Abstract
Precise spatial and temporal regulation of Rho GTPases is required in controlling F-actin-based changes in cell morphology. The molecular mechanisms through which microtubules (MTs) modulate the activity of RhoGTPases and regulate the actin cytoskeleton are unclear. Here we show that p21-activated-kinase 4 (PAK4) mediates morphological changes through its association with the Rho-family guanine nucleotide exchange factor (GEF), GEF-H1. We show that this association is dependent upon a novel GEF-H1 interaction domain (GID) within PAK4. Further, we show that PAK4-mediated phosphorylation of Ser810 acts as a switch to block GEF-H1-dependent stress fiber formation while promoting the formation of lamellipodia in NIH-3T3 cells. We found that the endogenous PAK4-GEF-H1 complex associates with MTs and that PAK4 phosphorylation of MT-bound GEF-H1 releases it into the cytoplasm of NIH-3T3 cells, which coincides with the dissolution of stress fibers. Our observations propose a novel role for PAK4 in GEF-H1-dependent crosstalk between MTs and the actin cytoskeleton.
Highlights
The typical cascade of F-actin-based morphological changes depends upon the temporal activation of Cdc42, Rac and Rho leading to the formation of filopodia, lamellipodia and stress fibers, respectively (Ridley and Hall, 1992; Nobes and Hall, 1995)
Together these results demonstrate that the guanine nucleotide exchange factor (GEF)-H1 interaction domain (GID) is not contained within the kinase domain that begins with the highly conserved ‘GxG’ motif at aa 323
The GID is localized between aa 276 and 324 of p21-activated-kinase 4 (PAK4) and probably involves a smaller region beginning around aa 291 of PAK4 (Fig. 1B,C) since, in the context of the kinase domain, aa 291-591 is necessary and sufficient to bind to GEF-H1
Summary
The typical cascade of F-actin-based morphological changes depends upon the temporal activation of Cdc, Rac and Rho leading to the formation of filopodia, lamellipodia and stress fibers, respectively (Ridley and Hall, 1992; Nobes and Hall, 1995). Rho family GTPases play a role in regulating the MT networks. In addition to its role in filopodia formation, Cdc has been linked to the reorientation of the microtubule organizing centre (MTOC) (Palazzo et al, 2001). Rho family GTPase-controlled morphological processes include cell migration, adhesion, morphogenesis, neurite outgrowth, axon guidance, phagocytosis, cell cycle progression and membrane transport (Chimini and Chavrier, 2000; Kaibuchi et al, 1999; Luo, 2000; Van Aelst and D’Souza-Schorey, 1997; Van Aelst and Symons, 2002)
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