Oxygen sensing by Prolyl-4-Hydroxylase PHD2 within the nuclear compartment and the influence of compartimentalisation on HIF-1 signalling

Abstract

SummaryHypoxia-inducible factors (HIFs) regulate more than 200 genes involved in cellular adaptation to reduced oxygen availability. HIFs are heterodimeric transcription factors that consist of one of three HIF-a subunits and a HIF-b subunit. Under normoxic conditions the HIFa subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1, PHD2 and PHD3, resulting in recognition by von-Hippel-Lindau protein, ubiquitylation and proteasomal degradation. It has been suggested that PHD2 is the key regulator of HIF-1a stability in vivo. Previous studies on the intracellular distribution of PHD2 have provided evidence for a predominant cytoplasmic localisation but also nuclear activity of PHD2. Here, we investigated functional nuclear transport signals in PHD2 and identified amino acids 196-205 as having a crucial role in nuclear import, whereas amino acids 6-20 are important for nuclear export. Fluorescence resonance energy transfer (FRET) showed that an interaction between PHD2 and HIF-1a occurs in both the nuclear and cytoplasmic compartments. However, a PHD2 mutant that is restricted to the cytoplasm does not interact with HIF-1a and shows less prolyl hydroxylase activity for its target HIF-1a than wild-type PHD2 located in the nucleus. Here, we present a new model by which PHD2-mediated hydroxylation of HIF-1a predominantly occurs in the cell nucleus and is dependent on very dynamic subcellular trafficking of PHD2.

Nuclear-cytoplasmatic shuttling of proteins in control of cellular oxygen sensing

“…Thus, nuclear export of PHD2 depends on the major exportin CRM1. Note that recent studies have not confirmed the NES between Leu-188 and Iso-198 that was predicted from in silico experiments [85]. Instead, immunofluorescence analyses have revealed that the nuclear export of PHD2 depends on an intact motif between aa Gly-6 and Tyr-20 without a classical leucine-rich NES consensus motif.…”

“…Nuclear import of PHD2 does not occur via classical importin α/β receptors. Although the primary sequence of PHD2 exhibits a motif with weak resemblance to a classical NLS between Arg-99 and Lys-115, PHD2 does not bind to importin α isoforms [85]. Moreover, coregulation of nuclear import of PHD2 and HIF-1α by importin α/β receptors was excluded.…”

“…It remains to be investigated whether PHD2 is imported by one of the importin β-like nuclear transport receptors described earlier [1]. Studies utilizing PHD2 deletion constructs identified aa 196-205 as a component of a putative non-classical NLS [85]. Importantly, the deleted aa 196-205 are not building a direct part of the active site of the enzyme, since the catalytic activity of PHD2 depends on His-313, Asp-315 and His-374 [86].…”

Progress on hypoxia-inducible factor-3: Its structure, gene regulation and biological function (Review)

“…PHD is a cellular sensor for low-oxygen. Under normal oxygen partial pressure and in the presence of Fe 2+ and acetone dicarboxylic acid, PHD catalyzes the hydroxylation of key amino acid residues in the HIF-α ODD domain (42,43). This is followed by VHL binding to HIF-α and inducing degradation via the ubiquitin-proteasome pathway (44,45).…”

Prolyl-4-hydroxylase Domain Protein 2 Controls NF-κB/p65 Transactivation and Enhances the Catabolic Effects of Inflammatory Cytokines on Cells of the Nucleus Pulposus

“…Commonly, the disc undergoes degeneration, a condition frequently linked to neck and back pain and requiring pharmacological or surgical intervention. Degenerative disc disease is characterized by elevated levels of proinflammatory cytokines including TNF-␣, IL-1␤, IL-6, and IL-8, as well as down-regulation of expression of the important matrix molecules aggrecan and collagen II (1)(2)(3)(4). TNF-␣ and IL-1␤ stimulate production of NGF, BDNF, and VEGF, molecules associated with nerve ingrowth and pain as well as angiogenesis (5).…”