CUL3-KBTBD6/KBTBD7 Ubiquitin Ligase Cooperates with GABARAP Proteins to Spatially Restrict TIAM1-RAC1 Signaling

Abstract

The small Rho GTPase RAC1 is an essential regulator of cellular signaling that controls actin rearrangements and cell motility. Here, we identify a novel CUL3 RING ubiquitin ligase complex, containing the substrate adaptors KBTBD6 and KBTBD7, that mediates ubiquitylation and proteasomal degradation of TIAM1, a RAC1-specific GEF. Increasing the abundance of TIAM1 by depletion of KBTBD6 and/or KBTBD7 leads to elevated RAC1 activity, changes in actin morphology, loss of focal adhesions, reduced proliferation, and enhanced invasion. KBTBD6 and KBTBD7 employ ATG8 family-interacting motifs to bind preferentially to GABARAP proteins. Surprisingly, ubiquitylation and degradation of TIAM1 by CUL3(KBTBD6/KBTBD7) depends on its binding to GABARAP proteins. Our study reveals that recruitment of CUL3(KBTBD6/KBTBD7) to GABARAP-containing vesicles regulates the abundance of membrane-associated TIAM1 and subsequently spatially restricted RAC1 signaling. Besides their role in autophagy and trafficking, we uncovered a previously unknown function of GABARAP proteins as membrane-localized signaling scaffolds.

TIAM1 Antagonizes TAZ/YAP Both in the Destruction Complex in the Cytoplasm and in the Nucleus to Inhibit Invasion of Intestinal Epithelial Cells

“…Previous work has shown that oncogenic RAS expression suppresses TIAM1 transcription (Zondag et al., 2000). Furthermore, we and others have identified E3 ubiquitin ligases targeting TIAM1 for degradation (Genau et al., 2015, Magliozzi et al., 2014, Vaughan et al., 2015, Zhu et al., 2014), which may be deregulated in intestinal carcinoma. Potentially, factors resulting in exclusion of TIAM1 from the nucleus might also result in downregulation of this critical suppressor function of TIAM1, and we are currently investigating the mechanisms regulating TIAM1 nuclear import and export.…”

Structural and Functional Analysis of a Novel Interaction Motif within UFM1-activating Enzyme 5 (UBA5) Required for Binding to Ubiquitin-like Proteins and Ufmylation

“…Preparation of Peptides and Proteins for Isothermal Titration Calorimetry (ITC), NMR, and X-ray Studies-Human LC3/ GABARAPs cloned under a modified Ub tag (21) were expressed and purified as described previously (20,22,23). UFM1 was cloned under a cleavable Ub19 leader (21) and expressed and purified in a similar manner.…”

FYCO1 Contains a C-terminally Extended, LC3A/B-preferring LC3-interacting Region (LIR) Motif Required for Efficient Maturation of Autophagosomes during Basal Autophagy

“…The N-terminal arms of the Atg8 molecules often engage in electrostatic interactions with acidic or phosphorylated residues preceding the aromatic residue of the LIR motif (5). The following structures of LIR peptides in complex with Atg8s have been reported: p62/SQSTM1-LC3B (11,12); yeast Atg19-Atg8 (12); ATG4B-LC3B (13); NBR1-GABARAP (14); NDP52-LC3C (15); Optineurin-LC3B (4); Bcl-2-GABARAP (16); ATG13-LC3A and ATG13-LC3C (17); ALFY-GABARAP (18); PLEKHM1-LC3B (19); and KBTBD6-GABARAP (20).…”