Distinct functional domains of PNMA5 mediate protein-protein interaction, nuclear localization, and apoptosis signaling in human cancer cells

Abstract

Purpose: Members of paraneoplastic Ma (PNMA) family have been identified as onconeuronal antigens, which aberrant expressions in cancer cells of patients with paraneoplastic disorder (PND) are closely linked to manifestation of auto-immunity, neuro-degeneration, and cancer. The purpose of present study was to determine the role of PNMA5 and its functional relationship to MOAP-1 (PNMA4) in human cancer cells.

Methods: PNMA5 mutants were generated through deletion or site-directed mutagenesis and transiently expressed in human cancer cell lines to investigate their role in apoptosis, subcellular localization, and potential interaction with MOAP-1 through apoptosis assays, fluorescence microscopy, and co-immunoprecipitation studies, respectively.

Results: Over-expressed human PNMA5 exhibited nuclear localization pattern in both MCF-7 and HeLa cells. Deletion mapping and mutagenesis studies showed that C-terminus of PNMA5 is responsible for nuclear localization, while the amino acid residues (391KRRR) within the C-terminus of PNMA5 are required for nuclear targeting. Deletion mapping and co-immunoprecipitation studies showed that PNMA5 interacts with MOAP-1 and N-terminal domain of PNMA5 is required for interaction with MOAP-1. Furthermore, co-expression of PNMA5 and MOAP-1 in MCF-7 cells significantly enhanced chemo-sensitivity of MCF-7 to Etoposide treatment, indicating that PNMA5 and MOAP-1 interact synergistically to promote apoptotic signaling in MCF-7 cells.

Conclusions: Our results show that PNMA5 promotes apoptosis signaling in HeLa and MCF-7 cells and interacts synergistically with MOAP-1 through its N-terminal domain to promote apoptosis and chemo-sensitivity in human cancer cells. The C-terminal domain of PNMA5 is required for nuclear localization; however, both N-and C-terminal domains of PNMA5 appear to be required for pro-apoptotic function.

Keywords: Apoptosis; Chemo-sensitivity; Functional domains; MOAP-1; PNMA5; Paraneoplastic Antigens.

Fig. 1

ClustalW2 sequence alignment of human MOAP-1 and PNMA5. BH-3-like domain of MOAP-1 is underlined. Sequence representing the C-terminal region of human PNMA5 is highlighted (bold-faced)

Fig. 2

Structure and sequence of human PNMA5. a Schematics showing deletion and point mutations of human PNMA5. b Western Blot analysis of Myc-tagged PNMA5 and its mutants. Expression constructs of PNMA5 and mutants were transiently transfected into 293T cells, and protein lysates were analyzed on Western Blot using anti-Myc antibody

Fig. 3

PNMA5 localizes to cell nucleus via its C-terminal domain. MCF-7 cells were transiently transfected with full-length Myc-tagged PNMA5 (a), DM1 (b), or DM2 (c) mutant, and immuno-stained with anti-Myc antibody, followed by detection with secondary antibody conjugated to FITC. In addition, the cells were stained with DAPI and Mito-Red fluorescence dyes. Fluorescence images were captured using 60× objective

Fig. 4

Critical amino acid residues within nuclear localization signal (NLS) of PNMA5 are required for nuclear localization. HeLa cells were transiently transfected with full-length Myc-tagged PNMA5 (a, upper panel), PM1 mutant (a, lower panel), or PM2 mutant (b, upper and lower panels), and immuno-stained with anti-Myc antibody, followed by detection with secondary antibody conjugated to FITC. In addition, the cells were stained with DAPI

Fig. 5

Over-expressed PNMA5 promotes apoptosis a. Cell morphology of PNMA5- or MOAP-1-transfected HeLa cells. The expression plasmids were co-transfected with β-Gal reporter gene and stained for β-Gal activity (left panel). β-Gal positive cells with round cell morphology were counted as described in Materials and Methods (right panel). b Over-expressed Myc-tagged PNMA5 induced nuclei condensation in MCF-7 cells. MCF-7 cells were transfected with Myc-tagged PNMA5, and transfected cells were stained with DAPI, and immuno-stained with anti-Myc antibody, followed by detection with secondary conjugated to DyLight550. c MCF-7 cells transfected with PNMA5 exhibited Annexin-V positive staining. MCF-7 cells were co-transfected with GFP and PNMA5, followed by 16 h of Etoposide treatment. Transfected cells were stained with Annexin-V-APC, and PI before flow cytometry analysis. GFP positive MCF-7 cells were selected and analyzed for Annexin-V-APC or PI staining through flow cytometer, and representative data were shown (left panel). The results from three independent experiments were analyzed (right panel). *, ^ Significantly different at p < 0.05 (Student’s t test)

Fig. 6

PNMA5 and MOAP-1 interact synergistically to promote apoptotic cell death. a PNMA5 and MOAP-1 interacted synergistically to promote caspase activation in transfected MCF-7 cells. MCF-7 cells were co-transfected with Myc-tagged expression construct together with pGloSensor–DEVD reporter gene, and relative luminescence (RLU) was measured. b Co-expression of PNMA5 and MOAP-1 promoted apoptotic cell death and chemo-sensitivity of MCF-7 cells. MCF-7 cells were transfected with the indicated expression constructs encoding either PNMA5, MOAP-1, or PNMA5 plus MOAP-1 followed by Etoposide treatment. Cell viability was measured as described in Materials and Methods. c Co-transfection of MCF-7 cells with PNMA5 and MOAP-1 increased apoptotic cell death as measured by Annexin-V staining. MCF-7 cells were co-transfected with indicated expression constructs and GFP expression construct, followed by Etoposide treatment, Annexin-V-APC and PI staining. Transfected cells were analyzed through flow cytometer to identify GFP, Annexin-V, and PI positive cells (left panel). The results from three independent experiments were analyzed (right panel). *, ^Significantly different at p < 0.05 (Student’s t test)

Fig. 7

PNMA5 interacts with MOAP-1 through its N-terminal domain to promote apoptosis. a PNMA5 and its N-terminal mutant interacted with MOAP-1. Expression constructs encoding the indicated Myc- and HA-tagged proteins were transiently transfected into 293T cells. 16 h after transfection, cell lysates were prepared and expressed proteins were immunoprecipitated with anti-HA antibody, and co-immunoprecipitated proteins were detected with anti-Myc antibody through Western Blot analysis. b PNMA5 induced caspase-dependent apoptosis; however, N- or C-terminal deletion of PNMA5 abolished apoptotic signaling in MCF-7 cells. Expression constructs encoding Myc-tagged proteins were transiently co-transfected with pGloSensor–DEVD into MCF-7 cells, and 12 h after transfection, the cells were incubated with caspase inhibitor for 24 h before harvesting cells for luciferase assay. Relative response ratio (RRR) was measured as described in Materials and Methods, using Bax, and Bcl-xL as positive and negatively control, respectively. CI, Caspase Inhibitor. c PNMA5 promoted while PNMA5 mutant, DM1, inhibited dose-dependent caspase activation through interaction with MOAP-1. Expression constructs encoding HA-tagged MOAP-1 and pGloSensor-DEVD were co-transfected alone or with increasing quantity of expression construct encoding Myc-tagged PNMA5 or DM1 mutant into MCF-7 cells. 24 h after transfection, luciferase activities of transfected cells were measured as described above. d PNMA5 mutant, DM1, inhibited MOAP-1-mediated apoptosis to promote cell viability. Expression constructs encoding the Myc-or HA-tagged proteins were transiently transfected into MCF-7, and cell viability was measured 24 h after transfection. #, ^, +,*, †, ŧSignificantly different at p < 0.05 (Student’s t test)