Asef2 and Neurabin2 cooperatively regulate actin cytoskeletal organization and are involved in HGF-induced cell migration

The tumor suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumors. APC interacts with the Rac1- and Cdc42-specific guanine-nucleotide exchange factors (GEF), Asef and Asef2, which contain an APC-binding region (ABR) in addition to Dbl homology, Pleckstrin homology (PH) and Src homology 3 (SH3) domains. APC stimulates the GEF activity of Asef and Asef2, and thereby regulates cell adhesion and migration. Here we show that Asef2, but not Asef, interacts with Neurabin2/Spinophilin, a scaffold protein that binds to Filamentous actin (F-actin). In response to hepatocyte growth factor (HGF) treatment of HeLa cells, Asef2, Neurabin2 and APC were induced to accumulate and colocalize in lamellipodia and membrane ruffles. Neurabin2 did not affect the GEF activity of Asef2. RNA interference experiments showed that Asef2, Neurabin2 and APC are involved in HGF-induced cell migration. Furthermore, knockdown of Neurabin2 resulted in the suppression of Asef2-induced filopodia formation. These results suggest that Asef2, Neurabin2 and APC cooperatively regulate actin cytoskeletal organization and are required for HGF-induced cell migration.

[1]  P. Comoglio,et al.  Invasive growth: a MET-driven genetic programme for cancer and stem cells , 2006, Nature Reviews Cancer.

[2]  T. Stossel,et al.  The Rac1- and RhoG-specific GEF domain of Trio targets filamin to remodel cytoskeletal actin , 2000, Nature Cell Biology.

[3]  P. Greengard,et al.  Spinophilin regulates the formation and function of dendritic spines. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[4]  K. Kinzler,et al.  Asef , a Link Between the Tumor Suppressor APC and G-Protein Signaling , 2022 .

[5]  Y. Kawasaki,et al.  Identification and characterization of Asef2, a guanine–nucleotide exchange factor specific for Rac1 and Cdc42 , 2007, Oncogene.

[6]  P. Polakis Wnt signaling and cancer. , 2000, Genes & development.

[7]  T. Akiyama,et al.  Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration. , 2004, Developmental cell.

[8]  V. Ladevèze,et al.  Spinophilin: from partners to functions. , 2006, Biochimie.

[9]  K. Kinzler,et al.  Lessons from Hereditary Colorectal Cancer , 1996, Cell.

[10]  R. Nusse,et al.  Wnt signaling: a common theme in animal development. , 1997, Genes & development.

[11]  Tohru Natsume,et al.  A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics. , 2002, Analytical chemistry.

[12]  Y. Kawasaki,et al.  Crystal Structure of the Rac Activator, Asef, Reveals Its Autoinhibitory Mechanism* , 2007, Journal of Biological Chemistry.

[13]  Tetsu Akiyama,et al.  Mutated APC and Asef are involved in the migration of colorectal tumour cells , 2003, Nature Cell Biology.

[14]  A. Hall,et al.  Cdc42 regulates GSK-3β and adenomatous polyposis coli to control cell polarity , 2003, Nature.

[15]  R. Buchsbaum,et al.  Regulation of p70 S6 Kinase by Complex Formation between the Rac Guanine Nucleotide Exchange Factor (Rac-GEF) Tiam1 and the Scaffold Spinophilin* , 2003, Journal of Biological Chemistry.

[16]  H. Clevers,et al.  APC, Signal transduction and genetic instability in colorectal cancer , 2001, Nature Reviews Cancer.

[17]  Tetsu Akiyama,et al.  Identification of a link between the tumour suppressor APC and the kinesin superfamily , 2002, Nature Cell Biology.

[18]  Y. Kawasaki,et al.  Wnt signalling and the actin cytoskeleton , 2006, Oncogene.

[19]  T. Akiyama,et al.  Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF , 2008, Journal of Cell Science.

[20]  Napoleone Ferrara,et al.  Angiogenesis as a therapeutic target , 2005, Nature.

[21]  Angus C Nairn,et al.  Phosphorylation of spinophilin by ERK and cyclin-dependent PK 5 (Cdk5). , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[22]  P. Greengard,et al.  The Rho-Specific GEF Lfc Interacts with Neurabin and Spinophilin to Regulate Dendritic Spine Morphology , 2005, Neuron.

[23]  W. Birchmeier,et al.  Met, metastasis, motility and more , 2003, Nature Reviews Molecular Cell Biology.

[24]  H. Clevers,et al.  Linking Colorectal Cancer to Wnt Signaling , 2000, Cell.

[25]  Gordon C Jayson,et al.  Angiogenesis as a therapeutic target in cancer. , 2008, Clinical medicine.

[26]  D. Billadeau,et al.  Asef2 Functions as a Cdc42 Exchange Factor and Is Stimulated by the Release of an Autoinhibitory Module from a Concealed C-Terminal Activation Element , 2006, Molecular and Cellular Biology.

[27]  R. Brent,et al.  APC binds to the novel protein EB1. , 1995, Cancer research.

[28]  P. Greengard,et al.  Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[29]  W. Peti,et al.  Structure–function analysis of the filamentous actin binding domain of the neuronal scaffolding protein spinophilin , 2008, The FEBS journal.

[30]  Y. Hata,et al.  Neurabin-II/Spinophilin , 1998, The Journal of Biological Chemistry.

[31]  C. Der,et al.  Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression , 2007, Nature Structural &Molecular Biology.