Abstract:

Objective

To investigate the subcellular localization and the biological characteristics of the novel FAK splicing mutant (FAK-Del33).

Methods

Basic experimental research applied. FAK wide-type and FAK-Del33 cDNA were cloned into pEGFP expression vector, and then transfected into breast cancer cell lines. The expression and subcellular localization of FAK wild-type and FAK-Del33 cDNA were analyzed. FAK or FAK-De133 overexpressing MDA-MB-468 were constructed. Its expression and in vitro colony forming ability was evaluated.

Results

FAK-GFP fusion protein expression plasmids were constructed and then transfected into MDA-MB-468. FAK-WT dispersed evenly in the cytoplasma while FAK-Del33 signal enriched into strong signal points at different subcellular sites; restriction enzyme analysis and sequencing confirmed the correct construction of lentivirus plasmids. The stable cell lines with expression of FAK were generated by lentivirus infection, puromycin screening and fluorescence quantitative PCR identification. Soft agar colony formation test showed that clone number from mutant FAK had significantly higher levels than FAK wide-type[(735±91)/1000 vs. (335±48)/1000, t=9.437, P＜0.01).

Conclusion

This work clearly indicates that the exon 33 deletion change the subcellular localization of FAK and can enhance tumor cell line' s in vitro colonyforming ability and the FAK mutant may participate in the occurrence and development of tumor.

Key words: Splicing mutant, Protein tyrosine kinase, Focal adhesion kinase, Tumor