Insulin gene is a target in activin receptor-like kinase 7 signaling pathway in pancreatic beta-cells

Activins regulate pancreatic development, differentiation and insulin secretion. Activin receptor-like kinase 7 (ALK7) has been identified as a receptor for Nodal and Activin AB and B, and is expressed in pancreatic islets and β-cell lines. In this study, human insulin promoter was activated by Smad2, Smad3 and the pancreatic and duodenal homeobox factor-1 (PDX-1) in the ALK7 pathway. A conserved Smad binding element was related to the promoter activation. Phosphorylated Smad2/Smad3 and PDX-1 were bound to insulin gene with Nodal and Activin AB, and the phosphorylated Smad2/Smad3 interacted with PDX-1. These results indicate that one of the direct target genes of Nodal and Activin AB signals is the insulin gene in pancreatic β-cells and that PDX-1 is directly involved in the ALK7-Smad pathway.     

Liver alcohol dehydrogenase is degraded by the ubiquitin-proteasome pathway.

Dihydrotestosterone (DHT) decreases rat liver alcohol dehydrogenase (ADH) due principally to an increased rate of degradation of the enzyme. The pathway of degradation of ADH was investigated. Exposure of hepatocytes in culture to lactacystin or to MG132, which are inhibitors of the ubiquitin-proteasome pathway of protein degradation, resulted in higher ADH. Furthermore, both lactacystin and MG132 prevented the decrease in ADH caused by DHT. By contrast, the lysosomal proteolytic inhibitors 3-methyladenine and leupeptin as well as inhibitors of the calcium-activated neutral protease calpain system had no effect on ADH in the absence or presence of DHT. ADH isolated by immunoprecipitation from hepatocytes exposed to DHT reacted specifically with anti-ubiquitin antibody. Ubiquitinated ADH was also demonstrated in hepatocytes exposed to MG132. The combination of DHT and MG132 resulted in more ubiquitinated ADH than exposure to either compound alone. These results suggest that the ubiquitin-proteasome pathway plays a role in the degradation of ADH and in the enhanced degradation of this enzyme by DHT.     

Role and regulation of nodal/activin receptor-like kinase 7 signaling pathway in the control of ovarian follicular atresia

Although the role of the TGF beta superfamily members in the regulation of ovarian folliculogenesis has been extensively studied, their involvement in follicular atresia is not well understood. In the present study, we have demonstrated for the first time that Nodal, a member of the TGF beta superfamily, is involved in promoting follicular atresia as evidenced by the following: 1) colocalization of Nodal and its type I receptor Activin receptor-like kinase 7 (ALK7) proteins in the granulosa cells was only observed in atretic antral follicles, whereas they were present in theca cells and granulosa cells of healthy follicles, respectively; 2) addition of recombinant Nodal or overexpression of Nodal by adenoviral infection induced apoptosis of otherwise healthy granulosa cells; 3) constitutively active ALK7 (ALK7-ca) overexpression mimicked the function of Nodal in the induction of granulosa cell apoptosis. Furthermore, overexpression of Nodal or ALK7-ca increased phosphorylation and nuclear translocation of Smad2, decreased X-linked inhibitor of apoptotic proteins (Xiap) expression at both mRNA and protein level and phospho-Akt content, as well as triggered mitochondrial release of death proteins Smac/DIABLO, Omi/HtrA2, and cytochrome c in the granulosa cells. Dominant-negative Smad2 significantly attenuated ALK7-ca-induced down-regulation of Xiap and thus rescued granulosa cells from undergoing apoptosis. In addition, whereas up-regulation of Xiap significantly attenuated ALK7-ca-induced apoptosis, down-regulation of Xiap sensitized granulosa cells to ALK7-ca-induced apoptosis. Furthermore, ALK7-ca-induced apoptosis was significantly attenuated by forced expression of activated Akt, and Akt rescued granulosa cells from undergoing apoptosis via proteasome-mediated ALK7 degradation. Taken together, Nodal plays an atretogenic role in the ovary where it induces granulosa cell apoptosis through activation of Smad2, down-regulation of the key survival molecules Xiap and phospho-Akt, as well as the activation of mitochondrial death pathway.     

Cyclin F is degraded during G2-M by mechanisms fundamentally different from other cyclins

Cyclin F, a cyclin that can form SCF complexes and bind to cyclin B, oscillates in the cell cycle with a pattern similar to cyclin A and cyclin B. Ectopic expression of cyclin F arrests the cell cycle in G(2)/M. How the level of cyclin F is regulated during the cell cycle is completely obscure. Here we show that, similar to cyclin A, cyclin F is degraded when the spindle assembly checkpoint is activated and accumulates when the DNA damage checkpoint is activated. Cyclin F is a very unstable protein throughout much of the cell cycle. Unlike other cyclins, degradation of cyclin F is independent of ubiquitination and proteasome-mediated pathways. Interestingly, proteolysis of cyclin F is likely to involve metalloproteases. Rapid destruction of cyclin F does not require the N-terminal F-box motif but requires the COOH-terminal PEST sequences. The PEST region alone is sufficient to interfere with the degradation of cyclin F and confer instability when fused to cyclin A. These data show that although cyclin F is degraded at similar time as the mitotic cyclins, the underlying mechanisms are entirely distinct.     

PINK1 is degraded through the N-end rule pathway

PINK1, a mitochondrial serine/threonine kinase, is the product of a gene mutated in an autosomal recessive form of Parkinson disease. PINK1 is constitutively degraded by an unknown mechanism and stabilized selectively on damaged mitochondria where it can recruit the E3 ligase PARK2/PARKIN to induce mitophagy. Here, we show that, under steady-state conditions, endogenous PINK1 is constitutively and rapidly degraded by E3 ubiquitin ligases UBR1, UBR2 and UBR4 through the N-end rule pathway. Following precursor import into mitochondria, PINK1 is cleaved in the transmembrane segment by a mitochondrial intramembrane protease PARL generating an N-terminal destabilizing amino acid and then retrotranslocates from mitochondria to the cytosol for N-end recognition and proteasomal degradation. Thus, sequential actions of mitochondrial import, PARL-processing, retrotranslocation and recognition by N-end rule E3 enzymes for the ubiquitin proteosomal degradation defines the rapid PINK1 turnover. PINK1 steady-state elimination by the N-end rule identifies a novel organelle to cytoplasm turnover pathway that yields a mechanism to flag damaged mitochondria for autophagic elimination.     

Mutant products of the NF2 tumor suppressor gene are degraded by the ubiquitin-proteasome pathway

Neurofibromatosis type 2 (NF2), a syndrome associated with multiple tumors of the nervous system, mostly schwannomas, is caused by mutations in the NF2 tumor suppressor gene that encodes schwannomin (Sch). Here we examined NF2 pathogenetic mutations that result in misfolding of the FERM domain. We found that these mutant forms of Sch were efficiently degraded by the ubiquitin-proteasome pathway. In transfected cells, Sch Delta F118 was 3-fold more efficiently degraded than the related molecule ezrin bearing the equivalent mutation. In heterozygous Nf2 knock-out mouse fibroblasts, endogenous mutant Sch Delta 81-121, but not wild type Sch, was also degraded by proteasomes. We further show that this degradation pathway is functional in primary Schwann cells. We analyzed Sch Delta 39-121 expressed in a transgenic mouse model of NF2 and found that Sch Delta 39-121, but not the endogenous wild type Sch, was unstable due to proteasome-mediated degradation. Altogether these results suggest that degradation of mutant Sch mediated by the ubiquitin-proteasome pathway is a physiopathological pathway contributing to the loss of Sch function in NF2 patients.     

MYO18B interacts with the proteasomal subunit Sug1 and is degraded by the ubiquitin-proteasome pathway

MYO18B is a class XVIIIB unconventional myosin encoded by a candidate tumor suppressor gene. To gain insights into the cellular function of this protein, we searched for MYO18B-interacting proteins by a yeast two-hybrid screen. Sug1, a 19S regulator subunit of the 26S proteasome, was identified as a binding partner of the C-terminal tail region of MYO18B. The association of MYO18B with Sug1 was further confirmed by GST pull-down, co-immunoprecipitation, and immunocytochemistry. Furthermore, proteasome dysfunction by a proteasome inhibitor or siRNA-mediated knock-down of Sug1 caused the up-regulation of MYO18B protein and MYO18B was polyubiquitinated in vivo. Collectively, these results suggested that MYO18B is a substrate for proteasomal degradation.     

Pim-1 kinase stability is regulated by heat shock proteins and the ubiquitin-proteasome pathway

Elevated expression of the serine/threonine kinase Pim-1 increases the incidence of lymphomas in Pim-1 transgenic mice and has also been found to occur in some human cancers. Pim-1 acts as a cell survival factor and may prevent apoptosis in malignant cells. It was therefore of interest to understand to what extent maintenance and degradation of Pim-1 protein is affected by heat shock proteins (Hsp) and the ubiquitin-proteasome pathway in K562 and BV173 human leukemic cells. The half-life of Pim-1 protein in these cells was found to increase from 1.7 to 3.1 hours when induced by heat shock or by treating the cells with the proteasome inhibitor PS-341 (bortezomib). The Hsp90 inhibitor geldanamycin prevented the stabilization of Pim-1 by heat shock. Using immunoprecipitation, it was determined that Pim-1 is targeted for degradation by ubiquitin and that Hsp70 is associated with Pim-1 under these circumstances. Conversely, Hsp90 was found to protect Pim-1 from proteasomal degradation. A luminescence-based kinase assay showed that Pim-1 kinase bound to Hsp70 or Hsp90 remains active, emphasizing the importance of its overall cellular levels. This study shows how Pim-1 levels can be modulated in cells through degradation and stabilization.     

EP4 mediates PGE2 dependent cell survival through the PI3 kinase/AKT pathway

The anti-apoptotic effect of PGE(2) was examined in Jurkat cells (human T-cell leukemia) by incubation with PGE(2) (5 nM) prior to treatment with the cancer chemotherapeutic agent camptothecin. Apoptosis was evaluated by caspase-3 activity in cell extracts and flow cytometry of propidium iodide-labeled cells. Pre-incubation with PGE(2) reduced camptothecin-induced caspase activity by 30% and apoptosis by 35%, respectively. Pharmacological data demonstrate that the EP4 receptor is responsible for mediating the protection from camptothecin-induced apoptosis. Pre-treatment of the cells with the EP4 antagonist (EP4A) prior to PGE(2) and camptothecin abolished the increased survival effect of PGE(2). Specific inhibition of the downstream of PI3 kinase or AKT/protein kinase but not protein kinase A prevents the observed increase in cell survival elicited by PGE(2). These findings have critical implications regarding the mechanism and potential application of PGE(2) receptor specific inhibition in cancer therapy.     

The activin receptor-like kinase 1 gene: genomic structure and mutations in hereditary hemorrhagic telangiectasia type 2.

The activin receptor-like kinase 1 gene (ALK-1) is the second locus for the autosomal dominant vascular disease hereditary hemorrhagic telangiectasia (HHT). In this paper we present the genomic structure of the ALK-1 gene, a type I serine-threonine kinase receptor expressed predominantly in endothelial cells. The coding region is contained within nine exons, spanning < 15 kb of genomic DNA. All introns follow the GT-AG rule, except for intron 6, which has a TAG/gcaag 5' splice junction. The positions of introns in the intracellular domain are almost identical to those of the mouse serine-threonine kinase receptor TSK-7L. By sequencing ALK-1 from genomic DNA, mutations were found in six of six families with HHT either shown to link to chromosome 12q13 or in which linkage of HHT to chromosome 9q33 had been excluded. Mutations were also found in three of six patients from families in which available linkage data were insufficient to allow certainty with regard to the locus involved. The high rate of detection of mutations by genomic sequencing of ALK-1 suggests that this will be a useful diagnostic test for HHT2, particularly where preliminary linkage to chromosome 12q13 can be established. In two cases in which premature termination codons were found in genomic DNA, the mutant mRNA was either not present or present at barely detectable levels. These data suggest that mutations in ALK-1 are functionally null alleles.     

ADRP/adipophilin is degraded through the proteasome-dependent pathway during regression of lipid-storing cells

Adipose differentiation-related protein (ADRP) is a major protein associated with lipid droplets in various types of cells, including macrophage-derived foam cells and liver cells. However, the role of ADRP in the processes of formation and regression of these cells is not understood. When J774 murine macrophages were incubated with either VLDL or oleic acid, their content of both ADRP and triacylglycerol (TG) increased 3- to 4-fold. Induction of ADRP during TG accumulation was also observed in oleic acid-treated HuH-7 human liver cells. Addition of triacsin C, a potent inhibitor of acyl-CoA synthase, for 6 h decreased the amount of TG in VLDL-induced foam cells and oleic acid-treated liver cells; it decreased the amount of ADRP protein in parallel, indicating the amount of ADRP reduced during regression of the lipid-storing cells. Addition of a proteasome inhibitor during triacsin C treatment abolished the ADRP decrease and accumulated polyubiquitinated ADRP. In addition, the proteasome inhibitor reversed not only the degradation of ADRP but also TG reduction by triacsin C. These results suggest that cellular amounts of ADRP and TG regulate each other and that the ubiquitin-proteasome system is involved in degradation of ADRP during regression of lipid-storing cells.     

Hypertrophic growth in cardiac myocytes is mediated by Myc through a Cyclin D2-dependent pathway

c-Myc (Myc) is highly expressed in developing embryos where it regulates body size by controlling proliferation but not cell size. However, Myc is also induced in many postmitotic tissues, including adult myocardium, in response to stress where the predominant form of growth is an increase in cell size (hypertrophy) and not number. The function of Myc induction in this setting is unproven. Therefore, to explore Myc's role in hypertrophic growth, we created mice where Myc can be inducibly inactivated, specifically in adult myocardium. Myc-deficient hearts demonstrated attenuated stress-induced hypertrophic growth, secondary to a reduction in cell growth of individual myocytes. To explore the dependence of Myc-induced cell growth on CycD2, we created bigenic mice where Myc can be selectively activated in CycD2-null adult myocardium. Myc-dependent hypertrophic growth and cell cycle reentry is blocked in CycD2-deficient hearts. However, in contrast to Myc-induced DNA synthesis, hypertrophic growth is independent of CycD2-induced Cdk2 activity. These data suggest that Myc is required for a normal hypertrophic response and that its growth-promoting effects are also mediated through a CycD2-dependent pathway.     

L166P mutant DJ-1, causative for recessive Parkinson's disease,is degraded through the ubiquitin-proteasome system

Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system. A double mutant (K130R, L166P) was more stable than L166P, suggesting that this lysine residue contributes to stability of the protein. Subcellular localization was broadly similar for both wild type and L166P forms of the protein, indicating that the effect of the mutation is predominantly on protein stability. These observations are reminiscent of other recessive gene mutations that produce an effective loss of function. The L166P mutation has the simple effect of promoting DJ-1 degradation, thereby reducing net DJ-1 protein within the cell.     

Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway

The neu (c-erbB-2) proto-oncogene encodes a tyrosine kinase receptor that is overexpressed in 20 to 30% of human breast tumors. Herein, cyclin D1 protein levels were increased in mammary tumors induced by overexpression of wild-type Neu or activating mutants of Neu in transgenic mice and in MCF7 cells overexpressing transforming Neu. Analyses of 12 Neu mutants in MCF7 cells indicated important roles for specific C-terminal autophosphorylation sites and the extracellular domain in cyclin D1 promoter activation. Induction of cyclin D1 by NeuT involved Ras, Rac, Rho, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, but not phosphatidylinositol 3-kinase. NeuT induction of the cyclin D1 promoter required the E2F and Sp1 DNA binding sites and was inhibited by dominant negative E2F-1 or DP-1. Neu-induced transformation was inhibited by a cyclin D1 antisense or dominant negative E2F-1 construct in Rat-1 cells. Growth of NeuT-transformed mammary adenocarcinoma cells in nude mice was blocked by the cyclin D1 antisense construct. These results demonstrate that E2F-1 mediates a Neu-signaling cascade to cyclin D1 and identify cyclin D1 as a critical downstream target of neu-induced transformation.     

Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway.

The Met tyrosine kinase receptor is a widely expressed molecule which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). In this communication we demonstrate that significant Met degradation is induced by HGF/SF and that this degradation can be blocked by lactacystin, an inhibitor of proteasome activity. We also show that Met is rapidly polyubiquitinated in response to ligand and that polyubiquitinated Met molecules, which are normally unstable, are stabilized by lactacystin. Both HGF/SF-induced degradation and polyubiquitination of Met were shown to be dependent on the receptor possessing intact tyrosine kinase activity. Finally, we found that a normally highly labile 55-kDa fragment of the Met receptor is stabilized by lactacystin and demonstrate that it represents a cell-associated remnant that is generated following the ligand-independent proteolytic cleavage of the Met receptor in its extracellular domain. This truncated Met molecule encompasses the kinase domain of the receptor and is itself tyrosine phosphorylated. We conclude that the ubiquitin-proteasome pathway plays a significant role in the degradation of the Met tyrosine kinase receptor as directed by ligand-dependent and -independent signals. We propose that this proteolytic pathway may be important for averting cellular transformation by desensitizing Met signaling following ligand stimulation and by eliminating potentially oncogenic fragments generated via extracellular cleavage of the Met receptor.