beta Pix-d promotes tubulin acetylation and neurite outgrowth through a PAK/Stathmin1 signaling pathway

Abstract

Microtubules are a major cytoskeletal component of neurites, and the regulation of microtubule stability is essential for neurite morphogenesis. beta Pix (ARHGEF7) is a guanine nucleotide exchange factor for the small GTPases Rac1 and Cdc42, which modulate the organization of actin filaments and microtubules. beta Pix is expressed as alternatively spliced variants, including the ubiquitous isoform beta Pix-a and the neuronal isoforms beta Pix-b and beta Pix-d, but the function of the neuronal isoforms remains unclear. Here, we reveal the novel role of beta Pix neuronal isoforms in regulating tubulin acetylation and neurite outgrowth. At DIV4, hippocampal neurons cultured from beta Pix neuronal isoform knockout (beta Pix-NIKO) mice exhibit defects in neurite morphology and tubulin acetylation, a type of tubulin modification which often labels stable microtubules. Treating beta Pix-NIKO neurons with paclitaxel, which stabilizes the microtubules, or reintroducing either neuronal beta Pix isoform to the KO neurons overcomes the impairment in neurite morphology and tubulin acetylation, suggesting that neuronal beta Pix isoforms may promote microtubule stabilization during neurite development. beta Pix-NIKO neurons also exhibit lower phosphorylation levels for Stathmin1, a microtubule-destabilizing protein, at Ser16. Expressing either beta Pix neuronal isoform in the beta Pix-NIKO neurons restores Stathmin1 phosphorylation levels, with beta Pix-d having a greater effect than beta Pix-b. Furthermore, we find that the recovery of neurite length and Stathmin1 phosphorylation via beta Pix-d expression requires PAK kinase activity. Taken together, our study demonstrates that beta Pix-d regulates the phosphorylation of Stathmin1 in a PAK-dependent manner and that neuronal beta Pix isoforms promote tubulin acetylation and neurite morphogenesis during neuronal development.