We surveyed proteins capable of binding to the cytoplasmic domain of Na(+)/H(+) exchanger (NHE)1 in a rat brain cDNA library with the yeast two-hybrid system. One clone obtained coded for a protein reported previously as a human calcineurin homologous protein (CHP). Since CHP is homologous to the regulatory subunit B of calcineurin, we expected a possible interacting partner of CHP like the catalytic subunit of calcineurin (calcineurin A), and surveyed this putative partner again with the yeast two-hybrid system. A clone thus obtained coded for a kinase, which is basically the same as that reported for human DRAK2. Overexpression of the rat homologue of DRAK2 caused apoptosis-like cell death of NIH3T3 cells, which was dependent on the kinase activity, confirming the previous result for DRAK2. The purified CHP and rat DRAK2 proteins synthesized in Escherichia coli could bind in vitro. CHP and rat DRAK2 expressed in COS-7 cells were found to be localized in the Golgi apparatus and nucleus, respectively. Some of them was also found in the membrane peripheral region. When they were co-expressed in the same cells, most of CHP moved to the nucleus where rat DRAK2 is located, suggesting in vivo interaction of these proteins. However, minor but significant fractions of both proteins were also found in the membrane peripheral region. Rat DRAK2 is expressed highly in thymus, spleen, and testis, where the apoptosis plays an important role in physiology.

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The Na ؉ /H؉ exchangers (NHEs) comprise a family of membrane proteins that catalyze the electroneutral exchange of Na ؉ and H ؉ . Calcineurin homologous protein (CHP) acts as a crucial cofactor for NHE activity through direct interaction with the carboxyl-terminal tail region of NHEs. We have cloned a new rat CHP isoform (rCHP2) and characterized the binding property to NHEs and the tissue distribution. rCHP2 binds to the juxtamembrane region of plasma membrane-type NHE isoforms (NHE1-5) in vivo and in vitro as well as rCHP1 (original rat CHP). Interestingly, CHP2 is predominantly expressed in the small and large intestine although rCHP1 shows relatively ubiquitous expression at both the mRNA and protein levels. In situ hybridization experiments demonstrated the abundant expression of CHP2 in the epithelial cell layer of villi of the small intestine in contrast with the expression of CHP1 in both the epithelial layer and connective tissues. These results suggest that CHP2 functions in the absorptive epithelium for the intestine with NHE(s).

We previously showed that calcineurin B homologous protein 1 (CHP1) interacts with nuclear apoptosis-inducing protein kinase DRAK2, and that overexpression of DRAK2 induces the nuclear accumulation of CHP1, although CHP1 usually resides in the cytoplasm [Matsumoto et al. (2001) J. Biochem. 130, 217-225]. Here we show that CHP1 has two functional nuclear export signal (NES) sequences in its carboxyl-terminal region. Treatment of several cell lines with leptomycin B, a specific inhibitor of CRM1-dependent nuclear export, induces the nuclear accumulation of CHP1. Moreover, CHP1-GFP fusion proteins with deletions or point mutations affecting the two putative NES sequences accumulate in the nucleus to a greater extent than wild-type CHP1-GFP. Tagging glutathione S-transferase-GFP fusion protein with each NES sequence caused a shift in their intracellular localization from all over the cells to the cytoplasm. These results suggest that after CHP1 has entered the nucleus, it is exported to the cytoplasm in an NES-dependent manner.