A serine/threonine kinase which causes apoptosis-like cell death interacts with a calcineurin B-like protein capable of binding Na(+)/H(+) exchanger.
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.
Matsumoto
M
M
Department of Biological Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Miyake
Y
Y
Nagita
M
M
Inoue
H
H
Shitakubo
D
D
Takemoto
K
K
Ohtsuka
C
C
Murakami
H
H
Nakamura
N
N
Kanazawa
H
H
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D029681:Arabidopsis Proteins; D001483:Base Sequence; D001665:Binding Sites; D019703:Calcineurin; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D014018:Tissue Distribution; D020798:Two-Hybrid System Techniques
D051017:Apoptosis Regulatory Proteins; D029681:Arabidopsis Proteins; D019703:Calcineurin; D002135:Calcium-Binding Proteins; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; C484078:CBL10 protein, Arabidopsis; C484079:CBL3 protein, Arabidopsis; C483419:calcineurin B-like protein 1, Arabidopsis; C069139:growth factor-activatable Na-H exchanger NHE-1; C071056:Ppp3r2 protein, rat; C441463:Stk17b protein, rat; C115489:STK17B protein, human; C480758:At5g55990 protein, Arabidopsis