Phosphorylation of Carbonic Anhydrase IX Controls Its Ability to Mediate Extracellular Acidification in Hypoxic Tumors

Abstract

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. Cancer Res; 71(24); 7558-67. Ó2011 AACR.

Interplay between a cytosolic and a cell surface carbonic anhydrase in pH homeostasis and acid tolerance of Leishmania

“…This was further validated by our experiment in which supplementation with bicarbonate rescued the heterozygous mutant parasites from growth defects and intracellular acidosis in low pH medium. Physical and functional interaction of the Na + /HCO 3 − cotransporter or Cl − /HCO 3 − anion exchanger with transmembrane carbonic anhydrase 9 has been shown to control cytosolic pH in cancer cells and cardiomyocytes (Ditte et al, 2011;Svastova et al, 2012;Swietach et al, 2007;Orlowski et al, 2012). It will be fascinating to verify if such a carbonic anhydrasebicarbonate transport metabolon is operational in Leishmania parasites.…”

Carbonic Anhydrase IX Interacts with Bicarbonate Transporters in Lamellipodia and Increases Cell Migration via Its Catalytic Domain

“…3). Indeed, data available so far indicate that the C-terminal intracellular tail of CA IX can transmit signals to the extracellular enzyme domain and that its PKAmediated phosphorylation at Thr-443 can activate the catalytic performance of CA IX in hypoxia and thereby influence the cell migration propensity of CA IX-expressing cells (7,8). On the other hand, the PG domain can affect cell-substrate adhesion and enhance enzymatic activity (24,25) so its contribution to migration is plausible.…”

“…Unlike other carbonic anhydrases, CA IX possesses a proteoglycan-like region (PG), which represents an N-terminal extension of the catalytic domain (6). A short intracellular tail at the C terminus of CA IX is regulated by phosphorylation of Thr-443 by protein kinase A and mediates inside-out signaling to extracellular catalytic domain (7,8).…”

Interplay between receptor tyrosine kinases and hypoxia signaling in cancer

“…One direct target gene whose expression significantly correlates in many tumor types with poor prognosis and resistance to chemotherapy in general is carbonic anhydrase IX (CAIX) [164], an enzyme which promotes conversion of carbon dioxide to bicarbonate ions and protons. CAIX plays an important role in pH regulation, a crucial step for survival of tumor cells in hypoxia and acidosis [165]. Its expression has been found almost exclusively in tumors and it is considered as one of the main intrinsic markers of tumor hypoxia [164,166].…”