Prolyl Hydroxylase PHD3 Activates Oxygen-dependent Protein Aggregation

Abstract

The HIF prolyl hydroxylases (PHDs/EGLNs) are central regulators of the molecular responses to oxygen availability. One isoform, PHD3, is expressed in response to hypoxia and causes apoptosis in oxygenated conditions in neural cells. Here we show that PHD3 forms subcellular aggregates in an oxygen-dependent manner. The aggregation of PHD3 was seen under normoxia and was strongly reduced under hypoxia or by the inactivation of the PHD3 hydroxylase activity. The PHD3 aggregates were dependent on microtubular integrity and contained components of the 26S proteasome, chaperones, and ubiquitin, thus demonstrating features that are characteristic for aggresome-like structures. Forced expression of the active PHD3 induced the aggregation of proteasomal components and activated apoptosis under normoxia in HeLa cells. The apoptosis was seen in cells prone to PHD3 aggregation and the PHD3 aggregation preceded apoptosis. The data demonstrates the cellular oxygen sensor PHD3 as a regulator of protein aggregation in response to varying oxygen availability. INTRODUCTIONHypoxia forms a key component of multiple diseases, including stroke, inflammatory diseases and the progression of solid tumors. Hypoxia and in some cases the following reoxygenation pose considerable stress to cells. These include increased production of reactive oxygen species (ROS) and changes in protein translation, as well as exposure of cells to protein damage and misfolding (Rifkind et al., 1991;Koumenis and Wouters, 2006;Liu et al., 2006;Thuerauf et al., 2006). Mammalian cells have evolved a molecular machinery to determine whether cells attempt to survive or go into apoptosis in these conditions. The best characterized molecular responses to hypoxia are mediated through a family of dioxygenases that use O 2 as a cosubstrate (Bruick and McKnight, 2001;Epstein et al., 2001) and are termed prolyl hydroxylase domain proteins (PHD), also known as HIF prolyl hydroxylases or Egl-9 homologues (EGLN). Three known PHD (PHD1-3) isoforms hydroxylate the ␣-subunits of hypoxia-inducible factors 1 and 2 (HIF-1 and -2) under normoxic conditions at two proline residues (Pro402 and 564; Ivan et al., 2001;Jaakkola et al., 2001;Yu et al., 2001;Masson et al., 2004). The hydroxylated HIF-1␣ is recognized by the von Hippel-Lindau tumor suppressor protein (pVHL) that subsequently leads to ubiquitination and proteosomal destruction of HIF-1␣ (Kallio et al., 1999;Maxwell et al., 1999;Cockman et al., 2000;Ohh et al., 2000;Tanimoto et al., 2000). Under restricted O 2 availability the PHD activity decreases and the degradation of HIF-1␣ is blocked, activating the transcription of a wide range of genes (Semenza, 2001;Harris, 2002;Pugh and Ratcliffe, 2003;Schofield and Ratcliffe, 2004).All PHD isoforms have been reported to hydroxylate HIF and to have similar requirements for O 2 and cosubstrates FeII and 2-oxoglutarate at least in vitro (Hirsila et al., 2003). However, their function and characteristics also differ in several aspects. Two isoforms, PHD2 and PHD3, are upregulated tra...

p62/SQSTM1 regulates cellular oxygen sensing by attenuating PHD3 activity through aggregate sequestration and enhanced degradation

“…We have previously described that exogenous PHD3 forms subcellular bodies in normoxic cells (Rantanen et al, 2008). To investigate the possible significance of PHD3 bodies in cancer the subcellular expression pattern of PHD3 was studied in clinical cancer samples using breast cancer and squamous cell carcinomas of head and neck region (HNSCC; Fig.…”

“…We have previously shown that under normoxia PHD3 aggregates in cytoplasmic/perinuclear speckles that contain chaperones and proteasomal components (Rantanen et al, 2008) and PHD3 can homomultimerize in complexes that might enhance its degradation by Siah2 E3 ubiquitin ligase (Nakayama et al, 2004;Nakayama et al, 2007). Noticeably, in some carcinoma cells the aggregation associates with enhanced apoptosis (Rantanen et al, 2008).…”

“…Knockout studies show that PHD3 increases apoptosis of sympathoadrenal ganglia, an event required for maintaining their normal development and function (Bishop et al, 2008;Lee et al, 2005;Schlisio et al, 2008). We have previously shown that under normoxia PHD3 aggregates in cytoplasmic/perinuclear speckles that contain chaperones and proteasomal components (Rantanen et al, 2008) and PHD3 can homomultimerize in complexes that might enhance its degradation by Siah2 E3 ubiquitin ligase (Nakayama et al, 2004;Nakayama et al, 2007). Noticeably, in some carcinoma cells the aggregation associates with enhanced apoptosis (Rantanen et al, 2008).…”

HIF Prolyl-4-hydroxylase Interacting Proteins: Consequences for Drug Targeting

“…Of note, functionally active PHD3 is particularly difficult to purify, even when expressed in eukaryotic cells (unpublished observations). This property of PHD3 might be related to its tendency to form protein aggregates [69,75]. In bacteria, coexpression of the bacterial chaperonins GroEL/ES substantially increased the yield of soluble PHD3 [18].…”

“…NGF withdrawal causes PHD3 accumulation [104], and PHD3 enzymatic activity is required for the induction of KIF1B . Of note, it has been shown that EGFP-tagged exogenous PHD3 clusters around microtubule-like structures upon inhibition of the proteasome, suggesting that PHD3 might be transported by a microtubular motor protein [75]. Whether KIF1B is hydroxylated by PHD3 and how this induces the KIF1B protein remains to be determined.…”