TY - JOUR
T1 - Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2 signaling in dictyostelium
AU - Van Der Wel, Hanke
AU - Johnson, Jennifer M.
AU - Xu, Yuechi
AU - Karunaratne, Chamini V.
AU - Wilson, Kyle D.
AU - Vohra, Yusuf
AU - Boons, Geert Jan
AU - Taylor, Carol M.
AU - Bendiak, Brad
AU - West, Christopher M.
PY - 2011/3/15
Y1 - 2011/3/15
N2 - The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O2 regulation of development, suggesting the existence of an ancient O2-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O2 signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. 1H NMR demonstrated formation of 4(trans)- hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFα by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3 SCFubiquitin ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily related E3 VBCubiquitin ligases.
AB - The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O2 regulation of development, suggesting the existence of an ancient O2-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O2 signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. 1H NMR demonstrated formation of 4(trans)- hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFα by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3 SCFubiquitin ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily related E3 VBCubiquitin ligases.
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U2 - 10.1021/bi101977w
DO - 10.1021/bi101977w
M3 - Article
C2 - 21247092
AN - SCOPUS:79952381729
SN - 0006-2960
VL - 50
SP - 1700
EP - 1713
JO - Biochemistry
JF - Biochemistry
IS - 10
ER -