Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression

Abstract

Autoinhibition of the Rho guanine nucleotide exchange factor ASEF is relieved by interaction with the APC tumor suppressor. Here we show that binding of the armadillo repeats of APC to a 'core APC-binding' (CAB) motif within ASEF, or truncation of the SH3 domain of ASEF, relieves autoinhibition, allowing the specific activation of CDC42. Structural determination of autoinhibited ASEF reveals that the SH3 domain forms an extensive interface with the catalytic DH and PH domains to obstruct binding and activation of CDC42, and the CAB motif is positioned adjacent to the SH3 domain to facilitate activation by APC. In colorectal cancer cell lines, full-length, but not truncated, APC activates CDC42 in an ASEF-dependent manner to suppress anchorage-independent growth. We therefore propose a model in which ASEF acts as a tumor suppressor when activated by APC and inactivation of ASEF by mutation or APC truncation promotes tumorigenesis.The adenomatous polyposis coli (APC) protein is a negative regulator of the Wnt signaling pathway and promotes the phosphorylation and degradation of β-catenin 1 . The majority of colorectal cancers (CRCs) harbor C-terminally truncated forms of APC that retain the Nterminal coiled-coil domain and the highly conserved armadillo repeat region (APC Arm ) 2 and are unable to regulate the degradation of β-catenin 3 . Recent studies suggest that APC regulates cytoskeletal dynamics to influence cellular migration, cell division, polarization and adhesion, and it seems increasingly likely that deregulated cytoskeletal dynamics, together with enhancement of transcription by β-catenin, potentiate tumor formation and progression upon APC truncation. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAPC may regulate cytoskeletal networks by binding to microtubules and to proteins implicated in reorganization of the actin cytoskeleton, such as the Rho effectors mDia and IQGAP1 and the Rho guanine nucleotide exchange factor (RhoGEF) 'APC-stimulated guanine nucleotide exchange factor' (ASEF, also called ARHGEF4) 4 . ASEF is a Dbl-family GEF that contains an Src-homology-3 (SH3) domain followed by the Dbl-homology (DH) and pleckstrinhomology (PH) domains characteristic of Dbl-family GEFs that specifically activate members of the Rho family of GTPases. DH and PH domains in Dbl proteins catalyze the exchange of GDP for GTP in Rho GTPases, allowing them to signal to downstream effectors 5 . ASEF is homologous to the Dbl proteins collybistin (also called PEM2) and SPATA13 (also called ASEF2), both of which have been shown to activate specifically CDC42 and not other Rhofamily GTPases 6,7 .Previous data suggest that ASEF exists in an autoinhibited form and is activated upon binding of APC Arm to the region termed the APC-binding region (ABR) 8 . Binding of APC Arm to the ABR stimulates activation of the Rho GTPase RAC1 by ASEF. Furthermore, truncation of the ABR is sufficient to relieve autoinhibition, rendering ASEF constitutively active. In addition, truncated, but not wild-...

Identification of a Negative Regulatory Region for the Exchange Activity and Characterization of T332I Mutant of Rho Guanine Nucleotide Exchange Factor 10 (ARHGEF10)

“…GEF activity of several RhoGEFs of the Dbl type is regulated by the intramolecular autoinhibition. For example, Vav, Dbl, Asef, Tim, and PDZRhoGEF are negatively regulated by their own N-terminal regions (21)(22)(23)(24)(25)(26). Our results demonstrated that the region containing amino acids 212-332 of ARHGEF10 is a negatively regulatory region for its GEF activity.…”

The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis

“…APC enhances their GEF activity and thereby regulates cell morphology, adhesion, and migration. In the absence of APC, the activity of Asef is inhibited by the intramolecular interaction between its Dbl homology (DH) and Src homology 3 (SH3) domains (17,18). However, a mutant form of Asef lacking the NH 2 -terminal region exhibits strong GEF activity even in the absence of APC.…”

Structural basis for the recognition of Asef by adenomatous polyposis coli

“…The crystal structures of Asef by itself show that its SH3 domain interacts with its DH and PH domains, obstructs the binding of Cdc42 to the DH domain, and autoinhibits its GEF activity [22,24]. On binding to APC, the autoinhibition of Asef is released and the GEF activity of Asef is stimulated.…”

“…In addition, the dissociation constant (K d ) between WT APC-PreARM-ARM and WT Asef-ABR-SH3 proteins was measured to be 17.8 nM by the ITC assay ( Figure 5A, Table 2 Asef-Ala186 and Asef-Asn184, respectively, which are in the center of the core APC-binding motif of Asef-ABR [22]. APC-Asn507 also makes van der Waals contacts with Asef-Leu185 ( Figure 4A).…”

“…On binding to APC, the autoinhibition of Asef is released and the GEF activity of Asef is stimulated. The activated Asef catalyzes the exchange of GDP for GTP in Cdc42, decreases cell-cell adhesion, and promotes cell migration [17,[20][21][22]. In colorectal cancers, truncated APC constitutively activates Asef and Cdc42, which upregulate the expression of matrix metalloproteinase 9 (MMP9) via the c-Jun N-terminal kinase (JNK) pathway, thus promotes cancercell migration and angiogenesis [25][26][27].…”