Jennifer Mariano

WW Domain HECT E3s Target Cbl RING Finger E3s for Proteasomal Degradation

Cbl proteins have RING finger-dependent ubiquitin ligase (E3) activity that is essential for down-regulation of tyrosine kinases. Here we establish that two WW domain HECT E3s, Nedd4 and Itch, bind Cbl proteins and target them for proteasomal degradation. This is dependent on the E3 activity of the HECT E3s but not on that of Cbl. Consistent with these observations, in cells expressing the epidermal growth factor receptor, Nedd4 reverses Cbl-b effects on receptor down-regulation, ubiquitylation, and proximal events in signaling. Cbl-b also targets active Src for degradation in cells, and Nedd4 similarly reverses Cbl-mediated Src degradation. These findings establish that RING finger E3s can be substrates, not only for autoubiquitylation but also for ubiquitylation by HECT E3s and suggest an additional level of regulation for Cbl substrates including protein-tyrosine kinases.

Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Defined Specific E2-Binding Region of gp78

The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an approximately 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and, consequently, ubiquitylation.

Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine

RING finger proteins constitute the large majority of ubiquitin ligases (E3s) and function by interacting with ubiquitin‐conjugating enzymes (E2s) charged with ubiquitin. How low‐affinity RING–E2 interactions result in highly processive substrate ubiquitination is largely unknown. The RING E3, gp78, represents an excellent model to study this process. gp78 includes a high‐affinity secondary binding region for its cognate E2, Ube2g2, the G2BR. The G2BR allosterically enhances RING:Ube2g2 binding and ubiquitination. Structural analysis of the RING:Ube2g2:G2BR complex reveals that a G2BR‐induced conformational effect at the RING:Ube2g2 interface is necessary for enhanced binding of RING to Ube2g2 or Ube2g2 conjugated to Ub. This conformational effect and a key ternary interaction with conjugated ubiquitin are required for ubiquitin transfer. Moreover, RING:Ube2g2 binding induces a second allosteric effect, disrupting Ube2g2:G2BR contacts, decreasing affinity and facilitating E2 exchange. Thus, gp78 is a ubiquitination machine where multiple E2‐binding sites coordinately facilitate processive ubiquitination.

Coordinate Expression of Transforming Growth Factor-β1 and Adrenomedullin in Rodent Embryogenesis

Transforming growth factor-β (TGFβ) and adrenomedullin (AM) are multifunctional regulatory peptides that are secreted by a variety of normal and malignant cells. The TGFβs are expressed in developing organs and adults, and their tissue distribution pattern has possible significance for signaling roles in many epithelial-mesenchymal interactions. AM is also expressed in a variety of embryonic and adult tissues. The present study reports a comparison of the patterns of expression of the proteins and messenger RNAs (mRNAs) for TGFβ1 and AM in the developing mouse embryo. Immunohistochemical and in situ hybridization analyses were performed on formalin-fixed paraffin-embedded sections of developing embryonic mouse tissues using specific antibodies and complementary RNA probes for TGFβ1 and AM. The early placenta, including the giant trophoblastic cells, showed high levels of staining and hybridization for TGFβ1 and AM proteins and mRNAs. The heart was the first organ that showed expression of TGFβ1 and AM dur...

Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion

Aryl hydrocarbon receptor (AHR)‐null mice display a liver fibrosis phenotype that is associated with a concomitant increase in liver retinoid concentration, tissue transglutaminase type II (TGaseII) activity, transforming growth factorβ (TGFβ) overexpression, and accumulation of collagen. To test the hypothesis that this phenotype might be triggered by the observed increase in liver retinoid content, we induced the condition of retinoid depletion by feeding AHR‐null mice a vitamin A‐ deficient diet with the purpose to reverse the phenotype. Liver retinoid content decreased sharply within the first few weeks on the retinoid‐deficient diet. Analysis of TGFβ1, TGFβ2, and TGFβ3 expression revealed a reduction to control levels in the AHR ‐/‐ mice accompanied by parallel changes in TGaseII protein levels. In addition, we observed an increase in the TGFβ receptors, TGFβ RI and TGFβ RII, as well as in Smad4, and their reduction to wild‐type mouse liver levels in AHR ‐/‐ mice fed the retinoid‐deficient diet. Reduction of peroxisomal proliferator‐activated receptor γ (PPARγ) messenger RNA (mRNA) and protein levels in AHR ‐/‐ mice was consistent with the presence of hepatic stellate cell (HSC) activation and liver fibrosis. Vitamin A deficiency normalized PPARγ expression in AHR ‐/‐ mice. In conclusion, livers from AHR ‐/‐ mice fed the vitamin A‐deficient diet showed a decrease in collagen deposition, consistent with the absence of liver fibrosis. (HEPATOLOGY 2004;39:157–166.)

A Ubc7p-binding domain in Cue1p activates ER-associated protein degradation.

Cue1p is an N-terminally anchored endoplasmic reticulum (ER) protein essential for the activity of the two major yeast RING finger ubiquitin ligases (E3s) implicated in ER-associated degradation (ERAD). Cue1p contains a CUE domain, which for several proteins is known to bind ubiquitin. We now establish that the CUE domain is dispensable for ERAD of substrates of both Hrd1p and Doa10p and that the Cue1p transmembrane domain is similarly not required for degradation of the Hrd1p substrate CPY*. Cue1p interacts with the ERAD E2 Ubc7p in vivo. We show that a discrete C-terminal Ubc7p binding region (U7BR) of Cue1p is required for ERAD and for Ubc7p-dependent ubiquitylation by Hrd1p in vitro. Strikingly, when Ubc7p is stabilized by direct anchoring to the ER membrane, the U7BR is sufficient to restore ERAD in cells lacking Cue1p. Thus, discrete E2 binding sites independent of ubiquitin ligase domains have the potential to activate ubiquitylation.

TRANSFORMING GROWTH FACTOR-b1 AND ITS RECEPTORS IN HUMAN LUNG CANCER AND MOUSE LUNG CARCINOGENESIS

The transforming growth factor-betas (TGF-beta s) are multifunctional proteins that inhibit the proliferation of many epithelial cells through a set of cell protein receptors that includes the TGF-beta type I (RI) and type II (RII) receptors. Loss of growth inhibition by TGF-beta is thought to contribute to the development of many types of tumors. In the present study, we have examined expression of the proteins and mRNAs for TGF-beta 1, TGF-beta RI, and TGF-beta RII in normal human lung, well-characterized non-small cell lung cancer (NSCLC) cell lines, and primary NSCLC specimens. Immunohistochemical staining for TGF-beta 1, TGF-beta RI, and TGF-beta RII using specific antibodies in normal human lung showed expression of the 3 proteins in the epithelium of bronchi and bronchioles as well as in alveoli. Differential expression of TGF-beta RI and TGF-beta RII proteins was detected in 5 NSCLC cell lines using Western blot analysis, with reduced levels in 3 cell lines. A panel of 45 formalin-fixed and paraffin-embedded NSCLC specimens showed positive immunostaining for TGF-beta 1, TGF-beta RI, and TGF-beta RII, with reduced TGF-beta RII in poorly differentiated adenocarcinomas and squamous cell carcinomas and some moderately differentiated adenocarcinomas. In situ hybridization studies conducted with specific riboprobes for TGF-beta 1, TGF-beta RI, and TGF-beta RII showed corresponding localization of expression of the mRNAs in the specimens that showed positive immunostaining for the proteins. To investigate the roles of TGF-beta 1, TGF-beta RI, and TGF-beta RII in chemically induced mouse lung tumorigenesis, we examined the expression of their proteins and mRNAs in 2 mouse model systems. Whereas expression of the proteins and mRNAs for TGF-beta 1 and TGF-beta RI was comparable in lung adenomas and bronchioles of A/J mice treated with benzo(alpha)pyrene, decreased immunostaining and hybridization for TGF-beta RII protein and mRNA was detected in 50% of lung adenomas in these mice. Interestingly, expression of TGF-beta 1 and the TGF-beta receptor proteins was similar to that of bronchioles in C57B1/6 mice and their littermates heterozygous for deletion of the TGF-beta 1 gene treated with diethylnitrosamine. These data show that reduced levels of expression of TGF-beta RII occur in some, but not all, human and mouse lung tumors. This suggests that different mechanisms of action, some of which may involve the TGF-beta signaling pathway, may contribute to the progression of lung tumorigenesis.

Differential regulation of protease and extracellular matrix protein expression by transforming growth factor-beta 1 in non-small cell lung cancer cells and normal human bronchial epithelial cells

In addition to autoregulating its own expression, transforming growth factor-beta 1 (TGF-beta1) also regulates the production of proteases, protease inhibitors and extracellular matrix proteins. To investigate the relationship between plasminogen activator (PA), plasminogen activator inhibitor-1 (PAI-1) and the extracellular matrix in malignant and normal lung epithelial cells and to determine whether malignant lung epithelial cells may be more invasive than normal lung epithelial cells because of differences in expression of these proteins in response to TGF-beta, the regulation of PA, PAI-1, fibronectin, laminin and thrombospondin by TGF-beta1 in human non-small cell lung cancer (NSCLC) cells was examined and compared with normal human bronchial epithelial (NHBE) cells. TGF-beta1 caused a persistent increase in expression of the mRNAs for both PA and PAI-1 in NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. By immunoprecipitation analysis, it was shown that TGF-beta1 also induced a corresponding increase in the amount of PAI-1 protein in these NSCLC cells as well. In contrast, while TGF-beta1 also increased expression of PAI-1 mRNA in NHBE cells, expression of PA mRNA decreased simultaneously. Treatment of NSCLC cells with TGF-beta1 resulted in a persistent increase in expression of the mRNAs for fibronectin, laminin and thrombospondin; expression of fibronectin protein also increased after treatment with TGF-beta1 in these cells. When NHBE cells were similarly cultured in the presence of TGF-beta1, expression of fibronectin mRNA also increased in a persistent manner; however, only an early transient increase in the level of the mRNAs for laminin and thrombospondin was detected in these cells. These data show that there is differential regulation of the genes for PA and PAI-1 and the extracellular matrix protein fibronectin in response to TGF-beta1 not only when NSCLC and NHBE cells are compared, but also when different NSCLC cells are compared with each other.

Reduction in transforming growth factor-β type II receptor in mouse lung carcinogenesis

Transforming growth factor‐β (TGF‐β) is a growth modulator that inhibits the proliferation of many epithelial cells through interaction with its receptors, the type I and type II receptors (TGF‐β RI and RII) by activating their serine/threonine kinase activities. Loss of growth inhibition by TGF‐β is thought to contribute to the development of many types of tumors. To examine the roles of TGF‐β1, ‐β2, and ‐β3 and TGF‐β RI and RII in chemically induced mouse lung tumorigenesis, we used immunohistochemical and in situ hybridization analyses to measure the expression of their proteins and mRNAs in A/J mice treated with the carcinogen urethane to induce lung adenomas. Immunostaining for the TGF‐β ligands and receptors was detected in the epithelia of the bronchioles of untreated and treated A/J mice at similar levels. Immunostaining for the TGF‐β ligands and receptors was also detected in adenomas by 2 mo. While immunostaining for TGF‐β1, ‐β2, and ‐β3 and TGF‐β RI in adenomas was detected at levels comparable to those in bronchioles, immunostaining for TGF‐β RII was less intense in adenomas than in bronchioles. Decreased immunostaining for TGF‐β RII in adenomas persisted for at least 8 mo after exposure to urethane, whereas immunostaining for TGF‐β1, ‐β2, and ‐β3 and TGF‐β RI persisted at levels comparable to those in normal bronchioles. In situ hybridization studies conducted with TGF‐β receptor riboprobes showed a corresponding reduction in expression of TGF‐β RII mRNA but not of TGF‐β RI mRNA in adenomas compared with expression in bronchioles. Expression of TGF‐β RII mRNA was also examined in non‐tumorigenic and tumorigenic mouse lung cells; expression of TGF‐β RII mRNA was lower in the tumorigenic cells derived from urethane‐induced lung tumors. These data suggest that a decrease in expression of TGF‐β RII may contribute to autonomous cell growth and may play an important role in mouse lung carcinogenesis induced by urethane. Mol. Carcinog. 22:46–56, 1998.

Enhanced tumorigenesis and reduced transforming growth factor-? type II receptor in lung tumors from mice with reduced gene dosage of transforming growth factor-?1

To elucidate the role of transforming growth factor‐β1 (TGF‐β1) and the TGF‐β type II receptor (TGF‐β RII) as tumor‐suppressor genes in lung carcinogenesis, we mated C57BL/6 mice heterozygous (HT) for deletion of the TGF‐β1 gene with A/J mice to produce AJBL6 TGF‐β1 HT progeny and their wild‐type (WT) littermates. Immunohistochemical staining, in situ hybridization, and northern blot analyses showed lower staining and hybridization for TGF‐β1 protein and mRNA, respectively, in the lungs of normal HT mice versus WT mice. Competitive reverse transcription–polymerase chain reaction (CRT‐PCR) amplification showed the level of TGF‐β1 mRNA in the lungs of HT mice to be fourfold lower than the level in WT lung. When challenged with ethyl carbamate, lung adenomas were detected in 55% of HT mice by 4 mo but only in 25% of WT littermates at this time. Whereas all HT mice had adenomas by 6 mo, it was not until 10 mo before all WT mice had adenomas. After 12 mo, the average number of adenomas was fivefold higher in HT lungs than in WT lungs. Most dramatic was the appearance of lung carcinomas in HT mice 8 mo before they were visible in WT mice. Thus, the AJBL6 TGF‐β1 HT mouse provides an excellent model system to examine carcinogen‐induced lung tumorigenesis by increasing progressive lesion incidence and multiplicity relative to their WT littermates. Immunohistochemical staining showed expression of the TGF‐β type I receptor (TGF‐β RI) at moderate to strong levels in lung adenomas and carcinomas in HT and WT mice. In contrast, whereas weak immunostaining for TGF‐β RII was detected in 67% of HT carcinomas at 12 mo, only 22% of WT carcinomas showed weak staining for this protein. Individual lung carcinomas showing reduced TGF‐β RII expression and adjacent normal bronchioles were excised from HT lungs using laser capture microdissection, and CRT‐PCR amplification of the extracted RNA showed 12‐fold less TGF‐β RII mRNA in these carcinomas compared with bronchioles. Decreasing TGF‐β RII mRNA levels occurred with increasing tumorigenesis in lung hyperplasias, adenomas, and carcinomas, with carcinomas having fourfold and sevenfold lower levels of TGF‐β RII mRNA than adenomas and hyperplasias, respectively. These data show enhanced ethyl carbamate–induced lung tumorigenesis in AJBL6 HT mice compared with WT mice, suggesting that both TGF‐β1 alleles are necessary for tumor‐suppressor activity. Reduction of TGF‐β RII mRNA expression in progressive stages of lung tumorigenesis in HT mice suggests that loss of TGF‐β RII may play an important role in the promotion of lung carcinogenesis in mice with reduced TGF‐β1 gene dosage when challenged with carcinogen. Mol. Carcinog. 29:112–126, 2000. Published 2000 Wiley‐Liss, Inc.