GABARAP proteins as scaffolds in localized TIAM1-RAC1 signaling

Abstract

Spatially restricted signaling is a hallmark of RAC1 signaling. Recent work has uncovered a novel role of gamma-aminobutyric acid receptor-associated proteins (GABARAPs), a subfamily of human ATG8 ubiquitin-like modifiers, in providing a scaffold for recruitment of an ubiquitin E3 ligase complex to its substrate, T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1), to enable ubiquitylation and thereby local control of RAC1 activity.

Local control of RAC1 activity is essential to allow a specific cellular response to stimuli such as growth factors or migratory impulses. Whereas GTPase-activating proteins (GAPs) render RAC1 inactive by facilitating GTP hydrolysis, guanine exchange factors (GEFs) such as T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1) activate RAC1 by stimulating the exchange of GDP with GTP. In contrast to the localized membrane-connected activation of RAC1 very little is known about the spatial restriction of TIAM1 activity. Our recent work further supports the concept of local restricted regulation of RAC1 activity and expands the function of gamma-aminobutyric acid receptor-associated proteins (GABARAPs) from autophagy to TIAM1-RAC1 signaling.¹ By establishing Kelch repeat and BTB domain-containing proteins 6 (KBTBD6) and 7 (KBTBD7), hereafter referred to as KBTBD6/7, as heterodimeric substrate adaptors for cullin-3 (CUL3), we characterized a new member of the family of cullin RING ubiquitin E3 ligases that critically control various cellular processes such as oxidative stress response, DNA repair, and cell cycle progression (reviewed in reference 2). Interestingly, out of 11 substrate candidates tested after proteomic analysis, only TIAM1 appeared to be ubiquitylated by CUL3^KBTBD6/7 at 2 distinct lysines (K1404 and K1420) close to its C-terminus. We confirmed the association of KBTBD6/7 with human ATG8 proteins and found that KBTBD6/7 employed a classic ATG8-interacting motif (AIM; also referred to as LC3-interacting region or LIR) as the binding interface. Structural and cell biological analysis revealed a preference for the GABARAP subfamily of human ATG8 proteins and, most importantly, a requirement of the GABARAP-KBTBD6/7 interaction for TIAM1 ubiquitylation. The finding that binding of TIAM1 to KBTBD6/7 AIM mutants was diminished raised the possibility that GABARAP binding mediates the recruitment of CUL3^KBTBD6/7 to membranes where TIAM1 is localized. Interestingly, TIAM1 ubiquitylation was reduced when GABARAP was depleted or when lipidation of GABARAP was prevented. Stabilization of TIAM1 upon KBTBD6/7 depletion led to elevated TIAM1-dependent RAC1 activity, altered actin morphology, and loss of vinculin foci. Re-introduction of wild-type KBTBD6/7, but not AIM mutants, reverted these phenotypes. Intriguingly, KBTBD6 and KBTBD7 were not subject to autophagosomal degradation thereby establishing a new function for GABARAP proteins beyond autophagy in providing a signaling platform for recruitment of the E3 ligase CUL3^KBTBD6/7 in close proximity to its substrate TIAM1 (Fig. 1).

To date, the best understood function of ATG8 proteins is their role during autophagy, where they act as docking sites for components of the autophagy machinery and modulators of autophagosome biogenesis. Despite their high similarity it is currently unclear to what extent human ATG8 proteins are functionally redundant or have unique roles. The subfamily of LC3 proteins has been demonstrated to act early in phagophore expansion whereas GABARAP proteins are implicated later in autophagosome closure and maturation.³ In our recent study we uncovered a non-autophagic function of the GABARAP subfamily as a signaling platform enabling the localized ubiquitylation of TIAM1 that is different from the known functions of the LC3 subfamily.

Local restricted control of RAC1 activity has previously been described for TIAM1-RAC1 signaling. Examples are HECT, UBA, and WWE domain-containing protein 1 (HUWE1)-mediated TIAM1 ubiquitylation that occurs predominantly at cell–cell junctions in response to hepatocyte growth factor stimulation in MDCKII cells⁴ or inhibition of RAC1 activity by the RAC1-GAP protein breakpoint cluster region (BCR) at the leading edge of astrocytes through binding to the TIAM1-Par (polarity) complex.⁵ SCF (SKP1-CUL1-F-box protein)^BTRC mediates ubiquitylation of TIAM1 in response to mitogens or DNA damage,^6,7 although whether this regulation is spatially restricted has not been explored. Thus, our study adds a novel layer of complexity to the local regulation of RAC1 signaling by implicating membrane-bound human ATG8 proteins in this process.

The crystal structure of GABARAP with bound KBTBD6 AIM peptide revealed a hydrogen bond between R670 in the AIM motif of KBTBD6 and Y25 of GABARAP.¹ Intriguingly, R670 was found to be mutated in human colon tumors. Based on our results we would predict that a reduction in KBTBD6–GABARAP association due to the R670 mutation is most probably linked to elevated TIAM1 levels and thereby RAC1 activity.⁸ Moreover, phosphorylation of serine residues preceding the AIM of the autophagy receptor optineurin (OPTN) was reported to promote selective autophagy of Salmonella enterica.⁹ The fact that KBTBD6/7 harbor several serine residues proximal to their AIMs makes phosphorylation of these residues a good candidate for differential regulation of KBTBD6/7 activity and/or their association with GABARAP.

In the context of localized signaling mediated by GABARAP proteins it will be interesting to explore whether this concept applies to other substrates of CUL3^KBTBD6/7 and signaling processes in which GABARAP proteins are involved. Controlling RAC1 activity at GABARAP-decorated membranes might also be important for trafficking events or autophagy as it was reported that RAC1 has an inhibitory function on autophagy.¹⁰ Therefore, spatially restricted ubiquitylation of TIAM1 resulting in specific deactivation of RAC1 could promote the autophagic process when locally needed. Although the catalytic mTOR inhibitor Torin1 and the lysosomal H⁺ ATPase inhibitor bafilomycinA1 promote TIAM1 ubiquitylation by increasing the pool of membrane-conjugated GABARAP,¹ other signals that stimulate GABARAP-KBTBD6/7 association and subsequent TIAM1 ubiquitylation remain to be identified. Additionally, determining the KBTBD6/7 binding site in TIAM1 or uncovering a deubiquitination enzyme (DUB) that locally counteracts the ubiquitylation of TIAM1 will enable better comprehension of the complete localized signaling cascade.

Taken together, results of our recent study establish a novel function for GABARAP in localized control of TIAM1-RAC1 signaling. Given that stabilization of TIAM1 by depletion of KBTBD6/7 increased invasiveness of the metastatic human breast cancer cell line MDA-MB-231 and reduced cell–cell adhesions, it will be of great importance to explore the function of KBTBD6/7 in epithelial-to-mesenchymal-transition or tumor formation. Concurrently, KBTBD6/7 were found to be deleted or mutated in several types of malignancy such as prostate cancer and leukemias. Since association of KBTBD6/7 with GABARAPs occurs via 2 hydrophobic pockets of GABARAP and this interaction is required for local TIAM1 ubiquitylation,¹ further research will be required to clarify whether this binding can be stabilized or disturbed by small molecules to respectively decrease or enhance TIAM1-mediated RAC1 activation.

Figure 1.

GABARAP coordinates localized TIAM1-RAC1 signaling through recruitment of CUL3^KBTBD6/7. To locally inactivate RAC1, CUL3^KBTBD6/7 is recruited to GABARAP-decorated membranes through its ATG8-interacting motif (AIM). Thereby, the E3 ligase is brought into close proximity of its substrate, the RAC1-GEF TIAM1, which binds PIP-containing membranes through its PH domain. This enables local ubiquitylation of TIAM1. As a consequence, ubiquitylated TIAM1 is degraded by the proteasome and RAC1 activity is locally reduced.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgements

H.G. and C.B. want to thank all authors of the original study and everybody who helped throughout the time of research by providing reagents, sharing unpublished data, and discussions.