Barry Kreutz

Regulation of RGS-RhoGEFs by Gα12 and Gα13 Proteins

Abstract Three mammalian Rho guanine nucleotide exchange factors (RhoGEFs), leukemia-associated RhoGEF (LARG), p115RhoGEF, and PDZ-RhoGEF, contain regulator of G-protein signaling (RGS) domains within their amino-terminal regions. These RhoGEFs link signals from heterotrimeric G12/13 protein-coupled receptors to Rho GTPase activation, leading to various cellular responses, such as actin reorganization and gene expression. The activity of these RhoGEFs is regulated by Gα12/13 through their RGS domains. Because RhoGEFs stimulate guanine nucleotide exchange by Rho GTPases, RhoGEF activation can be measured by monitoring GTP binding to or GDP dissociation from Rho GTPases. This article describes methods used to perform reconstitution assays to measure the activity of RhoGEFs regulated by Gα12/13.

VE-Cadherin Signaling Induces EB3 Phosphorylation to Suppress Microtubule Growth and Assemble Adherens Junctions

Vascular endothelial (VE)-cadherin homophilic adhesion controls endothelial barrier permeability through assembly of adherens junctions (AJs). We observed that loss of VE-cadherin-mediated adhesion induced the activation of Src and phospholipase C (PLC)γ2, which mediated Ca(2+) release from endoplasmic reticulum (ER) stores, resulting in activation of calcineurin (CaN), a Ca(2+)-dependent phosphatase. Downregulation of CaN activity induced phosphorylation of serine 162 in end binding (EB) protein 3. This phospho-switch was required to destabilize the EB3 dimer, suppress microtubule (MT) growth, and assemble AJs. The phospho-defective S162A EB3 mutant, in contrast, induced MT growth in confluent endothelial monolayers and disassembled AJs. Thus, VE-cadherin outside-in signaling regulates cytosolic Ca(2+) homeostasis and EB3 phosphorylation, which are required for assembly of AJs. These results identify a pivotal function of VE-cadherin homophilic interaction in modulating endothelial barrier through the tuning of MT dynamics.

The interaction of Gα13 with integrin β1 mediates cell migration by dynamic regulation of RhoA

Gα13 directly binds to the cytoplasmic-domain ExE motif of the integrin β1 subunit. Gα13–β1 interaction mediates β1 integrin–dependent Src activation and transient RhoA inhibition after adhesion. This binding is critical for cell migration on β1 integrin ligands.

Different Raf Protein Kinases Mediate Different Signaling Pathways to Stimulate E3 Ligase RFFL Gene Expression in Cell Migration Regulation

Background: Cell migration requires persistent PKC phosphorylation, which can be achieved through the RFFL-mTORC2 pathway. Results: We show how Gα12 specifically activates ARAF to stimulate RFFL expression, which can also be stimulated by EGF, via CRAF, and activated BRAF. Conclusion: Different signaling pathways, through different Raf proteins, stimulate RFFL expression to support cell migration. Significance: The RFFL-PKC pathway has a broad significance in cell migration regulation. We previously characterized a Gα12-specific signaling pathway that stimulates the transcription of the E3 ligase RFFL via the protein kinase ARAF and ERK. This pathway leads to persistent PKC activation and is important for sustaining fibroblast migration. However, questions remain regarding how Gα12 specifically activates ARAF, which transcription factor is involved in Gα12-mediated RFFL expression, and whether RFFL is important for cell migration stimulated by other signaling mechanisms that can activate ERK. In this study, we show that replacement of the Gα12 residue Arg-264 with Gln, which is the corresponding Gα13 residue, abrogates the ability of Gα12 to interact with or activate ARAF. We also show that Gα12 can no longer interact with and activate an ARAF mutant with its C-terminal sequence downstream of the kinase domain being replaced with the corresponding CRAF sequence. These results explain why Gα12, but not Gα13, specifically activates ARAF but not CRAF. Together with our finding that recombinant Gα12 is sufficient for stimulating the kinase activity of ARAF, this study reveals an ARAF activation mechanism that is different from that of CRAF. In addition, we show that this Gα12-ARAF-ERK pathway stimulates RFFL transcription through the transcription factor c-Myc. We further demonstrate that EGF, which signals through CRAF, and an activated BRAF mutant also activate PKC and stimulate cell migration through up-regulating RFFL expression. Thus, RFFL-mediated PKC activation has a broad significance in cell migration regulation.

Signaling through Galpha12/13 and RGS-RhoGEFs

The transforming heterotrimeric G proteins Gα12 and Gα13 have been implicated in the regulation of various cellular processes, such as gene transcription and rearrangement of the actin-based cytoskeleton. Many of these functions are dependent on the activation of the monomeric GTPase RhoA. The RGS-RhoGEFs, a distinct family of guanine nucleotide exchange factors, which contain RGS-like domains in their N-termini, serve as direct links between Gα12/13 and RhoA. The focus of this chapter is on the molecular mechanisms that underlie and regulate signaling from Gα12/13 to RhoA via the RGS-RhoGEFs. The physiological significance of this signaling pathway is also discussed.