| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 101 === p21-activated kinase 1 (PAK1) is a Rac/Cdc42-activated serine/threonine kinase involved in diverse cellular functions including cell growth, cell death, and cancer metastasis. In an attempt to identify novel interacting partner(s) for PAK1 by proteomic approach, our laboratory has previously found that collapsin response mediator protein 2 (CRMP2) could be co-immunoprecipitated with PAK1 antibody in the lysates extracted from human epidermoid carcinoma cell line A431. CRMP2 is known to participate in the processing of neural development and differentiation, via regulation of neuron axonal outgrowth, elongation, and neuronal polarization. In non neural cells, CRMP2 was recently reported to participate in cytoskeleton arrangement. Literature search reveals that both PAK1 and CRMP2 are overexpressed in colorectal cancer (CRC), and that PAK1 plays an important role in CRC tumor metastasis. In this study, to demonstrate the biological linkage between PAK1 and CRMP2, we found that CRMP2 and CRMP2L, a CRMP2 isoform known as the alternatively spliced, long form of CRMP2, are novel PAK1-binding partners in human CRC cell line SW480. Using confocal microscopy, we found that CRMP2, CRMP2L and PAK1 are colocalized in SW480 cells. By in vitro binding assay, we observed that CRMP2 can directly bind to PAK1 regardless of its kinase activity; however, the binding was weakened by CRMP2L. To map the binding domains of this interaction, we generated a series of CRMP2 deletion mutants, and our data showed that CRMP2 binds to PAK1 through its amino acid 480-488 (480KRIKARSRL488), and that CRMP2 interacts with CRMP2L or form homodimer by its amino acid 483-488 (483KARSRL488). To further evaluate the possible biological significance(s) of this interaction in CRC cells, we then designed a series of CRMP2 mutants including S486A, R487A, L488A, 3A, 6A and 9A mutants. The results from immunoprecipitation experiments indicated that CRMP2-9A mutant decreased CRMP2 dimerization in SW480 cells. Trans-well migration assays further revealed that overexpression of CRMP2 in SW480 cells significantly enhances their migration ability; however, compared to the CRMP2-overexpressed cells, overexpression of CRMP2-3A, -6A or -9A mutants decreases the migration ability of the cells. Moreover, overexpression of CRMP2 mutants including S486A, R487A, L488A , 3A, 6A and 9A in SW480 altered the cell morphology and the subcellular localization of CRMP2 and PAK1. Collectively, our findings indicate that CRMP2 is a novel binding partner of PAK1, and suggest that the interplay of PAK1/CRMP2/CRMP2L complex may play an important role in regulating epithelial cancer cell migration via affecting multiple cellular mechanisms.
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