Ubr3, a Novel Modulator of Hh Signaling Affects the Degradation of Costal-2 and Kif7 through Poly-ubiquitination

Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues.     

The type-2 N-end rule peptide recognition activity of Ubr11 ubiquitin ligase is required for the expression of peptide transporters

The Ubr1-like canonical N-recognins, widely conserved ubiquitin ligases in eukaryotes, play a role in the N-end rule pathway-mediated degradation of substrates harboring basic (type-1) or bulky hydrophobic (type-2) amino acids at the N-terminus. In this study, the roles of conserved domains were studied in the Schizosaccharomyces pombe Ubr11 protein. Mutations in the UBR box and the autoinhibitory domain blocked degradation of both type-1 and type-2 substrates, expression of peptide transporter genes, and the uptake of oligopeptides. An N-domain mutant was normal for the type-1-related function, but nevertheless failed to express peptide transporters. These data suggest the importance of the type-2-related activity of Ubr11 for its in vivo function.     

UBR1 promotes protein kinase quality control and sensitizes cells to Hsp90 inhibition

UBR1 and UBR2 are N-recognin ubiquitin ligases that function in the N-end rule degradation pathway. In yeast, the UBR1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated by treatment of cells with geldanamycin, a small molecule inhibitor of Hsp90. Based on these studies we examined the role of mammalian UBR1 and UBR2 in the degradation of protein kinase clients upon Hsp90 inhibition. Our findings show that protein kinase clients Akt and Cdk4 are still degraded in mouse Ubr1(-)/(-) cells treated with geldanamycin, but that their levels recover much more rapidly than is found in wild type cells. These findings correlate with increased induction of Hsp90 expression in the Ubr1(-)/(-) cells compared with wild type cells. We also observed a reduction of UBR1 protein levels in geldanamycin-treated mouse embryonic fibroblasts and human breast cancer cells, suggesting that UBR1 is an Hsp90 client. Further studies revealed a functional overlap between UBR1 and the quality control ubiquitin ligase, CHIP. Our findings show that UBR1 function is conserved in controlling the levels of Hsp90-dependent protein kinases upon geldanamycin treatment, and suggest that it plays a role in determining the sensitivity of cancer cells to the chemotherapeutic effects of Hsp90 inhibitors.     

The Substrate Recognition Domains of the N-end Rule Pathway

The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with K_d of ∼3.4 μm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The ∼80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway.The N-end rule pathway is a ubiquitin (Ub)²-dependent proteolytic system in which N-terminal residues of short-lived proteins function as an essential component of degradation signals (degrons) called N-degrons (Fig. 1A) (1-15). An N-degron can be created from a pre-N-degron through specific N-terminal modifications (12). Specifically, in mammals, N-terminal Asn and Gln are tertiary destabilizing residues that function through their deamidation by N-terminal amidohydrolases into the secondary destabilizing N-terminal residues Asp and Glu, respectively (6, 16) (Fig. 1A). N-terminal Asp and Glu are secondary destabilizing residues that function through their arginylation by ATE1 R-transferase, which creates the primary destabilizing residue Arg at the N terminus (4, 8) (Fig. 1A). N-terminal Cys can also function as a tertiary destabilizing residue through its oxidation in a manner depending on nitric oxide and oxygen (O₂); the oxidized Cys residue is subsequently arginylated by ATE1 (8, 13, 17).Open in a separate windowFIGURE 1.A, the mammalian N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. C^* denotes oxidized N-terminal Cys, either Cys-sulfinic acid [CysO₂(H)] or Cys-sulfonic acid [CysO₃(H)]. The Cys oxidation requires nitric oxide and oxygen (O₂) or its derivatives. The oxidized Cys is arginylated by ATE1 Arg-tRNA-protein transferase (R-transferase). N-recognins also recognize internal (non-N-terminal) degrons in other substrates of the N-end rule pathway. B, the X-peptide pull-down assay. Left, a 12-mer peptide bearing N-terminal Arg (type 1), Phe (type 2), Trp (type 2), or Gly (stabilizing control) residue was cross-linked through its C-terminal Cys residue to Ultralink Iodoacetyl beads. Right, the otherwise identical 12-mer peptide, bearing C-terminal biotinylated Lys instead of Cys, was conjugated, via biotin, to the streptavidin-Sepharose beads. C, the X-peptide pull-down assay of endogenous UBR proteins using testes extracts. Extracts from mouse testes were mixed with bead-conjugated X-peptides bearing N-terminal Phe (F), Gly (G), or Arg (R). After centrifugation, captured proteins were separated and subjected to anti-UBR immunoblotting. Mo, a pull-down reaction with mock beads. D, the X-peptide pull-down assays using rat testis extracts were performed in the presence of varying concentrations of NaCl. After incubation and washing, bound proteins were eluted by 10 mm Tyr-Ala for Phe-peptide, 10 mm Arg-Ala for Arg-peptide, and 5 mm Tyr-Ala and 5 mm Arg-Ala for Val-peptide. Eluted proteins were subjected to immunoblotting for UBR1 and UBR5. E, cytoplasmic fractions of wild-type (+/+), Ubr1^-/-, Ubr2^-/-, Ubr1^-/-Ubr2^-/-, and Ubr1^-/-Ubr2^-/-Ubr4^RNAi MEFs were subjected to X-peptide pull-down assay. Precipitated proteins were separated and analyzed by immunoblotting for UBR1 and UBR4.N-terminal Arg together with other primary destabilizing N-terminal residues are directly bound by specific E3 Ub ligases called N-recognins (3, 7, 9). Destabilizing N-terminal residues can be created through the removal of N-terminal Met or the endoproteolytic cleavage of a protein, which exposes a new amino acid at the N terminus (12, 13). N-terminal degradation signals can be divided into type-1 (basic; Arg, Lys, and His) and type-2 (bulky hydrophobic; Phe, Leu, Trp, Tyr, and Ile) destabilizing residues (2, 12). In addition to a destabilizing N-terminal residue, a functional N-degron requires at least one internal Lys residue (the site of a poly-Ub chain formation) and a conformational feature required for optimal ubiquitylation (1, 2, 18). UBR1 and UBR2 are functionally overlapping N-recognins (3, 7, 9). Our proteomic approach using synthetic peptides bearing destabilizing N-terminal residues captured a set of proteins (200-kDa UBR1, 200-kDa UBR2, 570-kDa UBR4, and 300-kDa UBR5/EDD) characterized by a 70-residue zinc finger-like domain termed the UBR box (10-12). UBR5 is a HECT E3 ligase known as EDD (E3 identified by differential display) (19) and a homolog of Drosophila hyperplastic discs (20). The mammalian genome encodes at least seven UBR box-containing proteins, termed UBR1 through UBR7 (10). UBR box proteins are generally heterogeneous in size and sequence but contain, with the exception of UBR4, specific signatures unique to E3s or a substrate recognition subunit of the E3 complex: the RING domain in UBR1, UBR2, and UBR3; the HECT domain in UBR5; the F-box in UBR6 and the plant homeodomain domain in UBR7 (Fig. 2B). The biochemical properties of more recently identified UBR box proteins, such as UBR3 through UBR7, are largely unknown.Open in a separate windowFIGURE 2.The binding properties of the UBR box family members to type-1 and type-2 destabilizing N-terminal residues. A, the X-peptide pull-down assay with overexpressed, full-length UBR proteins: UBR2, UBR3 (in S. cerevisiae cells), UBR4, UBR5 (in COS7 cells), and UBR6 and UBR7 (in the wheat germ lysates). Precipitates were analyzed by immunoblotting (for UBR2, UBR3, UBR4, and UBR5) with tag-specific antibodies as indicated in B or autoradiography (for UBR6 and UBR7). B, the structures of UBR box proteins. Shown are locations of the UBR box, the N-domain, and other E3-related domains. UBR, UBR box; RING, RING finger; UAIN, UBR-specific autoinhibitory domain; CRD, cysteine-rich domain; PHD, plant homeodomain; HECT, HECT domain.Studies using knock-out mice implicated the N-end rule pathway in cardiac development and signaling, angiogenesis (8, 15), meiosis (9), DNA repair (21), neurogenesis (15), pancreatic functions (22), learning and memory (23, 24), female development (9), muscle atrophy (25), and olfaction (11). Mutations in human UBR1 is a cause of Johanson-Blizzard syndrome (22), an autosomal recessive disorder with multiple developmental abnormalities (26). Other functions of the pathway include: (i) a nitric oxide and oxygen (O₂) sensor controlling the proteolysis of RGS4, RGS5, and RGS16 (8, 13, 17), (ii) a heme sensor through hemin-dependent inhibition of ATE1 function (27), (iii) the regulation of short peptide import through the peptide-modulated degradation of the repressor of the import (28, 29), (iv) the control of chromosome segregation through the degradation of a separate produced cohesin fragment (30), (v) the regulation of apoptosis through the degradation of a caspase-processed inhibitor of apoptosis (31, 32), (vi) the control of the human immunodeficiency virus replication cycle through the degradation of human immunodeficiency virus integrase (10, 33), and (vii) the regulation of leaf senescence in plants (34).In the present study we characterized substrate binding specificities and recognition domains of UBR proteins. In our binding assays, UBR1, UBR2, UBR4, and UBR5 were captured by N-terminal degradation determinants, whereas UBR3, UBR6, and UBR7 were not. We also report that in contrast to other E3 systems that usually recognize substrates through protein-protein interface, UBR1 and UBR2 have a general substrate recognition domain termed the UBR box. Remarkably, a 72-residue UBR box-only fragment fully retains its structural integrity and thereby the ability to recognize type-1 N-end rule substrates. We also report that the N-domain, structurally and functionally related with bacterial N-recognins, is required for recognizing type-2 N-end rule substrates. We discuss the evolutionary relationship between eukaryotic and prokaryotic N-recognins.     

Ubiquitin ligases of the N-end rule pathway: assessment of mutations in UBR1 that cause the Johanson-Blizzard syndrome

Background

Johanson-Blizzard syndrome (JBS; OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, facial dysmorphism with the characteristic nasal wing hypoplasia, multiple malformations, and frequent mental retardation. Our previous work has shown that JBS is caused by mutations in human UBR1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. One class of degradation signals (degrons) recognized by UBR1 are destabilizing N-terminal residues of protein substrates.

Methodology/Principal Findings

Most JBS-causing alterations of UBR1 are nonsense, frameshift or splice-site mutations that abolish UBR1 activity. We report here missense mutations of human UBR1 in patients with milder variants of JBS. These single-residue changes, including a previously reported missense mutation, involve positions in the RING-H2 and UBR domains of UBR1 that are conserved among eukaryotes. Taking advantage of this conservation, we constructed alleles of the yeast Saccharomyces cerevisiae UBR1 that were counterparts of missense JBS-UBR1 alleles. Among these yeast Ubr1 mutants, one of them (H160R) was inactive in yeast-based activity assays, the other one (Q1224E) had a detectable but weak activity, and the third one (V146L) exhibited a decreased but significant activity, in agreement with manifestations of JBS in the corresponding JBS patients.

Conclusions/Significance

These results, made possible by modeling defects of a human ubiquitin ligase in its yeast counterpart, verified and confirmed the relevance of specific missense UBR1 alleles to JBS, and suggested that a residual activity of a missense allele is causally associated with milder variants of JBS.     

Ubr1 and Ubr2 Function in a Quality Control Pathway for Degradation of Unfolded Cytosolic Proteins

Quality control systems facilitate polypeptide folding and degradation to maintain protein homeostasis. Molecular chaperones promote folding, whereas the ubiquitin/proteasome system mediates degradation. We show here that Saccharomyces cerevisiae Ubr1 and Ubr2 ubiquitin ligases promote degradation of unfolded or misfolded cytosolic polypeptides. Ubr1 also catalyzes ubiquitinylation of denatured but not native luciferase in a purified system. This activity is based on the direct interaction of denatured luciferase with Ubr1, although Hsp70 stimulates polyubiquitinylation of the denatured substrate. We also report that loss of Ubr1 and Ubr2 function suppressed the growth arrest phenotype resulting from chaperone mutation. This correlates with increased protein kinase maturation and indicates partitioning of foldable conformers toward the proteasome. Our findings, based on the efficiency of this quality control system, suggest that the cell trades growth potential to avert the potential toxicity associated with accumulation of unfolded or misfolded proteins. Ubr1 and Ubr2 therefore represent E3 components of a novel quality control pathway for proteins synthesized on cytosolic ribosomes.     

Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila

Apoptosis has essential roles in a variety of cellular and developmental processes. Although the pathway is well studied, how the activities of individual components in the pathway are regulated is less understood. In Drosophila, a key component in apoptosis is Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is required to prevent caspase activation. Here, we demonstrate that Drosophila CG42593 (ubr3), encoding the homolog of mammalian UBR3, has an essential role in regulating the apoptosis pathway. We show that loss of ubr3 activity causes caspase-dependent apoptosis in Drosophila eye and wing discs. Our genetic epistasis analyses show that the apoptosis induced by loss of ubr3 can be suppressed by loss of initiator caspase Drosophila Nedd2-like caspase (Dronc), or by ectopic expression of the apoptosis inhibitor p35, but cannot be rescued by overexpression of DIAP1. Importantly, we show that the activity of Ubr3 in the apoptosis pathway is not dependent on its Ring-domain, which is required for its E3 ligase activity. Furthermore, we find that through the UBR-box domain, Ubr3 physically interacts with the neo-epitope of DIAP1 that is exposed after caspase-mediated cleavage. This interaction promotes the recruitment and ubiquitination of substrate caspases by DIAP1. Together, our data indicate that Ubr3 interacts with DIAP1 and positively regulates DIAP1 activity, possibly by maintaining its active conformation in the apoptosis pathway.Morphogenesis in multicellular organisms is a process with a balanced control of cell proliferation and cell death. To maintain this homeostasis, superfluous or unwanted cells are usually removed promptly via programmed cell death or apoptosis.^{1, 2} Compelling evidence has shown that dysregulation of apoptosis results in a variety of diseases, such as cancer, neurodegenerative disorders and autoimmune diseases.^{3, 4, 5, 6} The apoptotic machinery is conserved from invertebrates to vertebrates. Drosophila has been used as an excellent model to study apoptosis because of its advantages in genetic manipulation. A crucial step in apoptosis is the cascade activation of initiator and effector caspases that eventually causes cell death. Under normal circumstances, the activities of caspases are kept in check by a conserved family of anti-apoptotic proteins termed inhibitor of apoptosis proteins (IAPs). The Drosophila genome encodes four IAPs, including Drosophila inhibitor of apoptosis protein 1 (DIAP1), DIAP2, DBruce and Deterin.^{7, 8, 9, 10} Among these four proteins, DIAP1 is stringently required to prevent caspase activation.^{11, 12} Although the requirement of DIAP1 in the apoptosis pathway is well documented, it is unclear how the activity of DIAP1 is regulated during development.The covalent attachment of ubiquitin to proteins is a crucial regulatory mechanism in many developmental and physiological processes.¹³ Ubiquitination is a catalytic cascade involving ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-ligating (E3) enzymes.¹⁴ The E3 proteins that specifically recognize a distinctive set of substrates for ubiquitination are an exceptionally large family.¹⁵ The RING domain of DIAP1 is an E3 ligase that inactivates caspases mainly through ubiquitination.¹⁶ Previous studies have shown that the anti-apoptotic activity of DIAP1 is negatively regulated by three pro-apoptotic proteins called Reaper, head involution defective (Hid) and Grim (RHG).^{2, 17} These proteins negatively regulate DIAP1 function through distinct mechanisms, either by disrupting interactions between DIAP1 and the initiator caspase Drosophila Nedd2-like caspase (Dronc), or by promoting the ubiquitination-dependent degradation of DIAP1.^{18, 19} In addition to regulation by RHG, DIAP1 has been considered a substrate of the N-end rule pathway. Ditzel et al.²⁰ first discovered that DIAP1 can be cleaved by effector caspases at its NH2 terminus, exposing a binding motif for UBR-box-containing E3 ligases and subsequently be degraded by the N-end rule-mediated degradation. Later, Yokokura et al.²¹ reported that the N-end rule pathway does not have a major role in the turnover of N-terminally truncated DIAP1. More recently, Ditzel et al.²² reported that the UBR-binding motif of DIAP1 is essential for its anti-apoptotic function, indicating UBR family proteins regulate apoptosis via ways other than destroying DIAP1. Although these reports highlight the importance of UBR-box-containing E3 ligases in regulating DIAP1, it is currently unknown which UBR family member is involved in apoptosis.The mammalian genome encodes seven evolutionarily conserved members of UBR E3 ligases (UBR1–UBR7).^{23, 24} All of the UBR E3 ligases share a ∼70-amino-acid UBR-box and can function as N-recognins, which are involved in the N-end rule pathway of the ubiquitination system.²⁵ Among them, UBR1, UBR2 and UBR3 are subfamily members containing both a UBR-box and a Ring domain, which is required for its E3 ligase activity.²⁶ Data from mouse models indicate that UBR1 and UBR2 are involved in the regulation of apoptosis in spermatocytes, skeletal muscle and cardiovascular development with partial redundancy.^{27, 28, 29} UBR3 was first characterized in mice as an E3 ligase involved in the regulation of olfactory and other sensory systems.³⁰ Recently, human UBR3 was also found to be required for genome stability by regulating the essential DNA repair protein APE1.³¹ As UBR3 does not bind to the known N-end rule substrates of UBR1 and UBR2,³⁰ it is currently unknown whether UBR3 is involved in the apoptosis pathway. Here, we have generated Drosophila ubr3 mutant and characterized its role in development. Our data suggest that Ubr3 is involved in the apoptosis pathway by regulating the activity of DIAP1 during development.     

Dual functions for WNT5A during cartilage development and in disease

Mouse and human genetic data suggests that Wnt5a is required for jaw development but the specific role in facial skeletogenesis is unknown. We mapped expression of WNT5A in the developing chicken skull and found that the highest expression was in early Meckel's cartilage but by stage 35 expression was decreased to background. We focused on chondrogenesis by targeting a retrovirus expressing WNT5A to the mandibular prominence prior to cell differentiation. Unexpectedly, there were no phenotypes in the first 6 days following injection; however later the mandibular bones and Meckel's cartilage were reduced or missing on the treated side. To examine the effects on cartilage differentiation we treated micromass cultures from mandibular mesenchyme with Wnt5a-conditioned media (CM). Similar to in vivo viral data, cartilage differentiates normally, but, after 6 days of culture, nearly all Alcian blue staining is lost. Collagen II and aggrecan were also decreased in treated cultures. The matrix loss was correlated with upregulation of metalloproteinases, MMP1, MMP13, and ADAMTS5 (codes for Aggrecanase). Moreover, Marimastat, an MMP and Aggrecanase inhibitor rescued cartilage matrix in Wnt5a-CM treated cultures. The pathways mediating these cartilage and RNA changes were investigated using luciferase assays. Wnt5a-CM was a potent inhibitor of the canonical pathway and strongly activated JNK/PCP signaling. To determine whether the matrix loss is mediated by repression of canonical signaling or activation of the JNK pathway we treated mandibular cultures with either DKK1, an antagonist of the canonical pathway, or a small molecule that antagonizes JNK signaling (TCS JNK 6o). DKK1 slightly increased cartilage formation and therefore suggested that the endogenous canonical signaling represses chondrogenesis. To test this further we added an excess of Wnt3a-CM and found that far fewer cartilage nodules differentiated. Since DKK1 did not mimic the effects of Wnt5a we excluded the canonical pathway from mediating the matrix loss phenotype. The JNK antagonist partially rescued the Wnt5a phenotype supporting this non-canonical pathway as the main mediator of the cartilage matrix degradation. Our study reveals two new roles for WNT5A in development and disease: 1) to repress canonical Wnt signaling in cartilage blastema in order to promote normal differentiation and 2) in conditions of excess to stimulate degradation of mature cartilage matrix via non-canonical pathways.     

Wnk kinases are positive regulators of canonical Wnt/β-catenin signalling

Wnt/β-catenin signalling is central to development and its regulation is essential in preventing cancer. Using phosphorylation of Dishevelled as readout of pathway activation, we identified Drosophila Wnk kinase as a new regulator of canonical Wnt/β-catenin signalling. WNK kinases are known for regulating ion co-transporters associated with hypertension disorders. We demonstrate that wnk loss-of-function phenotypes resemble canonical Wnt pathway mutants, while Wnk overexpression causes gain-of-function canonical Wnt-signalling phenotypes. Importantly, knockdown of human WNK1 and WNK2 also results in decreased Wnt signalling in mammalian cell culture, suggesting that Wnk kinases have a conserved function in ensuring peak levels of canonical Wnt signalling.     

Lgl2 Executes Its Function as a Tumor Suppressor by Regulating ErbB Signaling in the Zebrafish Epidermis

Changes in tissue homeostasis, acquisition of invasive cell characteristics, and tumor formation can often be linked to the loss of epithelial cell polarity. In carcinogenesis, the grade of neoplasia correlates with impaired cell polarity. In Drosophila, lethal giant larvae (lgl), discs large (dlg), and scribble, which are components of the epithelial apico-basal cell polarity machinery, act as tumor suppressors, and orthologs of this evolutionary conserved pathway are lost in human carcinoma with high frequency. However, a mechanistic link between neoplasia and vertebrate orthologs of these tumor-suppressor genes remains to be fully explored at the organismal level. Here, we show that the pen/lgl2 mutant phenotype shares two key cellular and molecular features of mammalian malignancy: cell autonomous epidermal neoplasia and epithelial-to-mesenchymal-transition (EMT) of basal epidermal cells including the differential expression of several regulators of EMT. Further, we found that epidermal neoplasia and EMT in pen/lgl2 mutant epidermal cells is promoted by ErbB signalling, a pathway of high significance in human carcinomas. Intriguingly, EMT in the pen/lgl2 mutant is facilitated specifically by ErbB2 mediated E-cadherin mislocalization and not via canonical snail–dependent down-regulation of E-cadherin expression. Our data reveal that pen/lgl2 functions as a tumor suppressor gene in vertebrates, establishing zebrafish pen/lgl2 mutants as a valuable cancer model.     

Presence of cartilage stem/progenitor cells in adult mice auricular perichondrium

Background

Based on evidence from several other tissues, cartilage stem/progenitor cells in the auricular cartilage presumably contribute to tissue development or homeostasis of the auricle. However, no definitive studies have identified or characterized a stem/progenitor population in mice auricle.

Methodology/Principal Findings

The 5-bromo-2′-deoxyuridine (BrdU) label-retaining technique was used to label dividing cells in fetal mice. Observations one year following the labeling revealed that label-retaining cells (LRCs) were present specifically in auricular perichondrium at a rate of 0.08±0.06%, but LRCs were not present in chondrium. Furthermore, LRCs were successfully isolated and cultivated from auricular cartilage. Immunocytochemical analyses showed that LRCs express CD44 and integrin-α₅. These LRCs, putative stem/progenitor cells, possess clonogenicity and chondrogenic capability in vitro.

Conclusions/Significance

We have identified a population of putative cartilage stem/progenitor cells in the auricular perichondrium of mice. Further characterization and utilization of the cell population should improve our understanding of basic cartilage biology and lead to advances in cartilage tissue engineering and novel therapeutic strategies for patients with craniofacial defects, including long-term tissue restoration.     

Functional Mapping of Protein Kinase A Reveals Its Importance in Adult Schistosoma mansoni Motor Activity

Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and ‘smart’ anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology.     

Negative Regulation of Bone Formation by the Transmembrane Wnt Antagonist Kremen-2

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.     

Calcineurin is part of a negative feedback loop in the InsP3/Ca signalling pathway in blowfly salivary glands

The ubiquitous InsP₃/Ca²⁺ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca²⁺ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca²⁺] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP₃-induced Ca²⁺ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP₃/Ca²⁺ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca²⁺ signals; (2) cyclosporin A and FK506, inhibitors of Ca²⁺/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca²⁺ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca²⁺ responses does not involve Ca²⁺ entry into the cells; (4) cyclosporin A increases InsP₃-dependent Ca²⁺ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca²⁺ responses, indicating that PKA and calcineurin act antagonistically on the InsP₃/Ca²⁺ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP₃/Ca²⁺ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations.     

Identification and characterization of RING-finger ubiquitin ligase UBR7 in mammalian spermatozoa

The ubiquitin-proteasome system (UPS) controls intracellular protein turnover in a substrate-specific manner via E3-type ubiquitin ligases. Mammalian fertilization and particularly sperm penetration through the oocyte vitelline coat, the zona pellucida (ZP), is regulated by UPS. We use an extrinsic substrate of the proteasome-dependent ubiquitin-fusion degradation pathway, the mutant ubiquitin UBB⁺¹, to provide evidence that an E3-type ligase activity exists in sperm-acrosomal fractions. Protein electrophoresis gels from such de novo ubiquitination experiments contained a unique protein band identified by tandem mass spectrometry as being similar to ubiquitin ligase UBR7 (alternative name: C14ORF130). Corresponding mRNA was amplified from boar testis and several variants of the UBR7 protein were detected in boar, mouse and human sperm extracts by Western blotting. Genomic analysis indicated a high degree of evolutionary conservation, remarkably constant purifying selection and conserved testis expression of the UBR7 gene. By immunofluorescence, UBR7 was localized to the spermatid acrosomal cap and sperm acrosome, in addition to hotspots of proteasomal activity in spermatids, such as the cytoplasmic lobe, caudal manchette, nucleus and centrosome. During fertilization, UBR7 remained with the ZP-bound acrosomal shroud following acrosomal exocytosis. Thus, UBR7 is present in the acrosomal cap of round spermatids and within the acrosomal matrix of mature boar spermatozoa. These data provide the first evidence of ubiquitin ligase activity in mammalian spermatozoa and indicate UBR7 involvement in spermiogenesis.     

TAK1 is an essential regulator of BMP signalling in cartilage

TGFβ activated kinase 1 (TAK1), a member of the MAPKKK family, controls diverse functions ranging from innate and adaptive immune system activation to vascular development and apoptosis. To analyse the in vivo function of TAK1 in cartilage, we generated mice with a conditional deletion of Tak1 driven by the collagen 2 promoter. Tak1^col2 mice displayed severe chondrodysplasia with runting, impaired formation of secondary centres of ossification, and joint abnormalities including elbow dislocation and tarsal fusion. This phenotype resembled that of bone morphogenetic protein receptor (BMPR)1 and Gdf5-deficient mice. BMPR signalling was markedly impaired in TAK1-deficient chondrocytes as evidenced by reduced expression of known BMP target genes as well as reduced phosphorylation of Smad1/5/8 and p38/Jnk/Erk MAP kinases. TAK1 mediates Smad1 phosphorylation at C-terminal serine residues. These findings provide the first in vivo evidence in a mammalian system that TAK1 is required for BMP signalling and functions as an upstream activating kinase for Smad1/5/8 in addition to its known role in regulating MAP kinase pathways. Our experiments reveal an essential role for TAK1 in the morphogenesis, growth, and maintenance of cartilage.