Cells have a multitude of controls to maintain their integrity and prevent random switching from one biological state to another. Raf Kinase Inhibitory Protein (RKIP), a member of the phosphatidylethanolamine binding protein (PEBP) family, is representative of a new class of modulators of signaling cascades that function to maintain the "yin yang" or balance of biological systems. RKIP inhibits MAP kinase (Raf-MEK-ERK), G protein-coupled receptor (GPCR) kinase and NFκB signaling cascades. Because RKIP targets different kinases dependent upon its state of phosphorylation, RKIP also acts to integrate crosstalk initiated by multiple environmental stimuli. Loss or depletion of RKIP results in disruption of the normal cellular stasis and can lead to chromosomal abnormalities and disease states such as cancer. Since RKIP and the PEBP family have been reviewed previously, the goal of this analysis is to provide an update and highlight some of the unique features of RKIP that make it a critical player in the regulation of cellular signaling processes.

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding-and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.Raf kinase inhibitory protein (RKIP/PEBP1) is a signaling modulator that regulates key signal transduction cascades in mammalian cells (reviewed in reference 16). A negative regulator of mitogen-activated protein kinase (MAPK) signaling (42), RKIP inhibits Raf kinase by binding directly to Raf-1, thereby preventing the phosphorylation and activation of 38). RKIP functions as a regulator of the spindle checkpoint and promotes genomic stability by preventing MAPK from inhibiting Aurora B kinase (10). Consistent with this role, RKIP suppresses lung metastasis by prostate tumor cells in an orthotopic murine model (15). RKIP may be a general metastasis suppressor for solid tumors, since RKIP expression is low or undetectable in prostate and breast tumors, melanoma, hepatocellular carcinoma, and colorectal tumors (1,2,14,15,19,34). Finally, RKIP suppresses NF-B activation (43), inhibits G protein-coupled receptor (GPCR) kinase 2 (GRK2)-mediated downregulation of GPCRs (28), and potentiates the efficacy of chemotherapeutic agents (5). Thus, RKIP regulates three key mammalian signaling pathways involving MAPK, GPCR, and NF-B signaling.RKIP is a member of the phosphatidylethanolamine binding protein (PEBP) family, which extends from bacteria to humans and consists of more than 400 proteins (16, 33). X-ray crystallographic studies have demonstrated that highly conserved sequences cluster around a pocket capable of binding anions, including o-phosphorylethanolamine (PE), acetate, and cacodylate (3,35). This pocket is the only clearly identifiable feature for potent...

Chimeric cGMP phosphodiesterases (PDEs) have been constructed using components of the cGMP-binding PDE (PDE5) and cone photoreceptor phosphodiesterase (PDE6␣) in order to study structure and function of the photoreceptor enzyme. A fully functional chimeric PDE6␣/PDE5 enzyme containing the PDE6␣ noncatalytic cGMP-binding sites, and the PDE5 catalytic domain has been efficiently expressed in the baculovirus/ High Five cell system. The catalytic properties of this chimera were practically indistinguishable from those of PDE5, whereas the noncatalytic cGMP binding was similar to that of native purified PDE6␣. The inhibitory Photoreceptor phosphodiesterases (PDEs) 1 serve as effector enzymes in the G protein-mediated visual transduction cascade (1-3). During transduction of the visual signal in vertebrate photoreceptor rod and cone cells, the activated G protein (transducin) ␣ subunit stimulates PDE catalytic activity by relieving the inhibitory constraint imposed by two identical inhibitory P␥ subunits. A recently adopted classification of cyclic nucleotide PDEs recognizes seven different families based on primary sequence and regulation (4). PDEs within each of the families have 60% or more homology while similarities between different families are 40% or less. According to this nomenclature, photoreceptor rod and cone PDEs comprise the PDE6 family (4). Rod photoreceptor PDE is composed of two large homologous catalytic ␣ and ␤ subunits of nearly identical size (molecular masses of 99.2 and 98.3 kDa) and two copies of an inhibitory ␥ subunit (molecular mass 9.7 kDa) (5-8). Cone PDE is composed of two identical ␣Ј subunits (molecular masses of 98.7 kDa) (9, 10), which share Ͼ60% homology with PDE6␣ and PDE6␤ (11). An inhibitory cone P␥ subunit that is highly homologous to rod P␥ and specific for a subset of cone photoreceptors has been identified (12). Recently, a rod-specific

The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory ␥-subunit of PDE (P␥), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin ␣-subunit, . Our data suggest that effects of hRGSr on transducin's GTPase activity are attenuated by P␥ but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade.In vertebrate photoreceptor cells, the signal is transduced from light-activated rhodopsin to the effector enzyme, cGMPphosphodiesterase (PDE), 1 via the heterotrimeric G-protein, transducin (G t␣␤␥ ). The GTP-bound ␣-subunit of transducin (G t␣ GTP) relieves the inhibition imposed by two inhibitory PDE ␥-subunits (P␥) on the enzyme catalytic ␣␤ subunits (P␣␤). Activation of PDE leads to a closure of cGMP-gated channels in the photoreceptor plasma membranes (1-3). The inactivation of PDE is a critical component of the turnoff mechanism in the visual transduction cascade. This inactivation is controlled by the intrinsic GTPase activity of transducin which hydrolyzes GTP to GDP. The GDP-bound G t␣ (G t␣ GDP) has a substantially reduced affinity for P␥ and releases P␥ to reinhibit P␣␤ (1, 4 -6). The rate of GTP hydrolysis by transducin measured in vitro (7, 8) is too slow to account for the fast photoresponse turnoff in vivo (9, 10). The P␥ subunit (11, 12) and a distinct membrane-associated protein factor (13,14) have been shown to enhance transducin GTPase activity in the activated membrane-bound transducin-PDE complex to a level comparable with the rate of transducin inactivation in vivo. A recent study has shown that a retinal specific member of the RGS family, RGSr, serves as a GTPase-activating protein (GAP) for transducin, providing an additional dimension to an already complex picture of the regulation of transducin GTPase activity (15). Functional relationships between RGSr, the ␥-subunit of PDE, and a putative membrane GAP factor are currently not understood. Here, we study the interaction between transducin and a human retinal specific RGS (hRGSr), and regulation of transducin GTPase activity by hRGSr. We examine the effects of P␥ and photoreceptor membrane concentration on modulation of the GTPase activity by hRGSr. EXPERIMENTAL PROCEDURESMaterials-GTP and GTP␥S were products of Boehringer Mannheim. Blue-Sepharose CL-6B was obtained from Pharmacia. 3-(Bromoacetyl)-7-diethylaminocoumarin (BC) was purchased from Molecular Probes, Inc. [␥-32 P]GTP (Ͼ5000 Ci/mmol) was obtained f...

Transducin is a photoreceptor-specific heterotrimeric GTP-binding protein that plays a key role in the vertebrate visual transduction cascade. Here, using scanning site-directed mutagenesis of the chimeric G␣ t /G␣ i1 ␣-subunit (G␣ t/i ), we identified G␣ t residues critical for interaction with the effector enzyme, rod cGMP phosphodiesterase (PDE). Our evidence suggests that residue Ile 208 in the switch II region directly interacts with the effector in the active GTP-bound conformation of G␣ t . Residues Arg 201 , Arg 204 , and Trp 207 are essential for the conformation-dependent G␣ t /effector interaction either via direct contacts with the inhibitory PDE ␥-subunit or by forming an effector-competent conformation through the communication network between switch II and the switch III/␣3-helix domain of G␣ t . Residues His 244 and Asn 247 in the ␣3 helix of G␣ t are responsible for the conformation-independent effector-specific interaction. Insertion of these residues rendered the G␣ t/i chimera with the ability to bind PDE ␥-subunit and stimulate PDE activity approaching that of native G␣ t . Comparative analysis of the interactions of G␣ t/i mutants with PDE and RGS16 revealed two adjacent but distinct interfaces on transducin. This indicates a possibility for a functional trimeric complex, RGS/G␣/effector, that may play a central role in turn-off mechanisms of G protein signaling systems, particularly in phototransduction.The visual transduction cascade in vertebrate photoreceptors represents a classical example of a G protein signaling system. In rod photoreceptor cells, light-activated rhodopsin stimulates GTP-GDP exchange on the retinal G protein, transducin, resulting in dissociation of G␣ t GTP 1 from G␤␥ t and rhodopsin. Liberated G␣ t in active GTP-bound conformation stimulates the effector enzyme, cGMP phosphodiesterase (PDE), by displacing the inhibitory ␥-subunits (P␥) from the PDE catalytic core (P␣␤). cGMP hydrolysis by active PDE results in closure of cGMP-gated channels in the plasma membrane (1-3). As in other G protein cascades, the lifetime of a transducin-mediated signal is linked to the intrinsic GTPase activity of G␣ t . Hydrolysis of GTP converts the G␣ t molecule to the inactive GDP-bound conformation allowing release of P␥ for re-inhibition of P␣␤. A member of the RGS family (4 -6), RGS9, and perhaps other retina-specific RGS proteins serve as GTPase-activating proteins (GAPs) for transducin (7-9). They target a transitional intermediate conformation of G␣ t during GTP hydrolysis to accelerate GTP hydrolysis and expedite the signal termination (8, 10, 11). The P␥ subunit assists RGS9 in its GAP function, thus providing an elegant feedback mechanism for the effector participation in quenching the visual excitation (7).Recent progress in understanding molecular mechanisms of G protein action has been advanced by solutions of crystal structures of several G protein ␣-subunits in active GTPbound, inactive GDP-bound, and transitional, AlF 4 Ϫ -complexed conformations (12-16). Three regions ...