Cell migration requires the coordinated spatiotemporal rules of actomyosin contraction and cell protrusion/adhesion. Binding to the GEFs required assembly of the MII into filaments and actin-stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of βPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between Rac1/Cdc42 and MII GTPases which may regulate protrusion/adhesion dynamics in migrating cells. Intro Nonmuscle myosin II (MII) contractility can be critically essential in cell motility (Vicente-Manzanares et al. 2007 MII consists of pairs of myosin weighty stores (MHCs) regulatory myosin light stores (MLCs) and important MLCs that assemble into bipolar filaments with actin-stimulated ATPase activity. The resultant contractility drives formation of actin tension fibers and focal adhesions. MII also cross-links actin which contributes to adhesion assembly and stabilization of actin filaments (Choi et al. 2008 Although MII is located away from the lamellipodium and nascent adhesions (Kolega 1998 2006 Gupton and Waterman-Storer 2006 its removal or inhibition induces ectopic lamellipodia and adhesions (Katsumi et al. 2002 Sandquist et al. 2006 Even-Ram et al. 2007 Vicente-Manzanares et al. 2007 MII might therefore control a diffusible factor(s) that affects processes at the leading edge. Rac1 Cdc42 and RhoA jointly control lamellipodial and filopodial protrusions adhesion dynamics and actin stress fibers during migration (Nobes and Hall 1995 Rho GTPases regulate MII through multiple pathways (Somlyo and Somlyo 2000 In general RhoA/Rho-kinase (ROCK) activates MII contractility whereas Rac1 and its effector PAK often negatively regulate MII and decrease contractility. Efficient cell motility requires that Rac1/Cdc42 RhoA and MII activity be coordinated; however the mechanisms of coordination remain incompletely understood. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) GNF 5837 most of which contain a tandem Dbl homology (DH)-pleckstrin homology (PH) domain as a catalytic core (Schmidt and Hall 2002 Recent studies have revealed a connection between MII and Dbl family GEFs suggesting their potential regulation by MII as well as a scaffold function (Wu et al. 2006 Conti and Adelstein GNF 5837 2008 However the molecular mechanism is unknown. We therefore investigated Rabbit polyclonal to YSA1H. how MII might regulate GEFs for Rho GTPases. Our studies reveal GNF 5837 that MII regulates multiple Dbl family members through direct binding which controls their activity and localization in GNF 5837 migrating cells. Results Identification of βPIX GEF as a novel MII-interacting protein To test whether MII regulates Rho GTPases through Dbl family GEFs we first examined whether MII could associate with βPIX a Rac1/Cdc42-specific GEF highly implicated in cell motility (Za et al. 2006 PC12 cells express βPIX and MIIA/MIIB at high levels so they were used for most immunoprecipitation (IP) experiments on this GEF. βPIX IPs in PC12 cells contained MIIA and MIIB whereas nonimmune IPs showed no association (Fig. 1 A). To test the specificity of the interaction we screened Jurkat T cells and C2C12 myoblasts that expressed MIB and MVa respectively (Fig. 1 A). No interaction between βPIX and myosin IB Va or VI was detected indicating that the MII-βPIX interaction is specific (Fig. 1 A). Figure 1. Identification and characterization of interaction between MII and βPIX. (A) Specific interaction of MII with βPIX. Cell lysates were immunoprecipitated with anti-βPIX antibody followed by immunoblotting for the indicated myosins … To identify the domain(s) involved in the βPIX-MII interaction multiple MIIB and βPIX constructs were examined (Fig. 1 B and C top). MIIB constructs were tagged with GFP and expressed in PC12 cells. IP with anti-GFP antibody followed by immunoblotting for endogenous βPIX showed that the MII head domain bound βPIX (Fig. 1 B bottom). Conversely analysis of βPIX constructs showed that only the N terminus of βPIX.