Trenton L. Place

Nedd4 controls animal growth by regulating IGF-1 signaling.

Nedd4 acts through Grb10 to enhance insulin-like growth factor signaling and control animal growth. A Growth-Promoting Ubiquitin Ligase Genetic knockout of the ubiquitin ligase Nedd4 decreases insulin-like growth factor 1 (IGF-1) and insulin signaling and causes delayed embryonic development, reduced growth and body weight, and neonatal lethality. Elevated Grb10 in the Nedd4-deficient cells appears to cause mislocalization of the IGF-1 receptor and prevent receptor signaling at the plasma membrane. Thus, by regulating the abundance of Grb10, a negative regulator of IGF-1 and insulin signaling, Nedd4 positively influences growth. The ubiquitin ligase Nedd4 has been proposed to regulate a number of signaling pathways, but its physiological role in mammals has not been characterized. Here we present an analysis of Nedd4-null mice to show that loss of Nedd4 results in reduced insulin-like growth factor 1 (IGF-1) and insulin signaling, delayed embryonic development, reduced growth and body weight, and neonatal lethality. In mouse embryonic fibroblasts, mitogenic activity was reduced, the abundance of the adaptor protein Grb10 was increased, and the IGF-1 receptor, which is normally present on the plasma membrane, was mislocalized. However, surface expression of IGF-1 receptor was restored in homozygous mutant mouse embryonic fibroblasts after knockdown of Grb10, and Nedd4−/− lethality was rescued by maternal inheritance of a disrupted Grb10 allele. Thus, in vivo, Nedd4 appears to positively control IGF-1 and insulin signaling partly through the regulation of Grb10 function.

Epidermal Growth Factor Receptor Fate Is Controlled by Hrs Tyrosine Phosphorylation Sites That Regulate Hrs Degradation

ABSTRACT Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is an endosomal protein essential for the efficient sorting of activated growth factor receptors into the lysosomal degradation pathway. Hrs undergoes ligand-induced tyrosine phosphorylation on residues Y329 and Y334 downstream of epidermal growth factor receptor (EGFR) activation. It has been difficult to investigate the functional roles of phosphoHrs, as only a small proportion of the cellular Hrs pool is detectably phosphorylated. Using an HEK 293 model system, we found that ectopic expression of the protein Cbl enhances Hrs ubiquitination and increases Hrs phosphorylation following cell stimulation with EGF. We exploited Cbls expansion of the phosphoHrs pool to determine whether Hrs tyrosine phosphorylation controls EGFR fate. In structure-function studies of Cbl and EGFR mutants, the level of Hrs phosphorylation and rapidity of apparent Hrs dephosphorylation correlated directly with EGFR degradation. Differential expression of wild-type versus Y329,334F mutant Hrs in Hrs-depleted cells revealed that one or both tyrosines regulate ligand-dependent Hrs degradation, as well as EGFR degradation. By modulating Hrs ubiquitination, phosphorylation, and protein levels, Cbl may control the composition of the endosomal sorting machinery and its ability to target EGFR for lysosomal degradation.

EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate

EGF-R [EGF (epidermal growth factor) receptor] ligands can promote or inhibit cell growth. The biological outcome of receptor activation is dictated, at least in part, by ligand-specified patterns of endocytic trafficking. EGF-R trafficking downstream of the ligands EGF and TGF-alpha (transforming growth factor-alpha) has been investigated extensively. However, less is known about EGF-R fates induced by the ligands BTC (betacellulin) and AR (amphiregulin). We undertook comparative analyses to identify ligand-specific molecular events that regulate EGF-R trafficking and degradation. EGF (17 nM) and BTC (8.5 nM) induced significant EGF-R degradation, with or without ectopic expression of the ubiquitin ligase Cbl. Human recombinant AR (17 nM) failed to affect receptor degradation in either case. Notably, levels of ligand-induced EGF-R ubiquitination did not correlate strictly with receptor degradation. Dose-response experiments revealed that AR at a saturating concentration was a partial agonist at the EGF-R, with approx. 40% efficacy (relative to EGF) at inducing receptor tyrosine phosphorylation, ubiquitination and association with Cbl. EGF-R down-regulation and degradation also were compromised upon cell stimulation with AR (136 nM). These outcomes correlated with decreased degradation of the Cbl substrate and internalization inhibitor hSprouty2. Downstream of the hSprouty2 checkpoint in AR-stimulated cells, Cbl-free EGF-R was incorporated into endosomes from which Cbl-EGF-R complexes were excluded. Our results suggest that the AR-specific EGF-R fate results from decreased hSprouty2 degradation and reduced Cbl recruitment to underphosphorylated EGF-R, two effects that impair EGF-R trafficking to lysosomes.

Cbl Controls EGFR Fate by Regulating Early Endosome Fusion

The E3 ubiquitin ligase Cbl mediates the fusion of early endosomes necessary to target EGFR for lysosomal degradation. Cbl and Endosomes The E3 ubiquitin ligase Cbl causes the mono- and polyubiquitination of receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR), thereby targeting these proteins for degradation in lysosomes. RTK trafficking also depends on the modification of other Cbl-associated proteins at the plasma membrane and in endosomes, but how these work together to control RTK trafficking is not well understood. Critical to the ubiquitination function of Cbl is the really interesting new gene (RING) finger (RF) tail region, which prompted Visser Smit et al. to investigate the effects of single substitution mutants in this region of Cbl on the ubiquitination, down-regulation, and degradation of EGFR. They found that individual amino acid residues in the RF tail contributed differently to these processes and that Cbl played a role in EGFR internalization independently of its ability to ubiquitinate the receptor. In particular, Cbl was required for the fusion of early endosomes that trafficked EGFR to lysosomes, which depended, in part, on Hrs, a regulator of EGFR trafficking. Given the role of Cbl in mediating the down-regulation of multiple RTKs, its ability to control endosomal maturation may have general implications for controlling RTK activity. Amino acid residues 1 to 434 of the E3 ubiquitin ligase Cbl control signaling of the epidermal growth factor receptor (EGFR) by enhancing its ubiquitination, down-regulation, and lysosomal degradation. This region of Cbl comprises a tyrosine kinase–binding domain, a linker region, a really interesting new gene finger (RF), and a subset of the residues of the RF tail. In experiments with full-length alanine substitution mutants, we demonstrated that the RF tail of Cbl regulated biochemically distinct checkpoints in the endocytosis of EGFR. The Cbl- and ubiquitin-dependent degradation of the regulator of internalization hSprouty2 was compromised by the Val431→ Ala mutation, whereas the Cbl- and EGFR-dependent dephosphorylation or degradation of the endosomal trafficking regulator Hrs was compromised by the Phe434→ Ala mutation. Deregulated phosphorylation of Hrs correlated with inhibition of the fusion of early endosomes and of the degradation of EGFR. This study provides the first evidence that Cbl regulates receptor fate by controlling the fusion of sorting endosomes. We postulate that it does so by modulating the abundance of tyrosine-phosphorylated Hrs.

Aberrant promoter CpG methylation is a mechanism for impaired PHD3 expression in a diverse set of malignant cells.

Background The prolyl-hydroxylase domain family of enzymes (PHD1-3) plays an important role in the cellular response to hypoxia by negatively regulating HIF-α proteins. Disruption of this process can lead to up-regulation of factors that promote tumorigenesis. We observed decreased basal expression of PHD3 in prostate cancer tissue and tumor cell lines representing diverse tissues of origin. Furthermore, some cancer lines displayed a failure of PHD3 mRNA induction when introduced to a hypoxic environment. This study explores the mechanism by which malignancies neither basally express PHD3 nor induce PHD3 under hypoxic conditions. Methodology/Principal Findings Using bisulfite sequencing and methylated DNA enrichment procedures, we identified human PHD3 promoter hypermethylation in prostate, breast, melanoma and renal carcinoma cell lines. In contrast, non-transformed human prostate and breast epithelial cell lines contained PHD3 CpG islands that were unmethylated and responded normally to hypoxia by upregulating PHD3 mRNA. Only treatment of cells lines containing PHD3 promoter hypermethylation with the demethylating drug 5-aza-2′-deoxycytidine significantly increased the expression of PHD3. Conclusions/Significance We conclude that expression of PHD3 is silenced by aberrant CpG methylation of the PHD3 promoter in a subset of human carcinoma cell lines of diverse origin and that this aberrant cytosine methylation status is the mechanism by which these cancer cell lines fail to upregulate PHD3 mRNA. We further show that a loss of PHD3 expression does not correlate with an increase in HIF-1α protein levels or an increase in the transcriptional activity of HIF, suggesting that loss of PHD3 may convey a selective advantage in some cancers by affecting pathway(s) other than HIF.

Prolyl-hydroxylase 3: evolving roles for an ancient signaling protein

The ability of cells to sense oxygen is a highly evolved process that facilitates adaptations to the local oxygen environment and is critical to energy homeostasis. In vertebrates, this process is largely controlled by three intracellular prolyl-4-hydroxylases (PHD) 1–3. These related enzymes share the ability to hydroxylate the hypoxia-inducible transcription factor (HIF), and therefore control the transcription of genes involved in metabolism and vascular recruitment. However, it is becoming increasingly apparent that PHD controls much more than HIF signaling, with PHD3 emerging as an exceptionally unique and functionally diverse PHD isoform. In fact, PHD3-mediated hydroxylation has recently been purported to function in such diverse roles as sympathetic neuronal and muscle development, sepsis, glycolytic metabolism, and cell fate. PHD3 expression is also highly distinct from that of the other PHD enzymes, and varies considerably between different cell types and oxygen concentrations. This review will examine the evolution of oxygen sensing by the HIF family of PHD enzymes, with a specific focus on the complex nature of PHD3 expression and function in mammalian cells.

Prolyl-4-Hydroxylase 3 (PHD3) Expression Is Downregulated during Epithelial-to-Mesenchymal Transition

Prolyl-4-hydroxylation by the intracellular prolyl-4-hydroxylase enzymes (PHD1-3) serves as a master regulator of environmental oxygen sensing. The activity of these enzymes is tightly tied to tumorigenesis, as they regulate cell metabolism and angiogenesis through their control of hypoxia-inducible factor (HIF) stability. PHD3 specifically, is gaining attention for its broad function and rapidly accumulating array of non-HIF target proteins. Data from several recent studies suggest a role for PHD3 in the regulation of cell morphology and cell migration. In this study, we aimed to investigate this role by closely examining the relationship between PHD3 expression and epithelial-to-mesenchymal transition (EMT); a transcriptional program that plays a major role in controlling cell morphology and migratory capacity. Using human pancreatic ductal adenocarcinoma (PDA) cell lines and Madin-Darby Canine Kidney (MDCK) cells, we examined the correlation between several markers of EMT and PHD3 expression. We demonstrated that loss of PHD3 expression in PDA cell lines is highly correlated with a mesenchymal-like morphology and an increase in cell migratory capacity. We also found that induction of EMT in MDCK cells resulted in the specific downregulation of PHD3, whereas the expression of the other HIF-PHD enzymes was not affected. The results of this study clearly support a model by which the basal expression and hypoxic induction of PHD3 is suppressed by the EMT transcriptional program. This may be a novel mechanism by which migratory or metastasizing cells alter signaling through specific pathways that are sensitive to regulation by O2. The identification of downstream pathways that are affected by the suppression of PHD3 expression during EMT may provide important insight into the crosstalk between O2 and the migratory and metastatic potential of tumor cells.

Molecular characterization of desmoid tumors: decryption of the enigma.

resolution characterization of chromosomal abnormalities down to the kilobase level, whereas previous studies on desmoid tumors have largely employed techniques suited for the detection of megabase or larger chromosomal alterations [14]. In conjunction with SNP-A, the authors performed traditional DNA sequencing on a number of genes known to be mutated in desmoid tumors, including β-catenin (CTNNB1) and APC. Using these combined methodologies, the authors validated existing data on the presence of mutations in CTNNB1, and losses in chromosome 8q, which contains the APC locus. In addition, they detected numerous other small deletions and insertions that have previously not been reported in desmoid tumors. The most significant of these novel lesions was a heterozygous, somatic deletion affecting a small region of chromosome 8p23, which was observed in 44% of the tumors tested. This deletion falls within the CUB and Sushi multiple domains1 (CSMD1) gene, a putative tumor suppressor that is also mutated or deleted in tumors of the head and neck, colon, breast, and prostate [15, 16]. Furthermore, the authors noted a correlation between CSMD1 deletion and relapse, as 3 of the 9 patients with disease recurrence at the time of publication had exhibited a CSMD1 deletion. The small sample size and short follow-up time limit the authors’ ability to make firm conclusions regarding the prognostic significance of these otherwise exceptional and novel data. The management of desmoid tumors remains a challenge to the patient and the clinician. However, studies such as this by Erben and colleagues add to the arsenal of data that will eventually lead to novel therapies for this disease. As for all patients presenting with complex malignancies such as desmoid tumors, continued efforts for tissue collection and multi-institution and protocol-based regimens are justified.

Aberrant Promoter CpG Methylation is a Mechanism for Lack of Hypoxic Induction of PHD3 in a Diverse Set of Malignant Cells

The prolyl-hydroxylase domain family of enzymes (PHD1-3) plays an important role in the cellular response to hypoxia by negatively regulating HIF-α proteins. Disruption of this process can lead to upregulation of factors that promote tumorigenesis. We observed decreased basal expression of PHD3 in tumor cell lines representing diverse tissues of origin. Furthermore, some cancer lines displayed a failure of PHD3 mRNA induction when introduced to a hypoxic environment. This study explores the mechanism by which malignancies neither basally express PHD3 nor induce PHD3 under hypoxic conditions. Using bisulfite sequencing and methylated DNA enrichment procedures, we identified human PHD3 promoter hypermethylation in prostate, breast, melanoma and renal carcinoma cell lines. In contrast, non-transformed human prostate and breast epithelial cell lines contained PHD3 CpG islands that were unmethylated and responded normally to hypoxia by upregulating PHD3 mRNA. We conclude that expression of PHD3 is silenced by aberrant CpG methylation of the PHD3 promoter in a subset of human carcinoma cell lines of diverse origin and that this aberrant cytosine methylation status is the mechanism by which these cancer cell lines fail to upregulate PHD3 mRNA. Introduction The cellular response to reduced oxygen availability (hypoxia) is controlled by a class of proteins called hypoxia-inducible factors (HIF-α). Transcriptionally active HIF-1α and 2α are heterodimers composed of the HIF-α subunit and aryl hydrocarbon nuclear translocator receptor (ARNT). HIF-1α activates the transcription of VEGF and several other critical intracellular responses to hypoxia [1]. HIF-α mRNA levels are generally stable in cells. It is not until after