Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-10581
Barth, S. Functional Characterization of the Interaction of PHD2 with FKBP38. 2008, University of Zurich, Faculty of Science.
Multicellular complex organisms utilize oxygen as the final electron acceptor in the cellular aerobic glucose catabolism and β-oxidation of fatty acids to efficiently
generate ATP. Hence, oxygen deprivation (hypoxia) requires adaptive mechanisms to respond to this stress stimulus. Response to hypoxia is governed by the transcription factor hypoxia-inducible factor (HIF) that regulates the expression of at least 70 known target genes that allow the organism to adapt on the cellular, local and systemic level. HIF is a heterodimeric protein complex that consists of an oxygen-dependently regulated HIF-α subunit and a constitutively expressed HIF-β subunit. The HIF-α subunit protein stability is tightly regulated by hydroxylation of
specific prolines in the oxygen-dependent degradation domain (ODDD) by prolyl-4-hydroxylases (PHDs) that require oxygen as a substrate for their enzymatic reaction.
Hydroxylated HIF-α is targeted by the von Hippel-Lindau tumor suppressor protein (pVHL), the recognition component of an E3 ubiquitin ligase complex, and subsequently degraded in the 26S proteasome. Besides HIF-α, only a few other PHD hydroxylation targets have been described so far. We investigated the possibility of further hydroxylation targets for the PHD2 isoform and if other regulatory
mechanisms apart from molecular oxygen might be important in the PHD2 protein regulation.
A yeast two-hybrid screening was carried out to identify novel PHD2 interacting partners. Among the identified interactors, we found the FK506-binding protein 38
(FKBP38). The specific interaction of FKBP38 with PHD2 but not PHD1 or PHD3 was confirmed in a variety of interaction assays, including yeast and mammalian twohybrid
analysis, GST pull-down and co-immunoprecipitation experiments and fluorescence resonance energy transfer (FRET) analysis. FKBP38 was shown to
bind with a linear minimal glutamate rich binding motif from amino acid (aa) 37 to 56 to PHD2. Conversely, PHD2 interacts with its N-terminal region from aa 1 to 114,
containing a MYND-type Zn2+ finger domain, with FKBP38.
Originally FKBP38 was identified as a target of the immunosuppressive drug FK506 and it has been proposed that FKBP38 regulates cell death or survival. FKBP38
belongs to the family of peptidyl prolyl cis/trans isomerases (PPIases) that participate in the conformational change of the aa proline from the cis to the trans position,
providing the basis for a potential regulatory switch. Thus, we speculated that FKBP38 might be a co-factor that is involved in the PHD-mediated enzymatic proline
hydroxylation of HIF-α subunits.
Stable RNA interference (RNAi)-mediated downregulation of FKBP38 increased PHD2 protein abundance and cellular hydroxylation activity. Conversely, elevated
PHD2 protein levels decreased hypoxic HIF-1α protein accumulation and attenuated HIF-dependent transcriptional regulation. Reconstitution of FKBP38 normalized
PHD2 protein levels and therefore cellular hydroxylation capacity and HIF-1 response. Strikingly, increased PHD2 protein abundance was due to prolonged PHD2 protein stability, suggesting that FKBP38 determines PHD2 protein levels.
However, we did not find any involvement of the FKBP38 enzymatic PPIase activity in the regulation of PHD2 protein abundance. So far, little is known about PHD2
protein regulation and we therefore further explored the molecular mechanism of FKBP38-dependent PHD2 protein regulation. PHD2 protein stability was neither
influenced by a variety of protease inhibitors nor by inhibition of the ubiquitindependent proteasomal degradation pathway. Interestingly, in cellular assays the Cterminal transmembrane domain of FKBP38 is required for a specific interaction with PHD2 in spite of the existence of a functional FKBP38 interaction domain at the Nterminus.
Consistent with these data, we could co-isolate FKBP38 and PHD2 from endoplasmatic reticulum and mitochondria membranes. Strikingly, regulation of PHD2 protein abundance requires the correct sub-cellular localization of FKBP38. As
an outlook, targeting FKBP38:PHD2 protein interaction by chemical compounds might be an attractive strategy to specifically increase PHD2 amount and therefore
attenuate HIF protein levels in tumors.
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|Referees:||Wenger R H, Camenisch G, Beck-Schimmer B, Marti H H|
|Communities & Collections:||04 Faculty of Medicine > Center for Integrative Human Physiology
07 Faculty of Science > Institute of Molecular Life Sciences
04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
|DDC:||570 Life sciences; biology
610 Medicine & health
|Deposited On:||19 Jan 2009 08:23|
|Last Modified:||16 Oct 2012 21:48|
|Number of Pages:||96|
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