Characterization of the βPix Family of Guanine Nucleotide Exchange Factor
βPix is a recently identified guanine nucleotide exchange factor for Rho family small G protein Rac1/Cdc42. The protein interacts with p21-activated protein kinase(Pak) through its SH3 domain. Pak is a major downstream effector of Rac1/Cdc42 and involved in morphological changes of the stimulated cells through actin cytoskeleton rearrangements. Previousely, we reported the cloning and focal localization of βPix(βPix1).
Recently, we have cloned two isoforms(named βPix2 and βPix3 respectively) of βPix1 that are most likely arised from alternative splicing of the same gene. Both isoforms containes the insertion of 58 amino acids in the carboxyl terminal region of βPix1. βPix3 differs from βPix2 by having a deletion of proline-rich region that resides in the central region of βPix1 and βPix2. We examined the effects of βPix-isoform expression on the activation of down stream effector protein, Pak. The extent of Pak activation decreased in the order of βPix3 ＞＞ βPix1 ＞ βPix2, suggesting a differential activation of Pak by βPix-isoforms.
We also examined the expression of βPix protein in human breast carcinomas obtained from 40 breast cancer patients. Immunoblot analysis revealed that high level of βPix expressions in 38 of 40 breast cancer tissues as compared to normal breast tissues. Immunohistochemical analysis showed that strong immunoreactivity to βPix antibodies in breast cancer tissues. However, the changes in the expression levels of Rac1/Cdc42 or Pak, which are known as the downstream signaling proteins of βPix, was not detected in breast cancer tissues. The NIH3T3 cells which stablely over-express βPix showed the enhanced mobilization through Transwell compare to control NIH3T3 cells, suggesting the role of βPix in metastasis of breast cancer cells.
In conclusion, we identified three isoforms of βPix which have differntial effects on the acivation of down stream effector protein, Pak. βPix1 expression is elevated in human breast cancer tissues and the enhanced expression of βPix1 in NIH3T3 cells induced the increase in cell mobility. These results implicate a possible role of βPix in human breast tumorigenesis, especially in metastasis.