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

Abstract

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.

FIGURE 1.

CHP1 localizes to the nucleus in quiescent cells. A, immunolabeling of endogenous CHP1 in CCL39 fibroblasts maintained for 18 h in medium without (Quiescent) or with 5% FBS (Growth Medium). B, immunoblots for the indicated proteins in subcellular fractions from lysates of quiescent CCL39 fibroblasts maintained in the absence of FBS (Quiescent) or for 4 h with FBS (+ FBS). Fractions include soluble (S100), particulate (P100), and nuclear (Nuc). Markers to confirm subcellular fractionation include lamin, a nuclear membrane protein, the MAP kinase MEKK4, a cytosolic protein, and the NHE1 isoform, an integral plasma membrane protein. C, confocal images of GFP fluorescence in quiescent and FBS-treated CCL39 fibroblasts transiently expressing GFP, GFP-CHP1-WT, and GFP-CHP1-NES. Images are of the midsection of cells fixed in paraformaldehyde and are representative of four separate cell preparations.

FIGURE 2.

CHP1 interacts with UBF in a Ca²⁺-sensitive manner. A, autoradiograph of proteins co-precipitating in CHP1 or IgG immune complexes in lysates prepared in the absence or presence of Ca²⁺ from quiescent CCL39 fibroblasts labeled with [³⁵S]methionine. Arrows indicate proteins of 97 and 94 kDa in CHP1 immune complexes in the absence but not presence of Ca²⁺ that are not in IgG immune complexes. MALDI-MS identified the 97-kDa protein as UBF1 and the 94-kDa protein as UBF2 (supplemental Fig. S1). B, immunoblot for UBF in CHP1 or IgG immune complexes isolated as described in A (upper panel) and in total cell lysate (lower panel). IP, immunoprecipitation. C, immunoblot for UBF in cell lysates incubated with GST or GST and in total cell lysates (bottom panel). D, immunoblots for UBF and for CHP1 in UBF immune complexes isolated from cells maintained in the absence of FBS (−) or for 4 h with FBS (+) (left), and in total cell lysate (right). E, immunoblots (IB) for UBF (upper panel) and for CHP1 (lower panel) prepared from CHP1 immune complexes isolated from quiescent cells.

FIGURE 3.

Nuclear retention of CHP1 inhibits rRNA synthesis. A and B, FUrd incorporation in quiescent and FBS-treated CCL39 cells, including images of immunolabeling (A) and quantification of fluorescence intensity (B). Data represent means ± S.D. (error bars) of three separate cell preparations, analyzing fluorescence in >20 cells/preparation. C, FUrd incorporation in quiescent and FBS-treated CCL39 cells transiently expressing GFP, GFP-CHP1-WT, and GFP-CHP1-NES. Data are expressed as intensity of FUrd signal normalized to the GFP signal and represent the indicated number of cells and means for three cell preparations with GFP-CHP1-WT, and GFP-CHP1-NES and two with GFP. D, 18 S ribosomal RNA synthesis in quiescent and FBS-treated CCL39 cells transiently expressing GFP, GFP-CHP1-WT, and GFP-CHP1-NES. Data are expressed as fold increase relative to quiescent treatment and are representative of two separate preparations.

FIGURE 4.

CHP1 associates with the promoter region of the rRNA genes. Quiescent CCL39 fibroblasts were used for ChIP assays with anti-UBF and anti-CHP1 antibodies, and with rabbit IgG. A, representative ethidium bromide gel showing PCR products for three regions of the hamster rDNA gene examined: the 5′-ETS; the 18 S ribosomal rDNA gene, and the 28 S ribosomal gene. B, ChIP of the 5′-ETS quantified by real time qPCR using immunoprecipitations from quiescent cells or after stimulating cells with 5% FBS for 4 h. Data are expressed as percent of DNA site immunoprecipitated with the indicated antibodies compared with total DNA input (based on 2.5% input). Shown are means ± S.E. of three independent experiments. The representative ethidium bromide gel shows the amount of ETS product present in the exponential phase of PCR using ChIP samples.

FIGURE 5.

Nuclear retention of CHP1 attenuates abundance of UBF in the nucleolus. A, immunolabeling of endogenous UBF in quiescent and FBS-treated CCL39 fibroblasts. B, quantified UBF signal in the nucleolus in quiescent and FBS-treated cells expressed as a percentage of total UBF in the nucleus. Data were obtained from 20 cells from three separate experiments. C, quantified UBF signal in the nucleolus of cells expressing GFP-CHP1-WT or GFP-CHP1-NES. Data are representative of three independent experiments.