Phosphorylation of carbonic anhydrase IX controls its ability to mediate extracellular acidification in hypoxic tumors.
In the hypoxic regions of a tumor, carbonic anhydrase IX (CA IX) is an important transmembrane component of the pH regulatory machinery that participates in bicarbonate transport. Because tumor pH has implications for growth, invasion, and therapy, determining the basis for the contributions of CA IX to the hypoxic tumor microenvironment could lead to new fundamental and practical insights. Here, we report that Thr443 phosphorylation at the intracellular domain of CA IX by protein kinase A (PKA) is critical for its activation in hypoxic cells, with the fullest activity of CA IX also requiring dephosphorylation of Ser448. PKA is activated by cAMP, which is elevated by hypoxia, and we found that attenuating PKA in cells disrupted CA IX-mediated extracellular acidification. Moreover, following hypoxia induction, CA IX colocalized with the sodium-bicarbonate cotransporter and other PKA substrates in the leading edge membranes of migrating tumor cells, in support of the concept that bicarbonate metabolism is spatially regulated at cell surface sites with high local ion transport and pH control. Using chimeric CA IX proteins containing heterologous catalytic domains derived from related CA enzymes, we showed that CA IX activity was modulated chiefly by the intracellular domain where Thr443 is located. Our findings indicate that CA IX is a pivotal mediator of the hypoxia-cAMP-PKA axis, which regulates pH in the hypoxic tumor microenvironment.
Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic.
Peter
P
Ditte
Franck
F
Dequiedt
Eliska
E
Svastova
Alzbeta
A
Hulikova
Anna
A
Ohradanova-Repic
Miriam
M
Zatovicova
Lucia
L
Csaderova
Juraj
J
Kopacek
Claudiu T
CT
Supuran
Silvia
S
Pastorekova
Jaromir
J
Pastorek
D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D015687:Cell Hypoxia; D002460:Cell Line; D002465:Cell Movement; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000737:chemistry*; D057809:HEK293 Cells; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D009154:Mutation; D009369:Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D010766:Phosphorylation; D011485:Protein Binding; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
D000951:Antigens, Neoplasm; D001639:Bicarbonates; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; D013912:Threonine; D012694:Serine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases