Hypoxia-activated Smad3-specific dephosphorylation by PP2A.
The transforming growth factor-beta (TGF-beta) maintains epithelial homeostasis and suppresses early tumor formation, but paradoxically at later stages of tumor progression, TGF-beta promotes malignancy. TGF-beta activates phosphorylation of Smad2 and -3 effectors. Smad2 and -3 are known to have different functions, but differential regulation of their phosphorylation has not been described. Here we show that upon hypoxia, the TGF-beta-induced phosphorylation of Smad3 was inhibited, although Smad2 remained phosphorylated. The inhibition of Smad3 phosphorylation was not due to TGF-beta receptor inactivation. We show that Smad3 was dephosphorylated by PP2A (protein phosphatase 2A) specifically under hypoxic conditions. The hypoxic Smad3 dephosphorylation required intact expression of the essential scaffold component PR65 of PP2A. PP2A physically interacted with Smad3 that occurred only in hypoxia. Accordingly, Smad3-associated PP2A activity was found under hypoxic conditions. Hypoxia attenuated the nuclear accumulation of TGF-beta-induced Smad3 but did not affect Smad2. Moreover, the influence of TGF-beta on a set of Smad3-activated genes was attenuated by hypoxia, and this was reversed by chemical PP2A inhibition. Our data demonstrate the existence of a Smad3-specific phosphatase and identify a novel role for PP2A. Moreover, our data implicate a novel mechanism by which hypoxia regulates growth factor responses.
Heikkinen
Pekka T
PT
From the Turku Centre for Biotechnology, Turku University and Åbo Akademi University, FI-20520 Turku, Finland; Turku University School of Biological Sciences, Turku University, FI-20520 Turku, Finland.
Nummela
Marika
M
From the Turku Centre for Biotechnology, Turku University and Åbo Akademi University, FI-20520 Turku, Finland.
Leivonen
Suvi-Katri
SK
Medicity Research Laboratories, Turku University, FI-20520 Turku, Finland.
Westermarck
Jukka
J
From the Turku Centre for Biotechnology, Turku University and Åbo Akademi University, FI-20520 Turku, Finland; the Institute of Medical Technology and University Hospital, Tampere University, FI-33014 Tampere, Finland.
Hill
Caroline S
CS
the Cancer Research UK London Research Institute, WC2A 3PX London, United Kingdom, and.
Kähäri
Veli-Matti
VM
Medicity Research Laboratories, Turku University, FI-20520 Turku, Finland; the Department of Dermatology, University of Turku and Turku University Hospital, FI-20520 Turku, Finland.
Jaakkola
Panu M
PM
From the Turku Centre for Biotechnology, Turku University and Åbo Akademi University, FI-20520 Turku, Finland; the Department of Oncology and Radiotherapy, Turku University Hospital, FI-20520 Turku, Finland. Electronic address: panjaa@utu.fi.
D000951:Antigens, Neoplasm / Q000235:genetics; D001665:Binding Sites; D015153:Blotting, Western; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics; D015687:Cell Hypoxia; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051899:Smad2 Protein / Q000378:metabolism*; D051900:Smad3 Protein / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
D000951:Antigens, Neoplasm; D018125:Receptors, Transforming Growth Factor beta; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D042461:Vascular Endothelial Growth Factor A; D054648:Protein Phosphatase 2; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases