GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation

Microtubule nucleation from gamma-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that gamma-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (pin and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, beta PIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of beta PIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and beta PIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and beta PIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, beta PIX and PAK1 in microtubule nucleation correlated with recruitment of gamma-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and beta PIX, but not gamma-tubulin, serve as substrates for PAK1. Finally, direct interaction of gamma-tubulin with the C-terminal domain of beta PIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/beta PIX signaling proteins with PAK1 lcinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells. (C) 2016 Elsevier B.V. All rights reserved.