LASS5 Interacts with SDHB and Synergistically Represses p53 and p21 Activity

Abstract

Longevity Assurance 5 (LASS5), a member of the LASS/Ceramide Synthases family, synthesizes C16-ceramide and is implicated in tumor biology. However, its precise role is not yet well understood. A yeast two-hybrid screen was performed using a human cDNA library to identify potential LASS5- interaction partners. One identified clone encodes succinate dehydrogenase subunit B (SDHB). Mammalian two-hybrid assays showed that LASS5 interacts with SDHB, and the result was also confirmed by GST pull-down and coimmunoprecipitation assays. The C-terminal fragment of SDHB was required for the interaction. LASS5 and SDHB were co-localized in COS-7 cells. LASS5 and SDHB expressions were found to be up-regulated in neuroglioma tissue. Transfection assays showed that LASS5 or SDHB expression repressed p53 or p21 reporter activity, respectively. Simultaneous LASS5 and SDHB expression resulted in stronger repression of p53 and p21 reporter activity, suggesting that LASS5 and SDHB interaction may synergistically affect transcriptional regulation of p53 and p21. Our data provide new molecular insights into potential roles of LASS5 and SDHB in tumor biology.

Fig. 1. LASS5 and SDHB mammalian cell two-hybrid assay, LASS5 and SDHB mRNA expression

A. LASS5 protein domains inferred from Smart program analysis. LC, low complexity region. TR, transmembrane region. HOX, hox domain. TLC, TRAM/LAG1/CLN8 domain. B. Interaction between LASS5 and SDHB in mammalian cell two-hybrid assay. pFR-Luc reporter plasmid co-transfected with pCMV-BD and pCMV-AD, or pCMV-BD–LASS5 (HOX) and pCMV-AD-SDHB-C, or pCMV-BD–LASS5 and pCMV-AD-SDHB into COS-7 cells. 48 h later luciferase activity was determined. Data shown are means of three repeats of a transfection experiment following normalization transfection efficiency (β-galactosidease activity). Each transfection experiment was repeated at least three times. C. RT-PCR analysis of LASS5 and SDHB mRNA expression in adult human tissues. GAPDH expression was used as internal control.

Fig. 2. LASS5 and SDHB co-immunoprecipitation assays and GST pull-down assays

A. COS-7 cells transfected with expression vectors encoding Myc-tagged LASS5 and Flag-tagged SDHB, alone or in combination as indicated. Upper two panels show immunoblot (IB) of cell lysates confirming expression of tagged proteins. Third panel: following immunoprecipitation (IP) using anti-Myc antibody, co-expressed Flag–SDHB was detected by anti-Flag IB. Fourth panel: following IP using anti-Flag antibody, co-expressed LASS5 was detected by anti-Myc IB. B. COS-7 cells transfected with expression vectors encoding LASS5 (HOX) and Flag–SDHB-C protein domains, alone or in combination as indicated. Upper two panels show immunoblot (IB) of cell lysates confirming expression of tagged proteins. Third panel: following immunoprecipitation (IP) using anti-Myc antibody, co-expressed Flag–SDHB-C was detected by anti-Flag IB. Fourth panel: following IP using anti-Flag antibody, co-expressed LASS5 (HOX) was detected by anti-Myc IB. C. 5 μg of GST or GST-LASS5 (HOX) fusion protein bound to glutathione sepharose beads (as indicated) was mixed with 100 μg of cell lysate containing Flag-SDHB. Bound protein was detected by SDS-PAGE and immunoblotting (IB) using anti-Flag antibody.

Fig. 3. LASS5 and SDHB colocalize in COS-7 cells

A. COS-7 cells co-transfected with LASS5-EGFP and SDHB-DsRed-Monomer expression vectors. Nuclei stained with DAPI. The full length proteins colocalized in the cell cytoplasm. B. LASS5(HOX)-EGFP protein domain and SDHB-DsRed-Monomer colocalized in the cell cytoplasm. C. LASS5(HOX)-EGFP protein domain and SDHB(N)-DsRed-Monomer N-terminal protein colocalized in cytoplasm and nucleus. D. LASS5(HOX)-EGFP protein domain and SDHB(C)-DsRed-MonomerC-terminal protein colocalized in the cytoplasm. These observations suggest that LASS5 (HOX) cytoplasmic localization is dependent upon interaction with SDHB-C domain. Scale bar 5μm.

Fig. 4. Expression level of LASS5 was up-regulaedin glioma tissue samples, and LASS5 and SDHB inhibited p53 and p21 promoter activity

A. RT-PCR analysis of LASS5 and SDHB mRNA expression levels in normal nervous ganglion (control, Cont) and four independent neuroglioma tissue samples. GAPDH expression assayed as internal control. Amplification products are 244 bp (LASS5) and 880-bp (SDHB). Bar graph shows quantitation of LASS5 and SDHB mRNA expression levels in tumor samples relative to control (CTRL). B. LASS5 protein expression in normal tissue and glioma samples analyzed by Western blot. Actin used as an internal control. Bar graph shows quantitation of LASS5 protein expression in tumor samples relative to control (CTRL). Unpaired student t test used to determine significance. *p<0.05. C. Cos-7 cells co-transfected with p53-Luc reporter and expression vectors pCMV-Tag2-LASS5, pCMV-Tag2-SDHB, pCMV-Tag2-LASS5(HOX), pCMV-Tag2-SDHB-C as indicated. D. Cos-7 cells co-transfected with p21-Luc reporter and expression vectors pCMV-Tag2-LASS5, pCMV-Tag2-SDHB, pCMV-Tag2-LASS5(HOX), pCMV-Tag2-SDHB-C as indicated. The data are the mean of three repeats of a transfection experiment. *p<0.05.