Two transactivation domains of hypoxia-inducible factor-1alpha regulated by the MEK-1/p42/p44 MAPK pathway

At a low-oxygen tension, cells increase the expression of several genes (such as erythropoietin, the vascular endothelial growth factor, and glycolytic enzymes) in order to adapt to hypoxic stress. A common transactivator, named the hypoxia-inducible factor 1 (HIF-1) activates these genes. HIF-1 is a heterodimeric transactivator that is composed of alpha and beta subunits. HIF-1 activity is primarily determined by the hypoxia-induced stabilization of the alpha subunit, whereas the HIF-1beta subunit is expressed constitutively. Our previous observation implied that the MEK-1/p42/p44 MAPK pathway is involved in the hypoxia-induced transactivation ability, but not in the stabilization and DNA binding of HIF-1alpha. In this paper, we dissected the transactivation domain of HIF-1alpha in more detail, and tested the correlation between specific domains of HIF-1alpha and specific signaling pathways. We designed several fusion proteins that contain deletion mutants of HIF-1alpha that is linked to the DNA binding domain of the yeast protein Gal4. By using the Gal4-driven reporter system, we tested the transactivation activities of the Gal4/HIF-1alpha fusion proteins in Hep3B cells. Our findings suggest that tyrosine kinases, the MEK-1/p42/p44 MAPK pathway, but not the PI-3 kinase/Akt pathway, are involved in the hypoxia-induced transactivation of HIF-1alpha. We have shown that the functional transactivation activities are located at both 522-649 and 650-822 amino acids of HIF-1alpha. Treatment of PD98059, a MEK-1 inhibitor, blocked the hypoxia-induced transactivation abilities of both the 522-649 and 650-822 amino acids of the C-terminal half of HIF-1alpha. This implies that the MEK-1/p42/p44 MAPK signaling pathway cannot distinguish between the two hypoxia-induced transactivation domains.     

Cytoplasmic localization of Wis1 MAPKK by nuclear export signal is important for nuclear targeting of Spc1/Sty1 MAPK in fission yeast

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus. Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells. Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain. Unexpectedly, an inactivating mutation to the NES of chromosomal wis1(+) does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1. However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress. Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress. These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus.     

Serine 396 of PDK1 is required for maximal PKB activation

Protein kinase B (PKB; also known as Akt) is important for mediating survival and proliferation signals. Following activation, PKB shuttles to various compartments of the cell, including the nucleus, where it phosphorylates an array of targets. PKB is phosphorylated at T308 by its activator PDK1. PDK1 is normally excluded from the nucleus via a nuclear exclusion sequence (NES), and our previous work suggested that nuclear exclusion can be attenuated by IGF-1-induced phosphorylation of S396 proximal to the NES. No studies have been done to test the significance of S396 phosphorylation or the impact of nuclear accumulation of PDK1 on PKB activation. To address these questions, we created isogenic embryonic stem cell (ESC) lines expressing various alleles of PDK1 within a PDK1-/- background. Disruption of the NES domain of PDK1 correlated with elevated PKB phosphorylation at both T308 and S473. In contrast, mutation of S396 to alanine reduced PDK1 nuclear localization and reduced PKB phosphorylation and activation. The loss of phosphorylation of PKB by S396A mutation was rescued by forcing nuclear PDK1 or by conversion of S396 to an aspartic acid. The phosphorylation of the PKB substrate FOXO3alpha was reduced in S396A PDK1 ESC. Other known and suspected PKB substrates, including GSK3 and Raf1, were unaffected. This study therefore reveals that S396 plays a role in the activation of PKB leading to the regulated phosphorylation of some PKB substrates including FOXO3alpha.     

Interaction of the PAS B domain with HSP90 accelerates hypoxia-inducible factor-1alpha stabilization.

Hypoxia-inducible factor (HIF) alpha subunits are induced under hypoxic conditions, when limited oxygen supply prevents prolyl hydroxylation-dependent binding of the ubiquitin ligase pVHL and subsequent proteasomal degradation. A short normoxic half-life of HIF-alpha and a very rapid hypoxic protein stabilization are crucial to the cellular adaptation to changing oxygen supply. However, the molecular requirements for the unusually rapid mechanisms of protein synthesis, folding and nuclear translocation are not well understood. We and others previously found that the chaperone heat-shock protein 90 (HSP90) can interact with HIF-1alpha in vitro. Here we show that HSP90 also interacts with HIF-2alpha and HIF-3alpha, suggesting a general involvement of HSP90 in HIF-alpha stabilization. The PAS B domain, common to all three alpha subunits, was required for HSP90 interaction. ARNT competed with HSP90 for binding to the PAS B domain since an excess of either component inhibited the activity of the other. HSP90 as well as the heterocomplex members HSP70 and p23, but not HSP40, were detected in immunoprecipitations of endogenous cellular HIF-1alpha. While HSP90 and HSP70 bound to HIF-1alpha predominantly under normoxic conditions, ARNT bound to HIF-1alpha primarily under hypoxic conditions, suggesting that ARNT displaced HSP90 from HIF-1alpha following nuclear translocation. Hypoxic accumulation of HIF-1alpha was delayed in a novel cell model deficient for HSP90beta as well as after treatment of wild-type cells with the HSP90 inhibitor geldanamycin, suggesting that HSP90 activity is involved in the rapid HIF-1alpha protein induction.