Hasem Habelhah

Siah2 Regulates Stability of Prolyl-Hydroxylases, Controls HIF1α Abundance, and Modulates Physiological Responses to Hypoxia

Hypoxia-inducible factor-1alpha (HIF1alpha) is a central regulator of the cellular response to hypoxia. Prolyl-hydroxylation of HIF1alpha by PHD enzymes is prerequisite for HIF1alpha degradation. Here, we demonstrate that the abundance of PHD1 and PHD3 are regulated via their targeting for proteasome-dependent degradation by the E3 ubiquitin ligases Siah1a/2, under hypoxia conditions. Siah2 null fibroblasts exhibit prolonged PHD3 half-life, resulting in lower levels of HIF1alpha expression during hypoxia. Significantly, hypoxia-induced HIF1alpha expression was completely inhibited in Siah1a/2 null cells, yet could be rescued upon inhibition of PHD3 by RNAi. Siah2 targeting of PHD3 for degradation increases upon exposure to even mild hypoxic conditions, which coincides with increased Siah2 transcription. Siah2 null mice subjected to hypoxia displayed an impaired hyperpneic respiratory response and reduced levels of hemoglobin. Thus, the control of PHD1/3 by Siah1a/2 constitutes another level of complexity in the regulation of HIF1alpha during hypoxia.

ERK phosphorylation drives cytoplasmic accumulation of hnRNP-K and inhibition of mRNA translation

Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) is one of a family of 20 proteins that are involved in transcription and post-transcriptional messenger RNA metabolism. The mechanisms that underlie regulation of hnRNP-K activities remain largely unknown. Here we show that cytoplasmic accumulation of hnRNP-K is phosphorylation-dependent. Mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) efficiently phosphorylates hnRNP-K both in vitro and in vivo at serines 284 and 353. Serum stimulation or constitutive activation of ERK kinase (MEK1) results in phosphorylation and cytoplasmic accumulation of hnRNP-K. Mutation at ERK phosphoacceptor sites in hnRNP-K abolishes the ability to accumulate in the cytoplasm and renders the protein incapable of regulating translation of mRNAs that have a differentiation-control element (DICE) in the 3′ untranslated region (UTR). Similarly, treatment with a pharmacological inhibitor of the ERK pathway abolishes cytoplasmic accumulation of hnRNP-K and attenuates inhibition of mRNA translation. Our results establish the role of MAPK/ERK in phosphorylation-dependent cellular localization of hnRNP-K, which is required for its ability to silence mRNA translation.

Ubiquitination and translocation of TRAF2 is required for activation of JNK but not of p38 or NF‐κB

TRAF2 is a RING finger protein that regulates the cellular response to stress and cytokines by controlling JNK, p38 and NF‐κB signaling cascades. Here, we demonstrate that TRAF2 ubiquitination is required for TNFα‐induced activation of JNK but not of p38 or NF‐κB. Intact RING and zinc finger domains are required for TNFα‐induced TRAF2 ubiquitination, which is also dependent on Ubc13. TRAF2 ubiquitination coincides with its translocation to the insoluble cellular fraction, resulting in selective activation of JNK. Inhibition of Ubc13 expression by RNAi resulted in inhibition of TNFα‐induced TRAF2 translocation and impaired activation of JNK but not of IKK or p38. TRAF2 aggregates in the cytoplasm, as seen in Hodgkin–Reed–Sternberg lymphoma cells, resulting in constitutive NF‐κB activity but failure to activate JNK. These findings demonstrate that the TRAF2 RING is required for Ubc13‐dependent ubiquitination, resulting in translocation of TRAF2 to an insoluble fraction and activation of JNK, but not of p38 or NF‐κB. Altogether, our findings highlight a novel mechanism of TRAF2‐dependent activation of diverse signaling cascades that is impaired in Hodgkin–Reed–Sternberg cells.

Identification of New JNK Substrate Using ATP Pocket Mutant JNK and a Corresponding ATP Analogue

Modification of the ATP pocket on protein kinases allows selective use of an ATP analogue that exhibits high affinity for the altered kinases. Using this approach, we altered the ATP-binding site on JNK and identifiedN 6-(2-phenythyl)-ATP, a modified form of ATP that exhibits high specificity and affinity for the modified, but not the wild type form, of JNK. Using modified JNK and its ATP analogue enables the detection of novel JNK substrates. Among substrates identified using this approach is heterogeneous nuclear ribonucleoprotein K, which is involved in transcription and post-transcriptional mRNA metabolism. The newly identified substrate can be phosphorylated by JNK on amino acids 216 and 353, which contribute to heterogeneous nuclear ribonucleoprotein K mediated transcriptional activities.

Stress-induced decrease in TRAF2 stability is mediated by Siah2

TRAF2 serves as a central regulator of the cellular response to stress and cytokines through the regulation of key stress‐signaling cascades. Here we demonstrate that wild‐type, but not RING mutant, Siah2 targets TRAF2 for ubiquitylation and degradation in vitro. Siah2 mediates equally efficient ubiquitylation of RING mutant TRAF2. In vivo, Siah2 primarily targets TRAF2 for degradation under stress conditions. Tumor necrosis factor‐α (TNF‐α) and actinomycin D treatment results in accelerated TRAF2 degradation in wild‐type mouse embryo fibroblasts (MEFs), as compared with Siah2−/− cells. Similarly, TRAF2 half‐life is prolonged in Siah2−/− compared with wild‐type MEFs subjected to stress stimuli. Siah2 efficiently decreases TNF‐α‐dependent induction of JNK activity and transcriptional activation of NF‐κB. Apoptosis induced by TNF‐α and actinomycin D treatment is increased upon expression of Siah2, or attenuated upon expression of TRAF2 or RING mutant Siah2. Identifying Siah2 as a regulator of TRAF2 stability reveals its role in the regulation of TRAF2 signaling following exposure to stress.

Distinct pattern of p53 phosphorylation in human tumors

The protein product of the tumor suppressor gene p53 is phosphorylated on multiple residues by several protein kinases. Using a battery of 10 antibodies developed against different phosphorylated and acetylated residues of p53, we compared the pattern of p53 phosphorylation and acetylation in tumor-derived cell lines, tumor samples, and non-neoplastic cells. Irrespective of tumor types or the presence of p53 mutation, phosphorylation and acetylation of p53 was substantially higher in samples obtained from tumor tissues than those found in non-transformed samples. Among the 10 sites analysed, phosphorylation of residues 15, 81, 392, and acetylation were among the more frequent modifications. Analysis of two of the more abundant phosphorylation or acetylation sites on p53 is sufficient to detect 72% of tumor-derived p53 proteins. The distinct pattern of p53 phosphorylation and acetylation in human tumors may offer a new means to monitor the status and activity of p53 in the course of tumor development and progression.

Conversion of Human Colonic Adenoma Cells to Adenocarcinoma Cells Through Inflammation in Nude Mice

The roles of inflammation in the malignant progression of tumors during multistep carcinogenesis have been much discussed but remain to be elucidated. To determine the direct contribution of inflammation to colon carcinogenesis, we established a new model of progression of human colonic adenoma cells using a nude mouse; the progression is accelerated by coimplantation of a plastic plate. The FPCK-1–1 cell line, derived from a colonic polyp in a patient with familial adenomatous polyposis, is nontumorigenic when injected subcutaneously into nude mice in a cell suspension of up to 5 × 106 cells per mouse. However implantation of 1 × 105 FPCK-1–1 cells attached to a plastic plate induced first acute and then chronic inflammation, and formed progressively growing tumors that were histologically determined as moderately differentiated adenocarcinoma in 65% of mice. Moreover cell lines established from the growing tumors were found to be tumorigenic when injected into mice even without a plastic plate. The tumor arising from the adenoma cells implanted attached to a plastic plate was surrounded by highly proliferating fibrous stroma. This fibrous tissue was considered essential for malignant progression, rather than for attachment to the plastic plate substrate, because the tumors were formed after injection of FPCK-1–1 cells into the fibrous tissue from which the plastic plate had been removed before the cell injection. The conditioned medium (CM) obtained from the fibroblasts derived from a plastic plate-associated stromal tissue was found to contain factors that stimulated growth of FPCK-1–1 cells, but not of the derivative progressor cell lines. The factor was stable to heating and neuraminidase treatment, but labile to trypsin treatment. The main growth-potentiating activity was contained in the fraction larger than 100 kDa. In contrast, the activity to promote FPCK-1–1 cell growth was not present in the CM of subcutaneous fibroblasts from untreated nude mice or the fibroblast cell lines C3H10T 1/2 and NIH3T3. These results demonstrated that inflammation-associated stroma promoted the conversion of colonic adenoma cells to adenocarcinoma cells.

Increased E1AF expression in mouse fibrosarcoma promotes metastasis through induction of MT1-MMP expression.

In this study, we investigated the role of E1AF, a member of ets family transcription factor, in the acquisition of metastatic capacity by non-metastatic mouse fibrosarcoma cell clone, QR-32. The QR-32 cell clone grows progressively after co-implantation with gelatin sponge in syngeneic C57BL/6 mice. The cell lines (QRsP) established from arising tumors after the co-implantation exhibited enhanced tumorigenicity and pulmonary metastasis in vivo as compared with parent QR-32 cells. The enhanced pulmonary metastasis of QRsP cells was correlated well with augmented production of matrix metalloproteinase-2 (MMP-2) and increased expression of membrane-type 1-MMP (MT1-MMP). The QRsP cells also acquired higher chemokinetic activities to fibronectin and higher invasive activities through a reconstituted basement membrane. Furthermore we observed the elevated mRNA expression of E1AF in QRsP cells compared to parent QR-32 cells. Therefore, we transfected QR-32 cells with E1AF cDNA. Overexpression of E1AF in the QR-32 cells resulted in the induction of MT1-MMP expression and converting an exogenously added precursor MMP-2 into active form. E1AF transfectants exhibited more motile and invasive activities, and moderately increased pulmonary metastatic activities than parental QR-32 cells in vivo, although their metastatic activities were lower than those of QRsP cells. These findings suggest that the increased expression of E1AF in fibrosarcoma contributes to invasive phenotypes including MT1-MMP expression and enhanced cell migration, but not sufficient for exhibiting highly metastatic activity in vivo.

TRAF2 Phosphorylation Modulates Tumor Necrosis Factor Alpha-Induced Gene Expression and Cell Resistance to Apoptosis

ABSTRACT TRAF2 is an adaptor protein that regulates the activation of the c-Jun N-terminal kinase (JNK) and IκB kinase (IKK) signaling cascades in response to tumor necrosis factor alpha (TNF-α) stimulation. Although the downstream events in TNF-α signaling are better understood, the membrane-proximal events are still elusive. Here, we demonstrate that TNF-α and cellular stresses induce TRAF2 phosphorylation at serine 11 and that this phosphorylation is required for the expression of a subset of NF-κB target genes. Although TRAF2 phosphorylation had a minimal effect on the TNF-α-induced rapid and transient IKK activation, it was essential for secondary and prolonged IKK activation. Consistent with this, TRAF2 phosphorylation is not required for its recruitment to the TNFR1 complex in response to TNF-α stimulation but is required for its association with a cytoplasmic complex containing RIP1 and IKK. In addition, TRAF2 phosphorylation was essential for the full TNF-α-induced activation of JNK. Notably, TRAF2 phosphorylation increased both basal and inducible c-Jun and NF-κB activities and rendered cells resistant to stress-induced apoptosis. Moreover, TRAF2 was found to be constitutively phosphorylated in some lymphomas. These results unveil a new, finely tuned mechanism for TNF-α-induced IKK activation modulated by TRAF2 phosphorylation and suggest that TRAF2 phosphorylation contributes to elevated levels of basal NF-κB activity in certain human cancers.

IκB Kinase ε Phosphorylates TRAF2 To Promote Mammary Epithelial Cell Transformation

ABSTRACT NF-κB transcription factors are central regulators of inflammation and when dysregulated contribute to malignant transformation. IκB kinase ε (IKKε; IKKi, encoded by IKBKE) is a breast oncogene that is amplified in 30% of breast cancers and drives transformation in an NF-κB-dependent manner. Here we demonstrate that IKKε interacts with and phosphorylates tumor necrosis factor receptor-associated factor 2 (TRAF2) at Ser11 in vitro and in vivo. This activity promotes Lys63-linked TRAF2 ubiquitination and NF-κB activation and is essential for IKKε transformation. Breast cancer cells that depend on IKKε expression for survival are also dependent on TRAF2. This work defines TRAF2 phosphorylation to be one key effector of IKKε-induced mammary epithelial cell transformation.