A Novel LZAP-binding Protein, NLBP, Inhibits Cell Invasion

Junhye Kwon,Hyun Jung Cho,Seung Hun Han,Jin Gu No,Jae Young Kwon,Hongtae Kim

doi:10.1074/jbc.m109.065920

Junhye Kwon, Hyun Jung Cho + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.065920

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Abstract

LXXLL/leucine zipper-containing alternative reading frame (ARF)-binding protein (LZAP) was recently shown to function as a tumor suppressor through inhibition of the NF-kappaB signaling pathway. LZAP is also known as a negative regulator of cell invasion, and its expression was demonstrated to be reduced in several tumor tissues. However, the molecular mechanism of the negative effect of LZAP on cell invasion is unclear. In this study, we identify NLBP as a novel LZAP-binding protein using tandem affinity purification. We demonstrate the negative effects of NLBP on cell invasion and the NF-kappaB signaling pathway. NLBP expression was not detected in hepatocellular carcinoma cells with strong invasive activity, whereas its expression was detected in a hepatocellular carcinoma cell line with no invasive activity. We also demonstrate that these two proteins mutually affect the stability of each other by inhibiting ubiquitination of the other protein. Based on these results, we suggest that NLBP may act as a novel tumor suppressor by inhibiting cell invasion, blocking NF-kappaB signaling, and increasing stability of the LZAP protein.

Highlights

Multistep progressions are involved in tumor formation
We show that NLBP is a novel LZAP-binding protein that may function as a tumor suppressor by inhibiting cell invasion, blocking NF-␬B signaling, and increasing the stability of the LZAP protein
We identified NLBP as a novel LZAP-binding protein using the tandem repeat affinity purification method

Summary

EXPERIMENTAL PROCEDURES

Plasmids—Human NLBP (KIAA0776), CT116, and LZAP cDNA were purchased from the American Type Culture Collection (Manassas, VA). Full-length and deletion mutants of human NLBP were generated by PCR and subcloned into a modified pIRES-EGFP mammalian expression vector, as described previously [8], to create constructs that are S-FLAGSBP (streptavidin-binding peptide)-tagged (SFB-tagged). Full-length and deletion mutants of human LZAP were generated by PCR and subcloned into a modified mammalian expression vector, as described previously [8], to create constructs encoding Myc-tagged full-length or deletion mutants of LZAP. To establish cell lines stably expressing epitope-tagged proteins, 293T cells were transfected with plasmids encoding SFB-LZAP and puromycin-resistant protein. 293T cells stably expressing SFB-LZAP protein were lysed with 4 ml of NETN buffer on ice for 10 min. Two ␮g of GST fusion protein or GST was immobilized on glutathione-Sepharose 4B beads and incubated with lysates prepared from cells that were transiently transfected with plasmids encoding the indicated proteins in NETN buffer for 2 h at 4 °C. List of proteins associated with SFB-LZAP identified by mass spectrometry analysis

60 S ribosomal protein I3

RESULTS

DISCUSSION