Abstract
Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous (WAVE) proteins play a major role in Rac-induced actin dynamics, but Rac does not bind directly to WAVE proteins. It has been proposed that either the insulin receptor substrate protein 53 (IRSp53) or a complex of proteins containing Abelson interactor protein 1 (Abi1) mediates the interaction of WAVE2 and Rac. Depletion of endogenous IRSp53 by RNA-mediated interference (RNAi) in a RAW/LR5 macrophage cell line resulted in a significant reduction of Rac1Q61L-induced surface ruffles and colony-stimulating factor 1 (CSF-1)-induced actin polymerization, protrusion and cell migration. However, IRSp53 was not essential for Fcgamma-R-mediated phagocytosis, formation of podosomes or for formation of Cdc42V12-induced filopodia. IRSp53 was found to be present in an immunoprecipitable complex with WAVE2 and Abi1 in a Rac1-activation-dependent manner in RAW/LR5 cells in vivo. Importantly, reduction of endogenous IRSp53 or expression of IRSp53 lacking the WAVE2-binding site (IRSp53DeltaSH3) resulted in a significant reduction in the association of Rac1 with WAVE2 and Abi1, indicating that the association of Rac1 with WAVE2 and Abi1 is IRSp53 dependent. While it has been proposed that WAVE2 activity is regulated by membrane recruitment, membrane targeting of WAVE2 in RAW/LR5 and Cos-7 cells did not induce actin polymerization or protrusion, suggesting that membrane recruitment was insufficient for regulation of WAVE2. Combined, these data suggest that IRSp53 links Rac1 to WAVE2 in vivo and its function is crucial for production of CSF-1-induced F-actin-rich protrusions and cell migration in macrophages. This study indicates that Rac1, along with IRSp53 and Abi1, is involved in a more complex and tight regulation of WAVE2 than one operating solely through membrane localization.
Highlights
Cell migration is a fundamental process required for normal immune system function, embryonic development, and tissue repair
The results shown provide evidence that insulin receptor substrate protein 53 (IRSp53) exists in a complex with WAVE2 and Abelson interactor protein 1 (Abi1) downstream of GTP-loaded Rac1 in vivo that is required for colony stimulating factor-1 (CSF-1)-induced protrusions and cell migration
Since it has been proposed that IRSp53 mediates the interaction between Rac1 and WAVE2, and since we showed that Rac1 interacts with IRSp53 (Fig. 3) and both WAVE2 and Abi1 exist as a stable unit in macrophages (Abou Kheir et al, 2005), this suggested that Rac1, IRSp53, WAVE2 and Abi1 might exist in the same complex in macrophages
Summary
Cell migration is a fundamental process required for normal immune system function, embryonic development, and tissue repair. This process contributes to the pathogenesis of several diseases, such as chronic inflammatory disease and tumor cell invasion (Raftopoulou and Hall, 2004; Ridley et al, 2003). Enhanced production of CSF-1 and the associated macrophage recruitment have been shown to promote the progression of diseases such as rheumatoid arthritis (Bischof et al, 2000; Campbell et al, 2000), atherosclerosis (Rajavashisth et al, 1998; Smith et al, 1995) and breast cancer (Aharinejad et al, 2004; Lin et al, 2001). Treatment of macrophages with CSF-1 induces a massive actin cytoskeleton reorganization leading to a formation of surface ruffling, followed by cell migration towards the source of CSF-1 (Allen et al, 1997; Boocock et al, 1989; Webb et al, 1996)
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