Degradation of RhoA by Smurf1 Ubiquitin Ligase

Abstract

The Rho family of small GTPases plays a key role in the dynamic regulation of the actin cytoskeleton that underlies various important cellular functions such as shape changes, migration, and polarity. We found that Smurf1, a HECT domain E3 ubiquitin ligase, could specifically target RhoA but not Cdc42 or Rac1 for degradation. Smurf1 interacts with the dominant inactive form of RhoA, RhoA N19, which binds constitutively to guanine nucleotide exchange factors (GEFs) in vivo. Smurf1 also interacts directly with either nucleotide‐free or GDP‐bound RhoA in vitro; however, loading with GTPγS inhibits the interaction. RhoA is ubiquitinated by wild‐type Smurf1 but not the catalytic mutant of Smurf1 (C699A) in vivo and in vitro, indicating that RhoA is a direct substrate of Smurf1. In this chapter, we summarize the systems and methods used in the analyses of Smurf1‐regulated RhoA ubiquitination and degradation.

Introduction

The Rho family of small GTPases is a subset of the Ras superfamily and is an important regulator of the cytoskeletal dynamics that control cell shape, motility, and polarity (Bar‐Sagi 2000, Bishop 2000, Etienne‐Manneville 2002, Hall 2000, Van Aelst 2002). The activity of Rho family members is regulated by their nucleotide‐bound state, cycling between an active GTP‐bound form and an inactive GDP‐bound form. This cycling is tightly controlled by associated cofactors such as GTPase activating proteins (GAPs), which stimulate the intrinsic GTPase activity to convert bound GTP to GDP and guanine nucleotide exchange factors (GEFs), which stimulate exchange of GDP for GTP. Current evidence suggests that GEF‐dependent nucleotide exchange is the key control point for regulating the biological function of GTPases (Bar‐Sagi 2000, Bishop 2000, Hall 2000, Van Aelst 2002).

Ubiquitin‐dependent proteolysis is a key regulatory mechanism that controls the degradation of intracellular and membrane proteins that have been tagged by ubiquitin for degradation by the proteasome or lysosome, respectively (Hershko and Ciechanover, 1998). Conjugation of ubiquitin to protein targets is mediated by an enzymatic cascade. An E1 ubiquitin–activating enzyme that transfers ubiquitin to an E2 ubiquitin–conjugating enzyme, which can then either conjugate ubiquitin directly onto protein targets using a set of substrate specific adaptors, or transfer ubiquitin to E3 ligases, which in turn transfer ubiquitin to the substrate.

The Smurfs belong to the HECT family ubiquitin ligases, which contain C2 and WW domains as well as a conserved C‐terminal HECT domain (Harvey and Kumar, 1999). The C2 domain can mediate interactions with membrane lipids and proteins, whereas the WW domains bind proline‐tyrosine (PY) motifs. The Smurf C2‐WW‐HECT domain ubiquitin ligases have been shown to regulate TGFβ signal transduction by targeting Smad signaling molecules for degradation (Lin 2000, Zhang 2001, Zhu 1999). In addition, Smurfs are recruited by Smads to other non‐PY containing substrates including TGFβ ser/thr kinase receptors (Ebisawa 2001, Kavsak 2000), as well as the nuclear oncoprotein SnoN (Bonni et al., 2001). In our recent study, we found that Smurf1 also controls RhoA levels at active cellular protrusions through ubiquitin‐mediated degradation (Wang et al., 2003). Our findings highlight an unexpected mechanism regulating RhoA activity by the ubiquitin‐dependent pathway.