Fgd1, the Cdc42 GEF responsible for Faciogenital Dysplasia, directly interacts with cortactin and mAbp1 to modulate cell shape

Abstract

FGD1 mutations result in Faciogenital Dysplasia (FGDY), an X-linked human disease that affects skeletal formation and embryonic morphogenesis. FGD1 and Fgd1, the mouse FGD1 ortholog, encode guanine nucleotide exchange factors (GEF) that specifically activate Cdc42, a Rho GTPase that controls the organization of the actin cytoskeleton. To further understand FGD1/Fgd1 signaling and begin to elucidate the molecular pathophysiology of FGDY, we demonstrate that Fgd1 directly interacts with cortactin and mouse actin-binding protein 1 (mAbp1), actin-binding proteins that regulate actin polymerization through the Arp2/3 complex. In yeast two-hybrid studies, cortactin and mAbp1 Src homology 3 (SH3) domains interact with a single Fgd1 SH3-binding domain (SH3-BD), and biochemical studies show that the Fgd1 SH3-BD directly binds to cortactin and mAbp1 in vitro. Immunoprecipitation studies show that Fgd1 interacts with cortactin and mAbp1 in vivo and that Fgd1 SH3-BD mutations disrupt binding. Immunocytochemical studies show that Fgd1 colocalizes with cortactin and mAbp1 in lamellipodia and membrane ruffles, and that Fgd1 subcellular targeting is dynamic. By using truncated cortactin proteins, immunocytochemical studies show that the cortactin SH3 domain targets Fgd1 to the subcortical actin cytoskeleton, and that abnormal Fgd1 localization results in actin cytoskeletal abnormalities and significant changes in cell shape and viability. Thus, this study provides novel in vitro and in vivo evidence that Fgd1 specifically and directly interacts with cortactin and mAbp1, and that these interactions play an important role in regulating the actin cytoskeleton and, subsequently, cell shape.

Faciogenital Dysplasia Protein (FGD1) Regulates Export of Cargo Proteins from the Golgi Complex via Cdc42 Activation

“…Apart from its interaction with Cdc42, FGD1 has been shown to interact directly with other proteins, including cortactin and actin binding protein 1 (Abp1), via its prolinerich domain (Hou et al, 2003). Given that cortactin appears to be involved in the regulation of export from the Golgi complex (Cao et al, 2005), this direct FGD1-cortactin interaction potentially has an important role in protein delivery from the Golgi complex to the cell surface.…”

Src-mediated phosphorylation of mammalian Abp1 (DBNL) regulates podosome rosette formation in transformed fibroblasts

“…mAbp1 was initially identified in a phage display screen for SH3-domain-containing proteins (Sparks et al, 1996), and two tyrosine residues in the proline-rich region were identified as the Src-phospho-acceptor sites (Larbolette, 1999;Lock et al, 1998). The SH3 domain has been shown to interact with proteins involved in diverse functions including synaptogenesis, endocytosis and cell motility (Pinyol, 2007;Fenster et al, 2003;Han et al, 2003;Kessels et al, 2001;Hou et al, 2003;Cortesio et al, 2010). Although mAbp1-knockout mice are viable, they lack motor coordination and display behavioral defects, which are in part due to aberrant synaptic vesicle recycling (Connert et al, 2006).…”

“…To our knowledge, no specific interacting proteins have been identified that bind to these residues. One attractive hypothesis is that mAbp1 regulates podosomes through its interaction with Rho GTPase regulatory proteins such as Fgd-1, a Cdc42 exchange factor (Hou et al, 2003). Expression of constitutively active Cdc42 (V12Cdc42) induces podosome formation and stimulates epithelial to mesenchymal transition of invasive cells (Bakin et al, 2000;Moreau et al, 2003).…”

FGD1 as a central regulator of extracellular matrix remodelling – lessons from faciogenital dysplasia

“…The PRD has potential binding sites for Src homology 3 (SH3)-, WW-and Ena/VASP-homology 1 (EVH1, also known as SPRE1)-domain-containing proteins and profilin. The only two known FGD1 interactors, however, are cortactin and mammalian actin-binding protein (mAbp1, also known as DBNL) (Hou et al, 2003). Obviously, the binding of FGD1 to other cytoskeletal or signal transduction protein ligands cannot be excluded at this time (Estrada et al, 2001).…”

“…Finally, FGD1 controls the export of cargo proteins from the Golgi complex through CDC42 activation (Egorov et al, 2009). The FGD1 SH3-binding domain binds directly to the SH3 domain of mAbp1 or cortactin (Hou et al, 2003), and FGD1-cortactin binding promotes CDC42-independent actin assembly by the Arp2/3 complex (Kim et al, 2004). Phosphorylation at serine 205 by glycogen synthase kinase 3-b (GSK3-b) mediates the degradation of FGD1 at the proteasome (Hayakawa et al, 2005).…”