The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, ⌬F508 CFTR, is retained in the endoplasmic reticulum (ER) and is consequently degraded by the ubiquitin-proteasome pathway known as ER-associated degradation (ERAD). Because the prolonged interaction of ⌬F508 CFTR with calnexin, an ER chaperone, results in the ERAD of ⌬F508 CFTR, calnexin seems to lead it to the ERAD pathway. However, the role of calnexin in the ERAD is controversial. In this study, we found that calnexin overexpression partially attenuated the ERAD of ⌬F508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the ⌬F508 CFTR but not the wild-type CFTR was retained in the concentric membranous bodies. Furthermore, we observed that calnexin overexpression moderately inhibited the formation of aggresomes accumulating the ubiquitinated ⌬F508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of ⌬F508 CFTR in the ER. INTRODUCTIONCystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic integral membrane protein that functions as a cAMP-dependent Cl Ϫ channel in the apical membrane of epithelial cells (Anderson et al., 1991;Drumm et al., 1991;Tabcharani et al., 1991;Bear et al., 1992). Mutations in the CFTR gene lead to the absence or malfunction of a regulated Cl Ϫ channel in the apical membrane of secretory epithelia, resulting in the clinical symptoms of cystic fibrosis (CF) (Kerem et al., 1989;Riordan et al., 1989;Rommens et al., 1989;Cheng et al., 1990). Therefore, potential CF therapies are aimed at overcoming the functional impairment of various mutant CFTRs, particularly ⌬F508 CFTR, in which a phenylalanine at position 508 is deleted from the first nucleotide-binding fold. This mutation is found in ϳ70% of CF chromosomes and results in a severe form of the disease; Ͼ90% of CF patients have at least one ⌬F508 allele (Tsui, 1992;Sferra and Collins, 1993). Although ⌬F508 CFTR is functionally competent as a cAMP-dependent Cl Ϫ channel in model situations where it reaches the plasma membrane (Drumm et al., 1991;Denning et al., 1992;Li et al., 1993), in mammalian cells ⌬F508 CFTR fails to reach the plasma membrane.CFTR biogenesis is inefficient. During insertion into the ER membrane, CFTR interacts with the cytosolic chaperones Hsc70/Hdj-2 and Hsp90, as well as the ER chaperone calnexin (CNX) (Yang et al., 1993;Pind et al., 1994;Loo et al., 1998;Meacham et al., 1999). After ATP-dependent conformational maturation, assisted by cytosolic and ER chaperones, the chaperones release CFTR, and only ϳ30% of the immature wild-type (wt) CFTR transits to the late secretory pathway, ultimately reaching the plasma membrane (Lukacs et al., 1994). However, most of the immature wt CFTR (ϳ70%), and nearly all of immature ⌬F508 CFTR, fail to mature and do not transit to the late secretory pathway (Lukacs et al., 1994). They are trapped in the ER as core-glycosylated products (ϳ140 kDa) and ar...

Toward the development of a novel cancer immunotherapy, we have previously identified several tumor-associated antigens (TAAs) and the epitopes recognized by human histocompatibility leukocyte (HLA)-A2/A24-restricted cytotoxic T lymphocyte (CTL). In this study, we tried to identify a TAA of lung cancer (LC) and its HLA-A2 restricted CTL epitopes to provide a target antigen useful for cancer immunotherapy of LC. We identified a novel cancer testis antigen, cell division cycle associated gene 1 (CDCA1), overexpressed in nonsmall cell LC using a cDNA microarray analysis. The expression levels of CDCA1 were also increased in the majority of small cell LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers. We used HLA-A2.1 transgenic mice to identify the HLA-A2 (A*0201)-restricted CDCA1 epitopes recognized by mouse CTL, and we investigated whether these peptides could induce CDCA1-reactive CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA-A2-positive donors and a NSCLC patient. Consequently, we found that the CDCA1 65-73 (YMMPVNSEV) peptide and CDCA1 [351][352][353][354][355][356][357][358][359] (KLATAQFKI) peptide could induce peptide-reactive CTLs in HLA-A2.1 transgenic mice. In HLA-A2 1 donors, in vitro stimulation of PBMC with these peptides could induce peptide-reactive CTLs which killed tumor cell lines endogenously expressing both HLA-A2 and CDCA1. As a result, CDCA1 is a novel cancer-testis antigen overexpressed in LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers, and CDCA1 may therefore be an ideal TAA useful for the diagnosis and immunotherapy of these cancers.

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When apoptosis is initiated by manganese (II) loading, hyperthermia or thapsigargin treatment, human HL-60 and AsPC-1 cells initiate de novo synthesis of the C5a receptor (C5aR) and generation of its ligand, the ribosomal protein S19 (RP S19) homodimer. The ligand-receptor interaction, in an autocrine/paracrine fashion, promotes apoptosis, which can be bypassed by exogenous administration of C5a, another ligand. The proapoptotic function of the RP S19 dimer is reproduced by a C5a/RPS19 chimera that contains the body of C5a and the C-terminal region (Ile134-His145) of RP S19. The RP S19 dimer or C5a/RPS19 and C5a inversely regulate the expression of Regulator of G protein Signaling 3 (RGS3) gene in the apoptosis-initiated cells. Namely, the RP S19-type proteins upregulate RGS3 expression, whereas the C5a reduce it. Transformation of HL-60 cells to overexpress RGS3 promotes apoptosis in association with the downregulation of the Extracellular signal-Regulated Kinase (ERK) signal, and vice versa in the RGS3 knocked-down cells. Consistent with this result, an inhibitor of ERK phosphorylation effectively enhances the apoptotic rate in wild-type HL-60 cells. Moreover, a dominant negative effect on the RP S19 dimer production encourages apoptosis-initiated HL-60 cells with a longer lifespan in mouse than the natural effect. Our data indicate that, in apoptosis-initiated cells, the ligand-dependent C5aR-mediated dual signal affects the fate of cells, either apoptosis execution or survival, through regulation of RGS3 gene expression and subsequent modulation of ERK signal.

Members of protein kinase D (PKD) family serine/threonine kinases (PKD1, PKD2 and PKD3) are expressed in wide range of cells and regulate various cellular responses including immune responses. We have previously shown that PKD is involved in the signaling pathways of a human CD4(+) T cell clone stimulated with its cognate antigen. Contrary to foregoing publications, PKD1 mRNA was not detected in human T cells, Jurkat cells and mouse thymocytes and splenocytes. Instead, mass-spectrometric and reverse transcription-PCR analyses revealed that PKD2 was predominant in T cells. To investigate the roles of PKD2, wild-type (WT) and constitutively active (CA) PKD2 were expressed in Jurkat cells together with IL-2 promoter-driven reporter gene. Expression of WT-PKD2 enhanced IL-2 promoter activity upon stimulation with anti-CD3 mAb, while expression of CA-PKD2 inhibited IL-2 promoter activity and induced cell death. Although the cell death was suppressed by the treatment with caspase inhibitor, the IL-2 promoter activity was rarely recovered in CA-PKD2-expressing cells upon TCR stimulation. WT-PKD2 localized mainly in the cytoplasm translocated into the nucleus after TCR stimulation, while CA-PKD2 was present in both the cytoplasm and the nuclei before and after stimulation. Proteomic analyses revealed that CA-PKD2 enhanced the amount of phosphorylated SET protein, a histone chaperon that regulates histone acetylation, in Jurkat cells and the recombinant SET protein was phosphorylated by CA-PKD2 in vitro. The data provide a renewing insight into the subset of PKD family kinases expressed in T cells and suggest that PKD2 is involved in IL-2 promoter regulation and cell death depending on its activity upon TCR stimulation.