The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-␣ subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-␣ for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione Stransferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.

Prolyl-4-hydroxylase domain (PHD) proteins are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor ␣ subunits. Whereas PHD1 and PHD3 proteolysis has been shown to be regulated by Siah2 ubiquitin E3 ligase-mediated polyubiquitylation and proteasomal destruction, protein regulation of the main oxygen sensor responsible for hypoxia-inducible factor ␣ regulation, PHD2, remained unknown. We recently reported that the FK506-binding protein (FKBP) 38 specifically interacts with PHD2 and determines PHD2 protein stability in a peptidylprolyl cis-trans isomerase-independent manner. Using peptide array binding assays, fluorescence spectroscopy, and fluorescence resonance energy transfer analysis, we defined a minimal linear glutamate-rich PHD2 binding domain in the N-terminal part of FKBP38 and showed that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a nonlinear N-terminal motif containing the MYND (myeloid, Nervy, and DEAF-1)-type Zn 2؉ finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum and mitochondria. An additional fraction of PHD2 was found in the cytoplasm. In cellulo PHD2/FKBP38 association, as well as regulation of PHD2 protein abundance by FKBP38, is dependent on membraneanchored FKBP38 localization mediated by the C-terminal transmembrane domain. Mechanistically our data indicate that PHD2 protein stability is regulated by a ubiquitin-independent proteasomal pathway involving FKBP38 as adaptor protein that mediates proteasomal interaction. We hypothesize that FKBP38-bound PHD2 is constantly degraded whereas cytosolic PHD2 is stable and able to function as an active prolyl-4-hydroxylase.The heterodimeric ␣/␤ transcription factor complexes of hypoxia-inducible factors (HIFs) 4 are central regulators of the cellular, local, and systemic response to reduced oxygen partial pressure (pO 2 ) (1, 2). Under normoxic conditions, two highly conserved prolyl residues within the oxygen-dependent degradation domain of HIF␣ subunits are hydroxylated by members of the prolyl-4-hydroxylase domain (PHD) family (also called egg laying-defective nine homolog (EGLN) or HIF prolyl hydroxylase) (3-5). Hydroxylated prolines are then bound by an E3 ubiquitin ligase complex containing the von Hippel-Lindau tumor suppressor protein (pVHL) as recognition subunit, mediating polyubiquitylation and proteasomal degradation of HIF␣ subunits (6 -8). In addition, factor inhibiting HIF hydroxylates under normoxic conditions an asparaginyl residue in the C-terminal transcriptional transactivation domain of HIF␣ subunits, preventing the association with the CH1 domain of the p300 and cAMP-responsive element-binding protein-binding protein (CBP) co-activators (9, 10). PHDs and factor inhibiting HIF belong to the 2-oxoglutarate-and irondependent dioxygenase superfamily and act as cellular oxygen sensors by correlating the ...

The hypoxia–inducible transcription factor (HIF) is a key component of the cellular adaptation mechanisms to hypoxic conditions. HIFα subunits are degraded by prolyl-4-hydroxylase domain (PHD) enzyme-dependent prolyl-4-hydroxylation of LxxLAP motifs that confer oxygen-dependent proteolytic degradation. Interestingly, only three non-HIFα proteins contain two conserved LxxLAP motifs, including the putative RNA helicase with a zinc finger domain HELZ. However, HELZ proteolytic regulation was found to be oxygen-independent, supporting the notion that a LxxLAP sequence motif alone is not sufficient for oxygen-dependent protein destruction. Since biochemical pathways involving RNA often require RNA helicases to modulate RNA structure and activity, we used luciferase reporter gene constructs and metabolic labeling to demonstrate that HELZ overexpression activates global protein translation whereas RNA-interference mediated HELZ suppression had the opposite effect. Although HELZ interacted with the poly(A)-binding protein (PABP) via its PAM2 motif, PABP was dispensable for HELZ function in protein translation. Importantly, downregulation of HELZ reduced translational initiation, resulting in the disassembly of polysomes, in a reduction of cell proliferation and hypophosphorylation of ribosomal protein S6.

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