Characterization of Carbonic Anhydrase IX Interactome Reveals Proteins Assisting Its Nuclear Localization in Hypoxic Cells

Abstract

Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface.

Probing the Surface of Human Carbonic Anhydrase for Clues towards the Design of Isoform Specific Inhibitors

“…Utilization of monoclonal antibodies, such as M75 and G250, to recognize the proteoglycan-like (PG) domain (the N-terminal extension unique to this isoform) of CA IX have shown effectiveness in disrupting the ability of the enzymes function in regulating tumor cell adhesion and motility [139, 140]. More recently, the monoclonal antibody 6A10 has been developed to mediate CA XII activity also acting as a potential anticancer therapeutic [124, 125].…”

Partial least squares based gene expression analysis in renal failure

“…Quantitative protein expression profiling revealed that overexpression of YWHAE prompt the proliferation of renal cancer cells [32]. CAND1 may also promote the progression of renal cell carcinoma through its interaction with carbonic anhydrase IX [33]. Whether the up regulation contributes to the pathogenesis of renal failure needs further investigation.…”

Modulation of cell surface density of carbonic anhydrase IX by shedding of the ectodomain and endocytosis

“…Interestingly enough, a recent proteomic study searching for the interacting partners of CA IX revealed a group of intracellular proteins that belong to nuclear transport machinery, including XPO1 exportin and TNPO1 importin (Buanne et al, 2013). Although the interaction occurs via the C-terminal region of CA IX, it involves entire (mature) CA IX molecule that can be detected in the nuclear and perinuclear regions of hypoxic cells through the catalytic domain-specific antibody.…”