A Serine/Threonine Kinase Which Causes Apoptosis-Like Cell Death Interacts with a Calcineurin B-Like Protein Capable of Binding Na+/H+ Exchanger

109

The calcineurin B homologous protein (designated CHP1) has been shown to be a common essential cofactor for the plasma membrane Na ؉ /H ؉ exchangers (NHEs) (Pang, T., Su, X., Wakabayashi, S., and Shigekawa, M. (2001) J. Biol. Chem. 276, 17367-17372). In this study, we characterized the function of another isoform of CHP (designated CHP2) that has a 61% amino acid identity with CHP1. CHP2, like CHP1, conferred the ability to NHEs 1-3 to express a high exchange activity by binding to the juxtamembrane region of the cytoplasmic domain of the exchanger, but it interacts more strongly (ϳ5-fold) with NHE1 than does CHP1. Although CHP1 is expressed ubiquitously at relatively high levels, CHP2 expression was extremely low in most human tissues but was higher in tumor cells. We produced stable cell clones overexpressing either CHP1 or CHP2 in which one of them is predominantly bound to NHE1. Serum (10%) induced a significant cytoplasmic alkalinization (0.1-0.2 pH unit) in cells co-expressing CHP1 and NHE1 but not in cells co-expressing CHP2 and NHE1. In the latter, pH i was high (7.4 -7.5) even in the absence of serum, suggesting that NHE1 was already activated. Surprisingly, most (>80%) of CHP2/NHE1 cells unlike CHP1/NHE1 cells were viable even after long serum starvation (>7 days). Thus, the expression of CHP2 appears to protect cells from serum deprivationinduced death by increasing pH i . These properties of CHP2/NHE1 cells are similar to those of malignantly transformed cells. We propose that serum-independent activation of NHE1 by bound CHP2 is one of the key mechanisms for the maintenance of high pH i and the resistance to serum deprivation-induced cell death in malignantly transformed cells.

Section: Discussionmentioning

confidence: 99%

Expression of Calcineurin B Homologous Protein 2 Protects Serum Deprivation-induced Cell Death by Serum-independent Activation of Na+/H+ Exchanger

Pang¹,

Wakabayashi

Shigekawa

2002

109

“…3B). The secondary structure consists of ␣A (residues [11][12][13][14][15][16][17][18][19][20][21][22], ␣B (residues 26 -37), ␣C (residues 48 -51), ␣D (residues 64 -70), ␣E (residues 80 -88), ␣F (residues 111-122), ␣G (residues 132-143), ␣H (residues 149 -162), ␣I (residues 174 -180), and ␣J (residues 185-188) (Figs. 1B and 3B).…”

Section: Journal Of Biological Chemistry 2743mentioning

confidence: 99%

Solution Structure of the Cytoplasmic Region of Na+/H+ Exchanger 1 Complexed with Essential Cofactor Calcineurin B Homologous Protein 1

Mishima

Wakabayashi

Kojima

2007

“…Although DAP, DRP-1, and DAPK2 have a calmodulin regulatory domain in their C terminus, ZIP, Drak1, and Drak2 do not (1)(2)(3)(4)(5). DAP, DAPK2, and DRP-1 are localized in the cytosol (1,2,4), ZIP kinase and Drak1 resides mainly in the nuclei (3,5), and Drak2 is found in both the cytosol and nuclei (5,6), suggesting different mechanisms of action. When DAP family kinases are overexpressed in various cells, apoptosis ensues, either directly or after cytokine stimulation (1)(2)(3)(4)(5), indicating their involvement in apoptosis.…”

mentioning

confidence: 99%

“…It autophosphorylates and phosphorylates myosin light chains as an exogenous substrate (5), although its endogenous substrates have not been identified. Drak2 interacts with a calcineurin homologous protein (6), but the biological significance of this interaction is not clear. According to DNA microarray (12) and real time reverse transcription (RT)-PCR analysis (13) of different tissues, Drak2 is considered exclusively expressed in the T cell compartment; yet such analyses are not precise, because these methods cannot reveal possible focal Drak2 expression in certain organs.…”

mentioning

confidence: 99%

Transgenic Drak2 Overexpression in Mice Leads to Increased T Cell Apoptosis and Compromised Memory T Cell Development

Mao

Qiao

Luo

et al. 2006

Drak2 is a death-associated protein family serine-threonine kinase. Its expression and roles in the immune system were investigated in this study. According to in situ hybridization, Drak2 expression was ubiquitous at the mid-gestation stage in embryos, followed by more focal expression in various organs in the perinatal period and adulthood, notably in the thymus, spleen, lymph nodes, cerebellum, suprachiasmatic nuclei, pituitary, olfactory lobes, adrenal medulla, stomach, skin, and testes. Drak2 transgenic (Tg) mice were generated using the human ␤-actin promoter. These Tg mice showed normal T cell versus B cell and CD4 versus CD8 populations in the spleen, but their spleen weight cellularity was lower in comparison with wild type mice. After TCR activation, the proliferation response in Drak2 Tg T cells was normal, although their interleukin (IL)-2 and IL-4 but not interferon-␥ production was augmented. Activated Drak2 Tg T cells demonstrated significantly enhanced apoptosis in the presence of exogenous IL-2. At the molecular level, Drak2 Tg T cells manifested a lower increase of anti-apoptotic factors during activation; such a change probably rendered the cells vulnerable to subsequent IL-2 insults. The compromised apoptosis in Drak2 Tg T cells was associated with reduced numbers of T cells with the memory cell phenotype (CD62L lo ) and repressed secondary T cell responses in delayed type hypersensitivity. Our study demonstrates that Drak2 expresses in the T cell compartment but is not T cell-specific; it plays critical roles in T cell apoptosis and memory T cell development.To elucidate the molecular mechanisms of T cell activation and differentiation, we conducted DNA microarray analysis employing the mouse 15,000 cDNA panel of the NIA, National Institutes of Health, to compare gene expression patterns of resting versus activated T cells (anti-CD3 and anti-CD28 stimulation for 24 h). Drak2 was one of the genes found to undergo significant changes after activation and was thus selected for further investigation.Drak2 is a serine/threonine kinase belonging to a family of deathassociated protein (DAP) 3 kinases that consists of DAP (1), DRP-1 (2), ZIP kinase (3), DAPK2 (4), Drak1, and Drak2 (5). Drak1 and Drak2 share 67.1% identity in their kinase domain and 24.2% identity in their noncatalytic regions (5). Drak2 also shares about 50% identity in the kinase domain with other members of the family (2). Although DAP, DRP-1, and DAPK2 have a calmodulin regulatory domain in their C terminus, ZIP, Drak1, and Drak2 do not (1-5). DAP, DAPK2, and DRP-1 are localized in the cytosol (1, 2, 4), ZIP kinase and Drak1 resides mainly in the nuclei (3, 5), and Drak2 is found in both the cytosol and nuclei (5, 6), suggesting different mechanisms of action. When DAP family kinases are overexpressed in various cells, apoptosis ensues, either directly or after cytokine stimulation (1-5), indicating their involvement in apoptosis.The mechanism of action and regulation of the prototype DAP family kinase, DAP, at the molecular le...