CaMKII protects MKP-1 from proteasome degradation in endothelial cells

CaMKs are a widely distributed family of kinases with multiple and often cell specific effects on intracellular signal transduction pathway. In endothelial cells, it has been recognized a role for CamKII in several pathways such as eNOS activation and nitric oxide production. It is not clear though, whether CaMKII interfere with other endothelial cell functions such as ERK activation and cell proliferation. We explored this issue in primary cultured rat endothelial cells and we evaluated the effect on endothelial cell proliferation and DNA synthesis. CaMKII inhibition through Cantide, conducted into the cell through Antoennapedia (ANT-CN), showed positive effects on proliferation and H³-thimdine incorporation similar to insulin stimulation. Accordingly, both CaMKII pharmacological inhibition and silencing through shRNA produced activation of the p44/42 MAPK. These observations leaded to the hypothesis that CamKII could regulate p44/p42 by interfering with specific ERK phosphatases. Indeed, we found that CaMKII interacts and protect the dual specific phosphatase MKP-1 from proteasome mediated degradation while this complex is disrupted by CaMKII inhibitors. This study reveals that CaMKII, besides phosphorylation through the known ras-raf-mek pathway, can regulate also dephosphorylation of p44/p42 by modulation of MKP-1 level. This novel finding opens to a novel scenario in regulation of endothelial cell functions.     

Activated c-Abl is degraded by the ubiquitin-dependent proteasome pathway.

C-Abl is a nonreceptor tyrosine kinase that is tightly regulated in the cell. Genetic data derived from studies in flies and mice strongly support a role for Abl kinases in the regulation of the cytoskeleton (reviewed in [1,2]). C-Abl can be activated by several stimuli, including oxidative stress [3], DNA damage [4], integrin engagement [5], growth factors, and Src family kinases [6]. Structural alterations elicit constitutive activation of the c-Abl tyrosine kinase, leading to oncogenic transformation. While the mechanisms that activate c-Abl are beginning to be elucidated, little is known regarding the mechanisms that downregulate activated c-Abl. Here, we show for the first time that activated c-Abl is downregulated by the ubiquitin-dependent degradation pathway. Activated forms of c-Abl are more unstable than wild-type and kinase-inactive forms. Moreover, inhibition of the 26S proteasome leads to increased c-Abl levels in vitro and in cells, and activated c-Abl proteins are ubiquitinated in vivo. Significantly, inhibition of the 26S proteasome in fibroblasts increases the levels of tyrosine-phosphorylated, endogenous c-Abl. Our data suggest a novel mechanism for irreversible downregulation of activated c-Abl, which is critical to prevent the deleterious consequences of c-Abl hyperactivation in mitogenic and cytoskeletal pathways.     

Cigarette smoke suppresses the ubiquitin-dependent degradation of OLC1

The newly identified gene, overexpressed in lung cancer 1 (OLC1), is highly expressed as OLC1 protein in the tumor tissues of lung cancer patients with histories of cigarette smoking. However, the underlying mechanisms of how the gene is affected by cigarette smoke have been poorly characterized. In this study, we investigated how OLC1 is regulated in lung cancer cells by cigarette smoke condensate (CSC).Compared to the controls, CSC treatment increased OLC1 protein levels in a dose- and time-dependent manner without affecting OLC1 mRNA levels in lung cancer cells. Ubiquitination of OLC1 protein was blocked upon CSC treatment. Biochemical analysis revealed that the ubiquitin E3 ligase anaphase promoting complex (APC) and its activators cell-division cycle protein 20 (CDC20) and cadherin-1 (CDH1) are responsible for the degradation of OLC1. However, upon introducing CSC the binding of OLC1 to the proteins CDC20, CDH1, and APC2 was impaired. These results demonstrate that CSC regulates OLC1 expression in lung cancer cells by compromising its ubiquitination and subsequent degradation through the ubiquitin E3 ligase APC.     

hSpry2 is targeted to the ubiquitin-dependent proteasome pathway by c-Cbl

Sprouty was originally identified in a genetic screen in Drosophila as an antagonist of fibroblast (FGF) and epidermal growth factor (EGF) signaling. Subsequently, four vertebrate homologs were discovered; among these, the human homolog Sprouty 2 (hSpry2) contains the highest degree of sequence homology to the Drosophila protein. It has been shown that hSpry2 interacts directly with c-Cbl, an E3-ubiquitin ligase, which promotes the downregulation of receptor tyrosine kinases (RTKs). In this study, we have investigated the functional consequences of the association between hSpry2 and c-Cbl. We have found that hSpry2 is ubiquitinated by c-Cbl in an EGF-dependent manner. EGF stimulation induces the tyrosine phosphorylation of hSpry2, which in turn enhances the interaction of hSpry2 with c-Cbl. The c-Cbl-mediated ubiquitination of hSpry2 targets the protein for degradation by the 26S proteasome. An enhanced proteolytic degradation of hSpry2 is also observed in response to FGF stimulation. The FGF-induced degradation of hSpry2 limits the duration of the inhibitory effect of hSpry2 on extracellular signal-regulated kinase (ERK) activation and enables the cells to recover their sensitivity to FGF stimulation. Our results indicate that the interaction of hSpry2 with c-Cbl might serve as a mechanism for the downregulation of hSpry2 during receptor tyrosine kinase signaling.     

Connexin37 protects against atherosclerosis by regulating monocyte adhesion

A genetic polymorphism in the human gene encoding connexin37 (CX37, encoded by GJA4, also known as CX37) has been reported as a potential prognostic marker for atherosclerosis. The expression of this gap-junction protein is altered in mouse and human atherosclerotic lesions: it disappears from the endothelium of advanced plaques but is detected in macrophages recruited to the lesions. The role of CX37 in atherogenesis, however, remains unknown. Here we have investigated the effect of deleting the mouse connexin37 (Cx37) gene (Gja4, also known as Cx37) on atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice, an animal model of this disease. We find that Gja4(-/-)Apoe(-/-) mice develop more aortic lesions than Gja4(+/+)Apoe(-/-) mice that express Cx37. Using in vivo adoptive transfer, we show that monocyte and macrophage recruitment is enhanced by eliminating expression of Cx37 in these leukocytes but not by eliminating its expression in the endothelium. We further show that Cx37 hemichannel activity in primary monocytes, macrophages and a macrophage cell line (H36.12j) inhibits leukocyte adhesion. This antiadhesive effect is mediated by release of ATP into the extracellular space. Thus, Cx37 hemichannels may control initiation of the development of atherosclerotic plaques by regulating monocyte adhesion. H36.12j macrophages expressing either of the two CX37 proteins encoded by a polymorphism in the human GJA4 gene show differential ATP-dependent adhesion. These results provide a potential mechanism by which a polymorphism in CX37 protects against atherosclerosis.     

The proteasome inhibitor MG132 protects against acute pancreatitis

The cell-permeant MG132 tripeptide (Z-Leu-Leu-Leu-aldehyde) is a peptide aldehyde proteasome inhibitor that also inhibits other proteases, including calpains and cathepsins. By blocking the proteasome, this tripeptide has been shown to induce the expression of cell-protective heat shock proteins (HSPs) in vitro. Effects of MG132 were studied in an in vivo model of acute pancreatitis. Pancreatitis was induced in male Wistar rats by injecting 2 x 100 microug/kg cholecystokinin octapeptide intraperitoneally (ip) at an interval of 1 h. Pretreating the animals with 10 mg/kg MG132 ip before the induction of pancreatitis significantly inhibited IkappaB degradation and subsequent activation of nuclear factor-kappaB (NF-kappaB). MG132 also increased HSP72 expression. Induction of HSP72 and inhibition of NF-kappaB improved parameters of acute pancreatitis. Thus MG132 significantly decreased serum amylase, pancreatic weight/body weight ratio, pancreatic myeloperoxidase activity, proinflammatory cytokine concentrations, and the expression of pancreatitis-associated protein. Parameters of oxidative stress (GSH, MDA, SOD, etc.) were improved in both the serum and the pancreas. Histopathological examinations revealed that pancreatic specimens of animals pretreated with the peptide demonstrated milder edema, cellular damage, and inflammatory activity. Our findings show that simultaneous inhibition of calpains, cathepsins, and the proteasome with MG132 prevents the onset of acute pancreatitis.     

Geldanamycin-induced degradation of Chk1 is mediated by proteasome

Checkpoint kinase 1 (Chk1) is a cell cycle regulator and a heat shock protein 90 (Hsp90) client. It is essential for cell proliferation and survival. In this report, we analyzed the mechanisms of Chk1 regulation in U87MG glioblastoma cells using Geldanamycin (GA), which interferes with the function of Hsp90. GA reduced Chk1 protein level but not its mRNA level in glioblastoma cells. Co-treatment with GA and cycloheximide (CHX), a protein synthesis inhibitor, induced a decrease of half-life of the Chk1 protein to 3h and resulted in Chk1 down-regulation. CHX alone induced only 32% reduction of Chk1 protein even after 24h. These findings indicated that reduction of Chk1 by GA was due to destabilization and degradation of the protein. In addition, GA-induced down-regulation of Chk1 was reversed by MG132, a specific proteasome inhibitor. And it was revealed that Chk1 was ubiquitinated by GA. These results have indicated that degradation of Chk1 by GA was mediated by the ubiquitin-proteasome pathway in U87MG glioblastoma cells.     

Human HRD1 protects against ER stress-induced apoptosis through ER-associated degradation

Stresses that impair the function of the endoplasmic reticulum (ER) lead to an accumulation of unfolded protein in the ER. Under these conditions, the expression of a variety of genes involved in preventing the accumulation of the unfolded proteins is induced. Yeast Hrd1p is an ER stress-inducible ER membrane protein that acts as a ubiquitin ligase (E3) with a RING finger motif and plays a role in the ubiquitination of proteins in the ER. We report here the identification and characterization of a human homolog to yeast Hrd1p. The predicted structures are highly conserved from yeast to humans. Indeed, human HRD1 was localized to the ER and ubiquitinated its substrates. Furthermore, it was found that human HRD1 was up-regulated by ER stress via IRE1 and ATF6, which are ER stress transducers. Interestingly, 293 cells stably expressing wild-type HRD1, but not the C329S mutant, afforded resistance to ER stress-induced apoptosis. These results suggest that the production of HRD1 is up-regulated to protect against ER stress-induced apoptosis by degrading unfolded proteins accumulated in the ER.     

Ubiquitin-independent degradation of proteins by the proteasome

The proteasome is the main proteolytic machinery of the cell and constitutes a recognized drugable target, in particular for treating cancer. It is involved in the elimination of misfolded, altered or aged proteins as well as in the generation of antigenic peptides presented by MHC class I molecules. It is also responsible for the proteolytic maturation of diverse polypeptide precursors and for the spatial and temporal regulation of the degradation of many key cell regulators whose destruction is necessary for progression through essential processes, such as cell division, differentiation and, more generally, adaptation to environmental signals. It is generally believed that proteins must undergo prior modification by polyubiquitin chains to be addressed to, and recognized by, the proteasome. In reality, however, there is accumulating evidence that ubiquitin-independent proteasomal degradation may have been largely underestimated. In particular, a number of proto-oncoproteins and oncosuppressive proteins are privileged ubiquitin-independent proteasomal substrates, the altered degradation of which may have tumorigenic consequences. The identification of ubiquitin-independent mechanisms for proteasomal degradation also poses the paramount question of the multiplicity of catabolic pathways targeting each protein substrate. As this may help design novel therapeutic strategies, the underlying mechanisms are critically reviewed here.     

DHHC protein-dependent palmitoylation protects regulator of G-protein signaling 4 from proteasome degradation

Regulator of G-protein signaling 4 (RGS4), an intracellular modulator of G-protein coupled receptor (GPCR)-mediated signaling, is regulated by multiple processes including palmitoylation and proteasome degradation. We found that co-expression of DHHC acyltransferases (DHHC3 or DHHC7), but not their acyltransferase-inactive mutants, increased expression levels of RGS4 but not its Cys2 to Ser mutant (RGS4C2S). DHHC3 interacts with and palmitoylates RGS4 but not RGS4C2S in vivo. Palmitoylation prolongs the half-life of RGS4 by over 8-fold and palmitoylated RGS4 blocked α_1A-adrenergic receptor-stimulated intracellular Ca²⁺ mobilization. Together, our findings revealed that DHHC proteins could regulate GPCR-mediated signaling by increasing RGS4 stability.

Structured summary

MINT-8049215: Rgs4 (uniprotkb:P49799) physically interacts (MI:0915) with DHHC3 (uniprotkb:Q8R173) by anti-tag coimmunoprecipitation (MI:0007)     

c-Cbl binding and ubiquitin-dependent lysosomal degradation of membrane-associated Notch1.

Regulation of Notch1 activity is critical for cell fate decisions and differentiation of skeletal myoblasts. We have employed the skeletal myoblast cell line C2C12 to study posttranslational regulation of Notch1 protein levels during myogenesis. Although the major degradation pathway of the activated intracellular Notch1 fragment appears to involve ubiquitination and degradation by the 26 S proteasome, we provide evidence for an alternative catalytic pathway where the endogenous, transmembrane form of Notch1 is targeted to the lysosomal compartment. Immunoprecipitation analysis revealed ubiquitin-dependent accumulation of transmembrane Notch1 protein after treatment with the lysosomal inhibitor chloroquine but not after treatment with various proteasome inhibitors. This finding was supported by the observation that the transmembrane form of Notch1 was tyrosine-phosphorylated and specifically coprecipitated with the ubiquitin ligase c-Cbl. Our data suggest a regulatory mechanism down-regulating Notch1 protein levels already at the cellular surface, possibly with consequences for Notch-dependent signal transduction during terminal differentiation processes.     

RAG2 is down-regulated by cytoplasmic sequestration and ubiquitin-dependent degradation

Periodic accumulation and degradation of RAG2 (recombination-activating gene 2) protein controls the cell-cycle-dependent V(D)J recombination of lymphocyte antigen receptor genes. Here we show the molecular mechanism of RAG2 degradation. The RAG2 protein is translocated from the nucleus to the cytoplasm and degraded through the ubiquitin/proteasome system. RAG2 translocation is mediated by the Thr-490 phosphorylation of RAG2. Inhibition of this phosphorylation by p27Kip1 stabilizes the RAG2 protein in the nucleus. These results suggest that RAG2 sequestration in the cytoplasm and its subsequent degradation by the ubiquitin/proteasome system upon entering the S phase is an integral part of G0/G1-specific V(D)J recombination.     

Synaptic protein degradation by the ubiquitin proteasome system

Synaptic plasticity -- the modulation of synaptic strength between a presynaptic terminal and a postsynaptic dendrite -- is thought to be a mechanism that underlies learning and memory. It has become increasingly clear that regulated protein synthesis is an important mechanism used to regulate the protein content of synapses that results in changes in synaptic strength. Recent experiments have highlighted a role for the opposing process, that is, regulated protein degradation via the ubiquitin-proteasome system, in synaptic plasticity. These recent findings raise exciting questions as to how proteasomal activity can regulate synapses over different temporal and spatial scales.     

A lipophilic heparin derivative protects elastin against degradation by leucocyte elastase.

An oleolylated derivative (I) of partially N-desulphated heparin was prepared containing an average number of three oleoyl residues for one molecule of heparin. The inhibitory capacity of I (IC50 = 0.55 microM) for leucocyte elastase resembles that of heparin (IC50 = 0.2 microM). In contrast to heparin, I is also an inhibitor of porcine pancreatic elastase (IC50 = 0.68 microM) and it also has the capacity to protect elastin fibres against the degradation by leucocyte elastase. When insoluble elastin is pretreated with I its degradation by leucocyte elastase is inhibited by almost 90% while pretreatment of elastin with heparin exhibited only a moderate effect on elastolysis (10% inhibition).     

The proteasome/multicatalytic-multifunctional proteinase. In vivo function in the ubiquitin-dependent N-end rule pathway of protein degradation in eukaryotes.

Proteinase yscE, the proteasome/multicatalytic-multifunctional proteinase of yeast had been shown to function in stress response and in the degradation of ubiquitinated proteins [(1991) EMBO J. 10, 555-562]. A well-defined set of proteins degraded via ubiquitin-mediated proteolysis are the substrates of the N-end rule pathway [(1986) Science 234, 179-186; (1989) Science 243, 1576-1583]. We show that mutants defective in the chymotryptic activity of proteinase yscE fail to degrade substrates of the N-end rule pathway. This gives further proof of the proteasome being a central catalyst in ubiquitin-mediated proteolysis.     

Discrimination between ubiquitin-dependent and ubiquitin-independent proteolytic pathways by the 26S proteasome subunit 5a.

The 26S proteasome subunit 5a binds polyubiquitin chains and has previously been shown to inhibit the degradation of mitotic cyclins. Presumably inhibition results from S5a binding and preventing recognition of Ub-cyclin conjugates by the 26S proteasome. Here we show that S5a does not inhibit the degradation of full-length ornithine decarboxylase (ODC) consistent with previous reports that the enzyme is degraded in an antizyme-dependent, but ubiquitin-independent reaction. S5a does, however, inhibit degradation of short ODC translation products generated by internal initiation events. Because in vitro translation often produces some shortened products, the existence of ubiquitin conjugated to a 35S-labeled protein is not necessarily evidence that the full-length protein is a substrate of the Ub-dependent proteolytic pathway.     

Hyperactivation of the proteasome in Caenorhabditis elegans protects against proteotoxic stress and extends lifespan

Virtually all age-related neurodegenerative diseases (NDs) can be characterized by the accumulation of proteins inside and outside the cell that are thought to significantly contribute to disease pathogenesis. One of the cell’s primary systems for the degradation of misfolded/damaged proteins is the ubiquitin proteasome system (UPS), and its impairment is implicated in essentially all NDs. Thus, upregulating this system to combat NDs has garnered a great deal of interest in recent years. Various animal models have focused on stimulating 26S activity and increasing 20S proteasome levels, but thus far, none have targeted intrinsic activation of the 20S proteasome itself. Therefore, we constructed an animal model that endogenously expresses a hyperactive, open gate proteasome in Caenorhabditis elegans. The gate-destabilizing mutation that we introduced into the nematode germline yielded a viable nematode population with enhanced proteasomal activity, including peptide, unstructured protein, and ubiquitin-dependent degradation activities. We determined these nematodes showed a significantly increased lifespan and substantial resistance to oxidative and proteotoxic stress but a significant decrease in fecundity. Our results show that introducing a constitutively active proteasome into a multicellular organism is feasible and suggests targeting the proteasome gating mechanism as a valid approach for future age-related disease research efforts in mammals.