Regulation of the ring finger E3 ligase Siah2 by p38 MAPK.
The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.
Khurana
Ashwani
A
Signal Transduction Program, Burnham Institute for Medical Research, La Jolla, California 92037, USA.
Nakayama
Koh
K
Williams
Scott
S
Davis
Roger J
RJ
Mustelin
Tomas
T
Ronai
Ze'ev
Z
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004249:DNA Damage / Q000502:physiology; D049308:Dioxygenases / Q000378:metabolism* / Q000528:radiation effects; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D017382:Reactive Oxygen Species; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D000098563:Seven in Absentia Proteins
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D017382:Reactive Oxygen Species; D049308:Dioxygenases; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins; D048051:p38 Mitogen-Activated Protein Kinases; D010100:Oxygen