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.

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.

TRAF2 is a RING finger protein that regulates the cellular response to stress and cytokines by controlling JNK, p38 and NF-jB signaling cascades. Here, we demonstrate that TRAF2 ubiquitination is required for TNFa-induced activation of JNK but not of p38 or NF-jB. Intact RING and zinc finger domains are required for TNFa-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 TNFa-induced TRAF2 translocation and impaired activation of JNK but not of IKK or p38. TRAF2 aggregates in the cytoplasm, as seen in HodgkinReed-Sternberg lymphoma cells, resulting in constitutive NF-jB 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-jB. Altogether, our findings highlight a novel mechanism of TRAF2-dependent activation of diverse signaling cascades that is impaired in HodgkinReed-Sternberg cells.

There was an error in the Experimental Procedures section under the subheading "Crystallization." The complex cocrystals were obtained with a final DNA concentration of 0.85 mM (not nM).

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 ef®cient ubiquitylation of RING mutant TRAF2. In vivo, Siah2 primarily targets TRAF2 for degradation under stress conditions. Tumor necrosis factor-a (TNF-a) and actinomycin D treatment results in accelerated TRAF2 degradation in wild-type mouse embryo ®broblasts (MEFs), as compared with Siah2 ±/± cells. Similarly, TRAF2 halflife is prolonged in Siah2 ±/± compared with wild-type MEFs subjected to stress stimuli. Siah2 ef®ciently decreases TNF-a-dependent induction of JNK activity and transcriptional activation of NF-kB. Apoptosis induced by TNF-a 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.