Nuclear-localized calcineurin homologous protein CHP1 interacts with upstream binding factor and inhibits ribosomal RNA synthesis.

Calcineurin homologous protein 1 (CHP1) is a widely expressed, 22-kDa myristoylated EF-hand Ca(2+)-binding protein that shares a high degree of similarity with the regulatory B subunit of calcineurin (65%) and with calmodulin (59%). CHP1 localizes to the plasma membrane, the Golgi apparatus, and the nucleus and functions to regulate trafficking of early secretory vesicles, activation of T cells, and expression and transport of the Na-H exchanger NHE1. Although CHP1 contains nuclear export signals, whether its nuclear and cytoplasmic localization is regulated and has distinct functions remain unknown. We show that CHP1 is predominantly in the nucleus in quiescent fibroblasts, is translocated to cytoplasmic compartments with growth medium, and that translocation is inhibited by mutations in the nuclear export motifs. In a screen for proteins co-precipitating with CHP1 in quiescent cells we identified the upstream binding factor UBF, a DNA-binding protein and component of the RNA polymerase I complex regulating RNA synthesis. The CHP1-UBF interaction is restricted to the nucleus and inhibited by Ca(2+). Nuclear retention of CHP1 attenuates the abundance of UBF in the nucleolus and inhibits RNA synthesis when quiescent cells are transferred to growth medium. These data show UBF as a newly identified CHP1-binding protein and regulation of RNA synthesis as a newly identified function for nuclear-localized CHP1, which is distinct from CHP1 functions in the cytosol.

Expression profile of genes regulated by activity of the Na-H exchanger NHE1.

BACKGROUND: In mammalian cells changes in intracellular pH (pHi), which are predominantly controlled by activity of plasma membrane ion exchangers, regulate a diverse range of normal and pathological cellular processes. How changes in pHi affect distinct cellular processes has primarily been determined by evaluating protein activities and we know little about how pHi regulates gene expression. RESULTS: A global profile of genes regulated in mammalian fibroblasts by decreased pHi induced by impaired activity of the plasma membrane Na-H exchanger NHE1 was characterized by using cDNA microarrays. Analysis of selected genes by quantitative RT-PCR, TaqMan, and immunoblot analyses confirmed results obtained from cDNA arrays. Consistent with established roles of pHi and NHE1 activity in cell proliferation and oncogenic transformation, grouping regulated genes into functional categories and biological pathways indicated a predominant number of genes with altered expression were associated with growth factor signaling, oncogenesis, and cell cycle progression. CONCLUSION: A comprehensive analysis of genes selectively regulated by pHi provides insight on candidate targets that might mediate established effects of pHi on a number of normal and pathological cell functions.

Na-H exchange-dependent increase in intracellular pH times G2/M entry and transition.

It is well established that activation of the Na-H exchanger NHE1 and increases in intracellular pH (pHi) are early and universal responses to mitogens and have permissive effects in promoting cell proliferation. Despite this evidence, a specific role for NHE1 or pHi in cell cycle progression remains undetermined. We now show that NHE1 activity and pHi regulate the timing of G2/M entry and transition. Prior to G2/M entry there is a rapid and transient increase in NHE1 activity and pHi, but in fibroblasts expressing a mutant NHE1 that lacks ion translocation activity, this increase in pHi is attenuated, S phase is delayed, and G2/M transition is impaired. In the absence of ion translocation by NHE1, expression of cyclin B1 and the kinase activity of Cdc2 are decreased and Wee1 kinase expression increases. Increasing pHi in the absence of NHE1 activity, however, is sufficient to restore Cdc2 activity and cyclin B1 expression and to promote G2/M entry and transition. These data indicate that a transient increase in pHi induced by NHE1 promotes the timing of G2/M, and they suggest that increases in pHi at the completion of S phase may constitute a previously unrecognized checkpoint for progression to G2 and mitosis.