Proline-Rich Domain Plays a Crucial Role in Extracellular Stimuli-Responsive Translocation of a Cdc42 Guanine Nucleotide Exchange Factor, FGD1

Abstract

The Rho family small guanosine 5Ј-triphosphate (GTP)-binding proteins (G-proteins), consisting mainly of the Rho, Rac, and Cdc42 subfamilies, regulates various actin cytoskeleton-dependent cell functions, including cell shape change, cell migration, cell adhesion, and cytokinesis. [1][2][3] Like all G-proteins, Rho family proteins act as binary switches by cycling between an inactive (GDP-bound) and an active (GTP-bound) conformation state.Guanine nucleotide exchange factors (GEFs) stimulate the exchange of GDP for GTP to generate active forms of Rho proteins. A family of candidate GEFs for Rho proteins has been identified based on the homology with CDC24, a protein genetically as an upstream activator of CDC42 in yeast. 4)Dbl, isolated as an oncogene in a focus formation assay using NIH 3T3 cells transfected with DNA from a human diffuse B-cell lymphoma, was identified as the first mammalian Rho GEF showing significant sequence similarity to CDC24. 5)The domain conserved in both Dbl and CDC24 is now recognized as the Dbl homology (DH) domain and almost 60 DHdomain-containing proteins have been found.6) The activities of GEFs must be tightly regulated and each member of the GEF family is likely to have a unique mechanism for activation and inactivation.In our previous reports, we revealed that FGD1, a GEF for Cdc42 and known as the faciogenital dysplasia (or AarskogScott syndrome; AAS) gene product, and FGD3, a homologue of FGD1, underwent proteasomal degradation through the polyubiquitination by the ubiquitin ligase SCF FWD1 . 7,8) FGD3 contains adjacent DH and pleckstrin homology (PH) domains, both of which are highly homologous to those of FGD1. Pasteris et al. reported that DHϩPH domains of FGD3 induced long finger-like protrusions, filopodia, as DHϩPH domains of FGD1 did, suggesting that both FGD1 and FGD3 share the similar GEF activity to induce Cdc42 activation.9) However, we demonstrated that the inducible expression of the full-length FGD1 led to the formation of striking filopodia structures whereas that of full-length FGD3 induced broad sheet-like protrusions, i.e., remarkable lamellipodia structures.8) These results indicate that FGD1 and FGD3 play different roles in cells but undergo the same destruction pathway.Although higher sequence similarity between FGD1 and FGD3 is observed in their DH and adjacent PH domains (i.e., amino acid residue identity is 70.0% and 60.6%, respectively), 9) there exist striking differences in their sequence in other regions; for example, most prominently, FGD3 lacks the N-terminal proline-rich domain conserved in FGD1.Proline-rich sequences are known to bind to specific protein interaction modules such as Src homology 3 (SH3), WW, and Ena/VASP homology 1 (EVH1) domains. [10][11][12] These different modules recognize different types of prolinerich sequences and play important roles in various signal transduction pathways. Phenotypic and molecular analyses revealed that a patient with missense mutation occurred in the proline-rich domain in FGD1 showed typical clinical f...

Role of FGD1, a Cdc42 Guanine Nucleotide Exchange Factor, in Epidermal Growth Factor-Stimulated c-Jun NH2-Terminal Kinase Activation and Cell Migration

“…14) During the wound-healing process, translocation of FGD1 to the leading edge membrane was observed in cells facing to the wound, 14) suggesting that the polarized intracellular signaling is triggered by FGD1 that translocates to the leading edge membrane. We previously established the HeLa Tet-Offderived stable cell lines that inducibly overexpress FGD1(SA), FGD3(SA), or FGD1(SAϩS 205 /I), respectively.…”

“…We previously established the HeLa Tet-Offderived stable cell lines that inducibly overexpress FGD1(SA), FGD3(SA), or FGD1(SAϩS 205 /I), respectively. 10,14) FGD1(SA) and FGD3(SA) bear Ser to Ala amino acid substitution within the SCF FWD1 putative consensus binding site. 10) In addition, a Ser to Ile substitution is made at position 205 in FGD1(SAϩS 205 /I).…”

“…10,14) To induce FGD1(SA), FGD3(SA) or FGD1(SAϩS 205 /I), cells were trypsinized and then cultured in the absence of Tc for 72 h.…”

“…[11][12][13] We have recently revealed that FGD1 translocated to the membrane fraction in response to epidermal growth factor (EGF) whereas FGD3 lacking the proline-rich domain did not. 14) Furthermore, FGD1 with a missense mutation in the proline-rich domain [FGD1(S 205 /I)] also failed to translocate to the membrane in response to extracellular stimuli. 14) Since a patient with same missense mutation in FGD1 showed typical clinical features as AAS, 8) the prolinerich domain may play a crucial role in the temporally and spatially regulated activation of FGD1.…”

“…14) Furthermore, FGD1 with a missense mutation in the proline-rich domain [FGD1(S 205 /I)] also failed to translocate to the membrane in response to extracellular stimuli. 14) Since a patient with same missense mutation in FGD1 showed typical clinical features as AAS, 8) the prolinerich domain may play a crucial role in the temporally and spatially regulated activation of FGD1.…”