RGS (regulator of G protein signaling) proteins areGTPase-activating proteins that attenuate signaling by heterotrimeric G proteins. Whether the biological functions of RGS proteins are governed by quantitative differences in GTPase-activating protein activity toward various classes of G␣ subunits and how G protein selectivity is achieved by differences in RGS protein structure are largely unknown. Here we provide evidence indicating that the function of RGS2 is determined in part by differences in potency toward G q versus G i family members. RGS2 was 5-fold more potent than RGS4 as an inhibitor of G q -stimulated phosphoinositide hydrolysis in vivo. In contrast, RGS4 was 8-fold more potent than RGS2 as an inhibitor of G i -mediated signaling. RGS2 mutants were identified that display increased potency toward G i family members without affecting potency toward G q . These mutations and the structure of RGS4-G i ␣ 1 complexes suggest that RGS2-G i ␣ interaction is unfavorable in part because of the geometry of the switch I binding pocket of RGS2 and a potential interaction between the ␣8-␣9 loop of RGS2 and ␣A of G i class ␣ subunits. The results suggest that the function of RGS2 relative to other RGS family members is governed in part by quantitative differences in activity toward different classes of G␣ subunits.Many hormones, neurotransmitters, and sensory stimuli exert their effect on target tissues by activating receptors that are coupled to heterotrimeric G proteins 1 (1, 2). Receptor activation results in exchange of GTP for GDP on G␣ subunits, dissociation of GTP-bound G␣ subunits from the G␤␥ heterodimers, and activation of downstream effector pathways. Signals are terminated following G␣-catalyzed hydrolysis of GTP and reformation of G protein heterotrimers. Thus, G proteins are molecular switches that coordinate physiological responses elicited by a variety of stimuli.RGS (regulator of G protein signaling) proteins are a family of more than 20 members that regulate G protein signaling in part by acting as GTPase-activating proteins (GAPs) for several classes of G protein ␣ subunits (3-6). The GAP activity of RGS proteins decreases the lifetime of active, GTP-bound G␣ subunits, thereby attenuating responses or accelerating the kinetics of signal termination (7,8). Binding of RGS proteins to active G␣ subunits can also antagonize effector activation, thereby blocking signal production (9). These activities are mediated by the conserved RGS domain of ϳ120 amino acids that is characteristic of this protein family.Higher eukaryotes express several types of RGS proteins, potentially to provide selective regulation of distinct types of G protein signaling pathways. Consistent with this hypothesis, RGS proteins are structurally diverse, distinguished by various domains that are likely to confer specific functions. For example, the N terminus of RGS4 confers receptor-selective regulation of G q -coupled responses (10, 11), the PDZ domain of RGS12 binds peptides from the C termini of certain G protein coupled r...

RGS4, a mammalian GTPase activating protein for G protein ␣ subunits, was identified by its ability to inhibit the pheromone response pathway in Saccharomyces cerevisiae. To define regions of RGS4 necessary for its function in vivo, we assayed mutants for activity in this system. Deletion of the N-terminal 33 aa of RGS4 (⌬1-33) yielded a nonfunctional protein and loss of plasma membrane localization. These functions were restored by addition of a C-terminal membrane-targeting sequence to RGS4 (⌬1-33). Thus, plasma membrane localization is tightly coupled with the ability of RGS4 to inhibit signaling. Fusion of the N-terminal 33 aa of RGS4 to green f luorescent protein was sufficient to localize an otherwise soluble protein to the plasma membrane, defining this N-terminal region as a plasma membrane anchorage domain. RGS4 is palmitoylated, with Cys-2 and Cys-12 the likely sites of palmitoylation. Surprisingly, mutation of the cysteine residues within the N-terminal domain of RGS4 did not affect plasma membrane localization in yeast or the ability to inhibit signaling. Features of the N-terminal domain other than palmitoylation are responsible for the plasma membrane association of RGS4 and its ability to inhibit pheromone response in yeast.Heterotrimeric G proteins couple receptors for hormones, neurotransmitters, and sensory signals to intracellular effector molecules, thereby eliciting cellular responses (1, 2). Guanine nucleotide exchange and hydrolysis on the G protein ␣ subunit drives the cycle of activation and deactivation of these signaling pathways. The duration of G protein-mediated responses is subject to the intrinsic GTPase rate of the G protein ␣ subunit, but is also modulated by extrinsic factors. A recently appreciated form of regulation has come from the discovery that members of a protein family called regulators of G protein signaling, or RGS proteins, stimulate the rate of GTP hydrolysis by G protein ␣ subunits (3-5). RGS proteins are found in species ranging from yeast to mammals and constitute a family of at least 20 mammalian proteins (6-8).All RGS family members share sequence similarity that extends over approximately 130 aa, separated in some cases by insertions of varying length (9-11). This conserved RGS domain is sufficient to stimulate GTPase activity of G protein ␣ subunits in vitro (12)(13)(14). Expression of the RGS homology domain of RET-RGS1 or RGS4 yields a recombinant protein that is a functional GAP (GTPase-activating protein). In the crystal structure of RGS4 bound to AlF 4 Ϫ -activated G i␣1 , only the core domain is visible (15). The RGS homology domain binds to the switch regions of G i␣1 and appears to catalyze GTP hydrolysis by stabilizing the switch regions of the G protein in a conformation that favors the transition state of the reactants (14, 15). Sequences outside the RGS homology domain exhibit considerable diversity among family members.Although rapid progress has been made in dissecting the biochemical mechanism by which RGS proteins regulate G protein a...

GTP hydrolysis by guanine nucleotide-binding proteins, an essential step in many biological processes, is stimulated by GTPase-activating proteins (GAPs). The mechanisms whereby GAPs stimulate GTP hydrolysis are unknown. We have used mutational, biochemical, and structural data to investigate how RGS4, a GAP for heterotrimeric G protein alpha subunits, stimulates GTP hydrolysis. Many of the residues of RGS4 that interact with Gi alpha 1 are important for GAP activity. Furthermore, optimal GAP activity appears to require the additive effects of interactions along the RGS4-G alpha interface. GAP-defective RGS4 mutants invariably were defective in binding G alpha subunits in their transition state; furthermore, the apparent strengths of GAP and binding defects were correlated. Thus, none of these residues of RGS4, including asparagine 128, the only residue positioned at the active site of Gi alpha 1, is required exclusively for catalyzing GTP hydrolysis. These results and structural data (Tesmer, J. G. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251-261) indicate that RGS4 stimulates GTP hydrolysis primarily by stabilizing the transition state conformation of the switch regions of the G protein, favoring the transition state of the reactants. Therefore, although monomeric and heterotrimeric G proteins are related, their GAPs have evolved distinct mechanisms of action.

Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling.

CDK (cyclin-dependent kinase) inhibitors have shown remarkable activity in CLL, where its efficacy has been linked to inhibition of the transcriptional CDKs (7 and 9) and deregulation of RNA polymerase and short-lived pro-survival proteins such as MCL1. Furthermore, ER (endoplasmic reticulum) stress has been implicated in CDK inhibition in CLL. Here we conducted a pre-clinical study of a novel orally active kinase inhibitor P1446A in CLL B-cells. P1446A inhibited CDKs at nanomolar concentrations and induced rapid apoptosis of CLL cells in vitro, irrespective of chromosomal abnormalities or IGHV mutational status. Apoptosis preceded inactivation of RNA polymerase, and was accompanied by phosphorylation of stress kinases JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase). Pharmacologic inhibitors of JNK/p38 MAPK conferred protection from P1446A-mediated apoptosis. Treatment with P1446A led to a dramatic induction of NOXA in a JNK-dependent manner, and sensitized CLL cells to ABT-737, a BH3-mimetic. We observed concurrent activation of apoptosis stress-inducing kinase 1 (ASK1) and its interaction with inositol-requiring enzyme 1 (IRE1) and tumor necrosis factor receptor-associated factor 2 (TRAF2) in CLL cells treated with P1446A, providing insights into upstream regulation of JNK in this setting. Consistent with previous reports on limited functionality of ER stress mechanism in CLL cells, treatment with P1446A failed to induce an extensive unfolded protein response. This study provides rationale for additional investigations of P1446A in CLL.