Interaction of microtubules and actin with the N-terminus of ßPix-b(L) directs cellular pinocytosis.

ABSTRACT

ßPix is a Rac/Cdc42 guanine nucleotide exchange factor (GEF) that is known to be a regulator of actin cytoskeleton remodeling. Recently, a novel splicing isoform, ßPix-b(L), was identified as an alternative translational product of the ßPix-b mRNA with an extended N-terminus comprising a partial calponin homology (CH) domain and a serine-rich (SR) domain. However, the cellular function of ßPix-b(L) is largely unknown. In the current study, we analyzed the genomic DNA structure and cellular functions of ßPix-b(L). The results of this study demonstrate that ßPix is composed of 24 exons and 21 introns spanning around 100 kb. RT-PCR experiments revealed that there are two forms of ßPix mRNA with distinct 5' UTRs that are the result of alternative splicing of exon 1 and 2 from ßPix genomic DNA. In addition, affinity chromatography analysis and a pull-down assay with the N-terminal region of ßPix-b(L) revealed that ßPix-b(L) interacts with tubulin and actin via its N-terminal CH and SR domains, respectively. Interaction with tubulin enabled ßPix-b(L) to bundle the microtubule and form membrane protrusions. Furthermore, the N-terminus of ßPix-b(L) was also critical for its localization to cellular vesicles. Functionally, ßPix-b(L) induced pinocytosis through cooperative action of the CH and Dbl homology (DH) domains, demonstrating the role of ßPix-b(L) in the regulation of membrane dynamics.