Hypoxia-inducible Factor Prolyl-4-hydroxylase PHD2 Protein Abundance Depends on Integral Membrane Anchoring of FKBP38

Abstract

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 ...

Hypoxia promotes glycogen synthesis and accumulation in human ovarian clear cell carcinoma

“…The rapid recovery by 48 h under hypoxic conditions and the unchanged level with DFO treatment in this study, suggested that FKBP38 is not a HIF target, as previously reported (37). These results indicated that the early and immediate compensatory bio-responses of HAC2 cells against sudden and severe hypoxic changes were almost the same as those described in previous reports (33,36). A decreased level of HSP90 and slight but detectable increased level of HSP70 were additionally found (Fig.…”

“…Our present study also demonstrated that a VHL mutation did not appear in OCCC, different from renal clear cell carcinoma. In contrast, in the initial 24 h after hypoxic culture, the occurrence was noted of continuous 48-h overexpression and stabilization of PHD2, which is a HIF1 downstream gene and the main HIF1α regulator via hydroxylation of ODDD (33,34), and also of transient downregulation of FKBP38, a key regulator in the stabilization and function of PHD2 by binding, followed by proteasomal degradation (35)(36)(37) (Fig. 2C).…”

Three-dimensional culture promotes reconstitution of the tumor-specific hypoxic microenvironment under TGFβ stimulation

“…These findings suggest that, assuming VHL exhibits complete functionality, HIF1 is able to induce PHD2-expression in a VHL-dependent manner in 3D culture under hypoxic conditions. As PHD2 is the main member of the PHD enzyme family that is involved in oxygen-sensing prolyl hydroxylation of the oxygen-dependent degradation domain in the HIF1 molecule (54,55), elevation and activation of PHD2 may have the compensatory effect of maintaining the HIF1 molecule as a mechanism leading to the hypoxic condition. In spite of equal levels of expression of mRNA between 2D and RFB cultured cells, there was a difference in the levels of PHD2 protein.…”

Role of the ANKMY2-FKBP38 Axis in Regulation of the Sonic Hedgehog (Shh) Signaling Pathway

“…In addition, FKBP38 appears to regulate protein degradation by anchoring the 26 S proteasome to organellar membranes (17). It thereby affects the stability of both Bcl-2 and the prolyl 4-hydroxylase domain-containing enzyme PHD2, the latter of which regulates the hypoxia-inducible transcription factor HIF1␣ (18). Although FKBP38 has been found to be expressed in all mammalian tissues examined, it is especially abundant in the central nervous system, both in neurons and in glial cells (19).…”