Abstract
The Rho GTPases are critical regulators of the actin cytoskeleton and are required for cell adhesion, migration, and polarity. Among the key Rho regulatory proteins in the context of cell migration are the p190 RhoGAPs (p190A and p190B), which function to modulate Rho signaling in response to integrin engagement. The p190 RhoGAPs undergo complex regulation, including phosphorylation by several identified kinases, interactions with phospholipids, and association with a variety of cellular proteins. Here, we have identified an additional regulatory mechanism unique to p190A RhoGAP that involves priming-dependent phosphorylation by glycogen synthase-3-beta (GSK-3beta), a kinase previously implicated in establishing cell polarity. We found that p190A-deficient fibroblasts exhibit a defect in directional cell migration reflecting a requirement for GSK-3beta-mediated phosphorylation of amino acids in the C-terminal "tail" of p190A. This phosphorylation leads to inhibition of p190A RhoGAP activity in vitro and in vivo. These studies identify p190A as a novel GSK-3beta substrate and reveal a mechanism by which GSK-3beta contributes to cellular polarization in directionally migrating cells via effects on Rho GTPase activity.
Highlights
Directional cell migration requires stringently coordinated regulation of the actin and microtubule cytoskeletal network
We found that p190A-deficient fibroblasts exhibit a defect in directional cell migration reflecting a requirement for GSK-3-mediated phosphorylation of amino acids in the C-terminal “tail” of p190A
We found that p190A-deficient fibroblasts exhibit a defect in directional cell migration and that GSK-3-mediated phosphorylation of amino acids in the C terminus of p190A is required for proper polarized migration
Summary
DNA Constructs—To generate the pGEX-KG-1252 and pGEX-KG-50AA constructs, DNA sequences corresponding to amino acids 1251–1500 and 1450 –1500 of p190A were PCRamplified and subcloned into the pGEX-KG. 360 l of cell lysate and 40 l of 10ϫ energyregenerating system (ERS) (20 mM Tris (pH 7.5), 10 mM ATP, 10 mM magnesium acetate, 300 mM creatine phosphate (Roche Applied Science), 0.5 mg/ml creatine phosphokinase (Roche Applied Science)) were added to the bacterially purified GST fusion proteins on glutathione beads, and the mixtures were incubated at 30 °C for 2–3 h before proceeding to the in vitro kinase assays. GAP Assays—In vivo assays of p190 RhoGAP activity were performed using COS-7 cells transiently transfected with pEGFP-p190A-WT or the indicated mutants as described previously [4]. The cells were lysed in TLB and incubated with glutathione-agarose beads at 4 °C for 2 h, washed three times with 50 mM Tris-HCl (pH 7.6), 0.1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and boiled in sample buffer. The proteins were resolved on a 10% SDS-polyacrylamide gel, and after Coomassie Blue staining the protein band was excised and sent for mass spectrometry analysis to identify phosphorylated peptides at the Taplin Mass Spectrometry Facility, Harvard Medical School
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