Prolyl-4-hydroxylase domain proteins (PHDs) are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor (HIF) α 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 HIFα 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 peptidyl prolyl cis/trans isomerase (PPIase)- independent manner. Using peptide array binding assays, fluorescence spectroscopy and fluorescence resonance energy transfer (FRET) analysis, we define a minimal linear glutamaterich PHD2 binding domain in the N-terminal part of FKBP38 and show that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a non-linear Nterminal motif containing the MYND-type Zn2+ finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum (ER) 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 membrane-anchored FKBP38 localization mediated by the C-terminal transmembrane domain. Mechanistically, our data indicate that PHD2 protein stability is regulated by an 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 active prolyl-4- hydroxylase.Prolyl-4-hydroxylase domain proteins (PHDs) are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor (HIF) α 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 HIFα 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 peptidyl prolyl cis/trans isomerase (PPIase)- independent manner. Using peptide array binding assays, fluorescence spectroscopy and fluorescence resonance energy transfer (FRET) analysis, we define a minimal linear glutamaterich PHD2 binding domain in the N-terminal part of FKBP38 and show that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a non-linear Nterminal motif containing the MYND-type Zn2+ finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum (ER) 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 membrane-anchored FKBP38 localization mediated by the C-terminal transmembrane domain. Mechanistically, our data indicate that PHD2 protein stability is regulated by an 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 active prolyl-4- hydroxylase.
Abstract
Prolyl-4-hydroxylase domain proteins (PHDs) are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor (HIF) α 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 HIFα 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 peptidyl prolyl cis/trans isomerase (PPIase)- independent manner. Using peptide array binding assays, fluorescence spectroscopy and fluorescence resonance energy transfer (FRET) analysis, we define a minimal linear glutamaterich PHD2 binding domain in the N-terminal part of FKBP38 and show that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a non-linear Nterminal motif containing the MYND-type Zn2+ finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum (ER) 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 membrane-anchored FKBP38 localization mediated by the C-terminal transmembrane domain. Mechanistically, our data indicate that PHD2 protein stability is regulated by an 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 active prolyl-4- hydroxylase.Prolyl-4-hydroxylase domain proteins (PHDs) are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor (HIF) α 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 HIFα 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 peptidyl prolyl cis/trans isomerase (PPIase)- independent manner. Using peptide array binding assays, fluorescence spectroscopy and fluorescence resonance energy transfer (FRET) analysis, we define a minimal linear glutamaterich PHD2 binding domain in the N-terminal part of FKBP38 and show that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a non-linear Nterminal motif containing the MYND-type Zn2+ finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum (ER) 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 membrane-anchored FKBP38 localization mediated by the C-terminal transmembrane domain. Mechanistically, our data indicate that PHD2 protein stability is regulated by an 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 active prolyl-4- hydroxylase.
American Society for Biochemistry and Molecular Biology
ISSN:
0021-9258
Funders:
Forschungskredit der Universität Zürich, Edoardo R., Giovanni, Giuseppe und Chiarina Sassella-Stiftung, Zürich, Hartmann Müller-Stiftung für Medizinische Forschung, Zürich, Krebsliga des Kantons Zürich, Olga Mayenfisch Stiftung, Zürich, 6th Framework Programme of the European Commission/SBF, Swiss National Foundation, Deutsche Forschungsgemeinschaft
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Barth, S