Nuclear accumulation of Calcineurin B Homologous Protein 2 (CHP2) results in enhanced proliferation of tumor cells

Abstract

The interaction between calcineurin B homologous protein 2 (CHP2) and Na + ⁄ H + exchanger 1 (NHE1), two membrane proteins, is essential for protecting cells from serum deprivationinduced death. Although four putative EF-hands in CHP2 had been predicted for years, Ca 2+ -binding activities of these motifs have not been tested yet, their role in this process remain poorly understood. To identify Ca 2+ -binding motifs required for the stable formation of CHP2 ⁄ NHE1 complexes, we developed a mutagenesis-based assay in PS120 cells. We found that 45 Ca 2+ bond to two EF-hand motifs (EF3 and 4) of CHP2 proteins with high affinity. Complex formation between CHP2 and the CHP2 binding domain of NHE1 resulted in a marked increase in the Ca 2+ -binding affinity of CHP2. Co-immunoprecipitation and distribution of GFP-tagged CHP2-EF3m ⁄ 4m also indicated that Ca 2+ affected the membrane location of CHP2 to interact with NHE1. The C-terminal region of CHP2 contains a nuclear export sequence (NES). When the six leucines of NES were mutated to alanines, the resulting CHP2 protein was predominantly localized to the nucleus. Furthermore, mutation of the NES resulted in enhanced proliferation and oncogenic potential of HeLa cells. Together, these results show that calcium and NES control the subcellular distribution of CHP2 and then distinctively regulate cell proliferation.

Calcineurin homologous protein: a multifunctional Ca2+-binding protein family

“…1A and 2), while CHP2 has only NES-1, with CHP3 not having any canonical NES signal. Simultaneous mutations in both NES-1 and NES-2 lead to nuclear localization of CHP1 (94), indicating that one NES is sufficient to transport CHP1 from the nucleus to the cytosol (71).…”

Regulation of the Na+/H+ Exchanger (NHE1) in Breast Cancer Metastasis

“…1B), where CHP1 is thought to be important for NHE1 activity and its stabilization and localization to the plasma membrane (9). CHP2 is highly expressed in tumor cells; it is protective against serum deprivation-induced cell death by increasing pH i (10) and may play a role in the enhanced proliferation of tumor cells (11). CHP3, or tescalin (binding site uncertain), is thought to promote maturation and cell surface stability of NHE1 (12,13).…”

A calcineurin homologous protein is required for sodium-proton exchange events in the C. elegans intestine

“…Biochemical studies have identified an array of CHP binding partners including microtubules, glyceraldehyde, kinesin-related motor KIF1B␤2, calcineurin, the DRAK2 protein kinases, and upstream binding factor (3,23,25,34,40). Studies designed to explore the significance of these biochemical interactions have found that CHPs can regulate calcineurin's phosphatase activity, constitutive vesicle secretion, cytoskeletal organization, proliferation, and cancer metastasis (5,17,26,27,29,49,62).…”

“…NHE activity is implicated broadly in the maintenance of cell volume, electrolyte transport, and cytoplasmic pH levels (10,33,57). More specialized functions of sodium-proton exchangers have been noted in particular cell lines, including contributions to cell migration, morphology, proliferation, apoptosis, and tumor metastasis (11,15,16,24,27,36,49,(52)(53)(54)63). The striking multiplicity of reported NHE functions suggests that these proteins may act in a highly context-dependent manner.…”