whereas the basal eIF4E phosphorylation levels were decreased in Mnk2(-/-) mice. In Mnk1-Mnk2 DKO mice, no phosphorylated eIF4E was detected in any tissue studied, even after LPS or insulin injection. However, neither general protein synthesis nor cap-dependent translation, as assayed by a bicistronic reporter assay system, was affected in Mnk-deficient embryonic fibroblasts, despite the absence of phosphorylated eIF4E. Thus, Mnk1 and Mnk2 are exclusive eIF4E kinases both in cultured fibroblasts and adult tissues, and they regulate inducible and constitutive eIF4E phosphorylation, respectively. These results strongly suggest that eIF4E phosphorylation at Ser209 is not essential for cell growth during development.     

Mechanism of cullin3 e3 ubiquitin ligase dimerization

Cullin E3 ligases are the largest family of ubiquitin ligases with diverse cellular functions. One of seven cullin proteins serves as a scaffold protein for the assembly of the multisubunit ubiquitin ligase complex. Cullin binds the RING domain protein Rbx1/Rbx2 via its C-terminus and a cullin-specific substrate adaptor protein via its N-terminus. In the Cul3 ubiquitin ligase complex, Cul3 substrate receptors contain a BTB/POZ domain. Several studies have established that Cul3-based E3 ubiquitin ligases exist in a dimeric state which is required for binding of a number of substrates and has been suggested to promote ubiquitin transfer. In two different models, Cul3 has been proposed to dimerize either via BTB/POZ domain dependent substrate receptor homodimerization or via direct interaction between two Cul3 proteins that is mediated by Nedd8 modification of one of the dimerization partners. In this study, we show that the majority of the Cul3 proteins in cells exist as dimers or multimers and that Cul3 self-association is mediated via the Cul3 N-terminus while the Cul3 C-terminus is not required. Furthermore, we show that Cul3 self-association is independent of its modification with Nedd8. Our results provide evidence for BTB substrate receptor dependent Cul3 dimerization which is likely to play an important role in promoting substrate ubiquitination.     

The N and C termini of the splice variants of the human mitogen-activated protein kinase-interacting kinase Mnk2 determine activity and localization

The cap-binding eukaryotic initiation factor eIF4E is phosphorylated by the mitogen-activated protein (MAP) kinase-interacting kinases (Mnk's). Three forms of the Mnk's exist in human cells: Mnk1, Mnk2a, and Mnk2b. These last two are derived from the same gene by alternative splicing and differ only at their C termini. While Mnk2a contains a MAP kinase-binding site in this region, Mnk2b lacks such a sequence and is much less readily activated by MAP kinases in vitro. Expression of Mnk2b in mammalian cells leads to increased phosphorylation of eIF4E, showing that it acts as an eIF4E kinase in vivo. While Mnk2a is cytoplasmic, a substantial amount of Mnk2b is found in the nucleus. Both enzymes contain a stretch of basic residues in their N termini that plays a role in binding to eIF4G and functions as a nuclear localization signal. Binding of eIF4G or nuclear import appears to be regulated by the C terminus of Mnk2a. Furthermore, the MAP kinase-binding site of Mnk2a regulates nuclear entry. Within the nucleus, Mnk2b and certain variants of Mnk2a that are present in the nucleus colocalize with the promyelocytic leukemia protein PML, which also binds to eIF4E.     

The SCF ubiquitin ligase: an extended look

The SCF E3 ubiquitin ligases select specific proteins for ubiquitination (and typically destruction) by coupling variable adaptor (F box) proteins that bind protein substrates to a conserved catalytic engine containing a cullin, Cul1, and the Rbx1/Roc1 RING finger protein. A new crystal structure of the SCF(Skp2) ubiquitin ligase shows the molecular organization of this complex and raises important questions as to how substrate ubiquitination is accomplished.     

The role of cullin 5-containing ubiquitin ligases

The suppressor of cytokine signaling (SOCS) box consists of the BC box and the cullin 5 (Cul5) box, which interact with Elongin BC and Cul5, respectively. SOCS box-containing proteins have ubiquitin ligase activity mediated by the formation of a complex with the scaffold protein Cul5 and the RING domain protein Rbx2, and are thereby members of the cullin RING ligase superfamily. Cul5-type ubiquitin ligases have a variety of substrates that are targeted for polyubiquitination and proteasomal degradation. Here, we review the current knowledge on the identification of Cul5 and the regulation of its expression, as well as the signaling pathways regulated by Cul5 and how viruses highjack the Cul5 system to overcome antiviral responses.     

Drosophila von Hippel-Lindau tumor suppressor complex possesses E3 ubiquitin ligase activity

Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene predispose individuals to a variety of human tumors, including renal cell carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. Here we report on the identification and characterization of the Drosophila homolog of VHL. The predicted amino acid sequence of Drosophila VHL protein shows 29% identity and 44% similarity to that of human VHL protein. Biochemical studies have shown that Drosophila VHL protein binds to Elongins B and C directly, and via this Elongin BC complex, associates with Cul-2 and Rbx1. Like human VHL, Drosophila VHL complex containing Cul-2, Rbx1, Elongins B and C, exhibits E3 ubiquitin ligase activity. In addition, we provide evidence that hypoxia-inducible factor (HIF)-1alpha is the ubiquitination target of both human and Drosophila VHL complexes.     

Flexible cullins in cullin-RING E3 ligases allosterically regulate ubiquitination

How do the cullins, with conserved structures, accommodate substrate-binding proteins with distinct shapes and sizes? Cullin-RING E3 ubiquitin ligases facilitate ubiquitin transfer from E2 to the substrate, tagging the substrate for degradation. They contain substrate-binding, adaptor, cullin, and Rbx proteins. Previously, we showed that substrate-binding and Rbx proteins are flexible. This allows shortening of the E2-substrate distance for initiation of ubiquitination or increasing the distance to accommodate the polyubiquitin chain. However, the role of the cullin remained unclear. Is cullin a rigid scaffold, or is it flexible and actively assists in the ubiquitin transfer reaction? Why are there different cullins, and how do these cullins specifically facilitate ubiquitination for different substrates? To answer these questions, we performed structural analysis and molecular dynamics simulations based on Cul1, Cul4A, and Cul5 crystal structures. Our results show that these three cullins are not rigid scaffolds but are flexible with conserved hinges in the N-terminal domain. However, the degrees of flexibilities are distinct among the different cullins. Of interest, Cul1 flexibility can also be changed by deletion of the long loop (which is absent in Cul4A) in the N-terminal domain, suggesting that the loop may have an allosteric functional role. In all three cases, these conformational changes increase the E2-substrate distance to a specific range to facilitate polyubiquitination, suggesting that rather than being inert scaffold proteins, cullins allosterically regulate ubiquitination.