SHORT COMMUNICATION The tumour suppressor HACE1 controls cell migration by regulating Rac1 degradation S Castillo-Lluva 1 , C-T Tan 1 , M Daugaard 2 , PHB Sorensen 2 and A Malliri 1 The small GTPase Rac1 is a key regulator of cell motility. Multiple mechanisms regulate Rac1 activity including its ubiquitylation and subsequent degradation. Here, we identify the tumour suppressor HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) as an E3 ubiquitin ligase responsible for Rac1 degradation following activation by a migration stimulus. We show that HACE1 and Rac1 interaction is enhanced by hepatocyte growth factor (HGF) signalling, a Rac activator and potent stimulus of cell migration. Furthermore, HACE1 catalyses the poly-ubiquitylation of Rac1 at lysine 147 following its activation by HGF, resulting in its proteasomal degradation. This negative feedback mechanism likely restricts cell motility. Consistent with this, HACE1 depletion is accompanied by increased total Rac1 levels and accumulation of Rac1 in membrane ruffles. Moreover, HACE1- depletion enhances cell migration independently of growth factor stimulation, which may have significance for malignant conversion. A non-ubiquitylatable Rac1 rescues the migration defect of Rac1-null cells to a greater extent than wild-type Rac1. These findings identify HACE1 as an antagonist of cell migration through its ability to degrade active Rac1. Oncogene (2013) 32, 1735–1742; doi:10.1038/onc.2012.189; published online 21 May 2012 Keywords: HACE1; Rac1; ubiquitylation; migration; HGF INTRODUCTION The Rho-family GTPase Rac1 cycles between a GDP- and a GTP- bound state. When GTP-bound, it interacts with various molecules that elicit downstream responses. 1 Multiple mechanisms control Rac1 activity including nucleotide binding and hydrolysis catalysed by guanine nucleotide exchange factors and GTPase activating proteins (GAPs), regulation of subcellular localization, modulation of protein levels and post-translational modifications such as prenylation 2 as well as ubiquitin-like (Ubl)-type modifications, including ubiquitylation 3–5 and SUMOylation. 6 Rac1 degradation by the ubiquitin–proteasome system occurs early during hepatocyte growth factor (HGF)-induced cell scattering 4 and in response to cytotoxic necrotizing factor-1 (CNF-1), 7 both of which activate Rac1. Rac1 ubiquitylation at Lys147 is also stimulated by downstream signalling through JNK, creating a negative feedback loop to terminate Rac1 signalling. 3 More recently, it was shown that ubiquitylation and subsequent degradation of active Rac1 is regulated by caveolin1. 5 Together, these studies imply a mechanism for terminating Rac1 signalling downstream of multiple stimuli. Here, we identify the tumour suppressor HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) as a Rac1-binding protein whose interaction is enhanced by HGF stimulation. Recently, this ligase was also shown to ubiquitylate Rac1 following CNF-1 stimulation. 8 We find that HACE1 antagonizes cell migration through its ability to poly-ubiquitylate active Rac1 at Lys147 triggering degradation. Significantly, HACE1-depletion enhances cell migration independently of growth factor stimulation, which may have significance for malignant conversion. RESULTS AND DISCUSSION Identification of HACE1 as an E3 ubiquitin ligase for Rac1 following a migration stimulus To identify novel regulators of Rac1 activity during cell migration, we previously performed tandem affinity purification of protein complexes from MDCKII cells expressing tandem affinity purifica- tion-tagged Rac1 following HGF treatment. This identified proteins interacting with Rac1 not only in the presence of HGF 6 but also proteins whose interaction with Rac1 appeared enriched in response to HGF (Supplementary Table I). Amongst putative Rac1-binding proteins, we identified HACE1, which had previously been identified as an E3 ubiquitin ligase deleted in sporadic Wilms’ tumours. 9 HACE1 interacts with Rac1 in cells (Figure 1a and Supplementary Figures S1A, B) and this interaction was more efficient when Rac1 was in its active form (bound to GTP, Figure 1b). In addition, activation of the HGF-signalling pathway using an oncogenic form of the Met receptor (Tpr-Met), that does not require addition of HGF for activation, 10,11 increased this interaction (Supplementary Figure S1C). Furthermore, the inter- action between Rac1 and HACE1 was direct (Figure 1c). HACE1 comprises a 103-kDa protein containing six N-terminal ankyrin repeats connected via a linker region to a C-terminal HECT domain (Figure 1d). Rac1 was unable to interact with HACE1-lacking ankyrin repeats (Figure 1e). To determine whether HACE1 can act as an E3 ubiquitin ligase for Rac1 in cells we performed ubiquitylation experiments in HEK293T cells. Previous studies have shown that the active form of Rac1 is predisposed to ubiquitin-dependent degradation. 5,12 Co-expression of Rac1V12 (active form of Rac1) and HACE1 promoted the poly-ubiquitylation of Rac1 but the ligase dead 1 Cell Signalling Group, Cancer Research UK Paterson Institute for Cancer Research, The University of Manchester, Manchester, UK and 2 Department of Molecular Oncology, BC Cancer Research Centre and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4. Correspondence: Dr A Malliri, Cell Signalling Group, Cancer Research UK Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. E-mail: amalliri@picr.man.ac.uk Received 12 December 2011; revised 12 April 2012; accepted 13 April 2012; published online 21 May 2012 Oncogene (2013) 32, 1735–1742 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc