Stephen Gutowski

ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity

The hydrolysis of phosphatidylcholine by phospholipase D (PLD) results in the production of phosphatidic acid and choline. An assay that uses an exogenous substrate was developed to measure this activity in membranes and solubilized preparations from HL60 cells. A cytosolic factor markedly enhanced PLD activity in membranes and was essential for GTP gamma S-dependent stimulation of an enriched preparation of PLD. The factor was purified to homogeneity from bovine brain cytosol and identified as a member of the ADP-Ribosylation Factor (ARF) subfamily of small G proteins. Subsequently, recombinant myristoylated ARF1 was found to be a better activator of PLD activity than was the nonmyristoylated form. ARF proteins have been implicated recently as factors for regulation of intracellular vesicle traffic. The current finding suggests that PLD activity plays a prominent role in the action of ARF and that ARF may be a key component in the generation of second messengers via phospholipase D.

RhoA Binds to the Amino Terminus of MEKK1 and Regulates Its Kinase Activity

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that can regulate the c-Jun amino-terminal kinase (JNK) MAP kinase cascade. MEKK1 is comprised of a kinase domain and a long amino-terminal regulatory domain. This amino-terminal domain has a scaffold function in that it can assemble modules of the JNK and ERK MAP kinase cascades. Recently, we have demonstrated that MEKK1 binds to p115 Rho GTPase-activating protein, which has GTPase-activating protein activity toward RhoA. Thus, we tested whether Rho GTPases interact with the regulatory domain of MEKK1. RhoA, but not Rac or Cdc42, binds to a site in the aminoterminal one-third of MEKK1, which includes its PHD domain. The interaction is prevented by mutation of the essential cysteine in the MEKK1 PHD domain. Rho-GTP stimulates the kinase activity of full-length MEKK1 as much as 10-fold toward MEK4 but does not appear to be ubiquitinated by MEKK1 under conditions that result in modification of ERK2. In summary, we have characterized a novel point at which Rho GTPases impinge upon the regulation and function of MEKK1.

Bradykinin modulates potassium and calcium currents in neuroblastoma hybrid cells via different pertussis toxin-insensitive pathways

In NG108-15 cells, bradykinin (BK) activates a potassium current (IK,BK) and inhibits the voltage-dependent calcium current (ICa,V). BK also stimulates a phosphatidylinositol-specific phospholipase C (PI-PLC). The subsequent release of inositol 1,4,5-trisphosphate and increase in intracellular calcium contribute to IK,BK, through activation of a calcium-dependent potassium current. In membranes from these cells, stimulation of PI-PLC by BK is mediated by Gq and/or G11, two homologous, pertussis toxin-insensitive G proteins. Here, we have investigated the role of Gq/11 in the electrical responses to BK. GTP gamma S mimicked and occluded both actions of BK, and both effects were insensitive to pertussis toxin. Perfusion of an anti-Gq/11 alpha antibody into the pipette suppressed IK,BK, but not the inhibition of ICa,V by BK. Thus, BK couples to IK,BK via Gq/11, but coupling to ICa,V is most likely via a different, pertussis toxin-insensitive G protein.

The G protein G13 mediates inhibition of voltage-dependent calcium current by bradykinin

In neuroblastoma-glioma hybrid cells, bradykinin has dual modulatory effects on ion channels: it activates a K+ current as well as inhibits the voltage-dependent Ca2+ current (ICa,V). Both of these actions are mediated by pertussis toxin-insensitive G proteins. Antibodies raised against the homologous Gq and G11 proteins suppress only the activation of the K+ current; this suggested that at least two distinct G protein pathways transduce diverse effects of this transmitter. Here, we show that the inhibition of ICa,V by bradykinin is suppressed selectively by intracellular application of antibodies specific for G13. This novel G protein may play a general role in the inhibition of ICa,V by pathways resistant to pertussis toxin.

Activated RhoA Is a Positive Feedback Regulator of the Lbc Family of Rho Guanine Nucleotide Exchange Factor Proteins

Background: Activated RhoA binds to the PH domain of its own GEF, PDZRhoGEF. Results: Activated RhoA binds to all Lbc RhoGEFs and provides positive feedback regulation to these regulators. Conclusion: Allosteric binding of activated RhoA to its own regulators facilitates other regulatory stimuli. Significance: This autoregulatory mechanism could mediate robust localized signaling and may represent a general paradigm for regulation of other monomeric GTPases. The monomeric Rho GTPases are essential for cellular regulation including cell architecture and movement. A direct mechanism for hormonal regulation of the RhoA-type GTPases is their modulation by the G12 and G13 proteins via RH (RGS homology) containing RhoGEFs. In addition to the interaction of the G protein α subunits with the RH domain, activated RhoA also binds to the pleckstrin homology (PH) domain of PDZRhoGEF. The latter interaction is now extended to all seven members of the homologous Lbc family of RhoGEFs which includes the RH-RhoGEFs. This is evinced by direct measurements of binding or through effects on selected signaling pathways in cells. Overexpression of these PH domains alone can block RhoA-dependent signaling in cells to various extents. Whereas activated RhoA does not modulate the intrinsic activity of the RhoGEFs, activated RhoA associated with phospholipid vesicles can facilitate increased activity of soluble RhoGEFs on vesicle-delimited substrate (RhoA-GDP). This demonstrates feasibility of the hypothesis that binding of activated RhoA to the PH domains acts as a positive feedback mechanism. This is supported by cellular studies in which mutation of this binding site on PH strongly attenuates the stimulation of RhoA observed by overexpression of five of the RhoGEF DH-PH domains. This mutation is even more dramatic in the context of full-length p115RhoGEF. The utilization of this mechanism by multiple RhoGEFs suggests that this regulatory paradigm may be a common feature in the broader family of RhoGEFs.

Activation of p115-RhoGEF Requires Direct Association of Gα13 and the Dbl Homology Domain

Background: p115-RhoGEF can be regulated by activated Gα13. Results: Both RGS and DH domains of p115-RhoGEF interact with Gα13. Conclusion: The binding of RGS to Gα13 facilitates direct association of Gα13 to DH to regulate its exchange activity. Significance: RGS domains can act cooperatively with other domains to mediate effector regulation by G proteins. RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G12 class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated α subunits of G12 and G13. Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by Gα13, the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated Gα13 in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of Gα13 docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the α3b helix of DH reduces binding to activated Gα13 and ablates the stimulation of p115 by Gα13. Complementary mutations at the predicted DH-binding site in the αB-αC loop of the helical domain of Gα13 also affect stimulation of p115 by Gα13. Although the GAP activity of p115 is not required for stimulation by Gα13, two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of Gα13 to the RH domain facilitates direct association of Gα13 to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.

Functional characterization of p115 RhoGEF

The Rho family of monomeric GTPases plays a prominent role in the regulation of cell shape and movement. The activity of these proteins is dependent on a nucleotide cycle that is regulated by a combination of guanine nucleotide exchange factors (GEFs) that facilitate exchange of GTP for GDP on the Rho proteins, and GTPase-activating proteins (GAPs) that stimulate hydrolysis of GTP bound to Rho. The GEFs promote activation, that is, formation of the GTP-bound state, whereas proteins with GAP function serve to inactivate the GTPases. The heterotrimeric G proteins that mediate regulation by a variety of extracellular stimuli are also controlled by a GDP/GTP cycle. Integral membrane receptors for detection of hormones and other stimuli act as the GEFs to provide stimulatory inputs and a family of RGS (regulator of G protein signaling) proteins as GAPs that can effect either inhibitory or downstream regulation. The GEFs for the Rho family of GTPases compose a large and growing family of proteins characterized by conserved, tandem DH (Dbl homology) and PH (pleckstrin homology) domains. These exchange factors stimulate Rho proteins with various selectivities for three defined functional groups represented by RhoA, Racl, and Cdc42. p115 RhoGEF has been purified and cloned as a GEF that is selective for Rho. The subsequent identification of an RGS domain in the N terminus of p115, which has specificity for the G12 family of heterotrimeric G proteins, and the regulation of GEF activity by the activated Ga∼3 subunit, defines this protein as a direct link for coupling regulation between these two G protein pathways.

Assays and characterization of mammalian phosphatidylinositol 4,5-bisphosphate-sensitive phospholipase D.

Phospholipase D (PLD) hydrolyzes phospholipids into phosphatidic acid (PA) and the base head groups. In mammalian cells, this activity plays an active role in signal transduction by acting as a mediator for a variety of extracellular stimuli. Two mammalian PLD genes have been identified and the encoded enzymes expressed and characterized. The activity of PLD1 is regulated by the ADPribosylation factor (ARF) and Rho families of monomeric GTPases, classic isoforms of protein kinase C (PKC), and the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ). PLD2, although highly sensitive to PIP 2 has shown only modest response to ARF and appears unresponsive to Rho and PKC, in vitro . Characterization of PLD activity and identification of various regulatory molecules were greatly facilitated by the establishment of an assay for in vitro studies. Alternative methods for the assay of PLD described in this chapter facilitate measurement of basal activity and regulation in the absence of PIP 2 .

Regulated Localization Is Sufficient for Hormonal Control of Regulator of G Protein Signaling Homology Rho Guanine Nucleotide Exchange Factors (RH-RhoGEFs)

Background: Constitutive localization of RH-RhoGEFs to the plasma membrane elevates levels of active RhoA. Results: Acute localization of RH-RhoGEFs activates RhoA to extents that are comparable with those observed during hormone signaling. Conclusion: Regulated localization is a viable mechanism for controlling the activity of RhoGEFs. Significance: Multiple membrane-associated binding partners of RH-RhoGEFs could mediate robust localized signaling by anchoring the RhoGEFs in the proximity of their substrate. The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. The RH domains interact specifically with the α subunits of G12 heterotrimeric GTPases. Activated Gα13 modestly stimulates the exchange activity of both p115RhoGEF and leukemia-associated RhoGEF but not PDZ-RhoGEF. Because all three RH-RhoGEFs can localize to the plasma membrane upon expression of activated Gα13, cellular localization of these RhoGEFs has been proposed as a mechanism for controlling their activity. We use a small molecule-regulated heterodimerization system to rapidly control the localization of RH-RhoGEFs. Acute localization of the proteins to the plasma membrane activates RhoA within minutes and to levels that are comparable with activation of RhoA by hormonal stimulation of G protein-coupled receptors. The catalytic activity of membrane-localized RhoGEFs is not dependent on activated Gα13. We further show that the conserved RH domains can rewire two different RacGEFs to activate Rac1 in response to a traditional activator of RhoA. Thus, RH domains act as independent detectors for activated Gα13 and are sufficient to modulate the activity of RhoGEFs by hormones via mediating their localization to substrate, membrane-associated RhoA.

Secondary PDZ domain-binding site on class B plexins enhances the affinity for PDZ–RhoGEF

Significance Protein interactions mediated by modular domains, such as PDZ and SH2 domains, play critical roles in biology. The modules typically recognize a linear motif in their ligands, with a few residues in the motif determining the specificity. We report a crystal structure of the complex between the cytoplasmic region of PlexinB2 and the PDZ domain of PDZ–RhoGEF. The structure shows that, in addition to the PDZ/motif interaction, a secondary interface is formed between the three-dimensional domains of the two proteins. We further show that the secondary interface enhances the affinity between plexin and PDZ–RhoGEF and is important for plexin signaling. Our analyses suggest that secondary interface-mediated interactions may be a broadly used mechanism for modular domains to achieve high specificity. PDZ domains are abundant protein interaction modules and typically recognize a short motif at the C terminus of their ligands, with a few residues in the motif endowing the binding specificity. The sequence-based rules, however, cannot fully account for the specificity between the vast number of PDZ domains and ligands in the cell. Plexins are transmembrane receptors that regulate processes such as axon guidance and angiogenesis. Two related guanine nucleotide exchange factors (GEFs), PDZ–RhoGEF and leukemia-associated RhoGEF (LARG), use their PDZ domains to bind class B plexins and play critical roles in signaling. Here, we present the crystal structure of the full-length cytoplasmic region of PlexinB2 in complex with the PDZ domain of PDZ–RhoGEF. The structure reveals that, in addition to the canonical C-terminal motif/PDZ interaction, the 3D domain of PlexinB2 forms a secondary interface with the PDZ domain. Our biophysical and cell-based assays show that the secondary interface contributes to the specific interaction between plexin and PDZ–RhoGEF and to signaling by plexin in the cell. Formation of secondary interfaces may be a general mechanism for increasing affinity and specificity of modular domain-mediated interactions.