Ana Ruiz-Gómez

Phosphorylation of p38 by GRK2 at the Docking Groove Unveils a Novel Mechanism for Inactivating p38MAPK

p38 Mitogen-activated protein kinases (MAPK) are a family of Ser/Thr kinases that regulate important cellular processes such as stress responses, differentiation, and cell-cycle control . Activation of MAPK is achieved through a linear signaling cascade in which upstream kinases (MAPKKs) dually phosphorylate MAPKs at a conserved 3-amino-acid motif (Thr-X-Tyr) . G-protein-coupled receptor kinases (GRKs) are known to selectively phosphorylate G-protein-coupled receptors (GPCRs) and thus trigger desensitization . We report that GRK2 is a novel inactivating kinase of p38MAPK. p38 associates with GRK2 endogenously and is phosphorylated by GRK2 at Thr-123, a residue located at its docking groove. Mimicking phosphorylation at this site impairs the binding and activation of p38 by MKK6 and diminishes the capacity of p38 to bind and phosphorylate its substrates. Accordingly, p38 activation is decreased or increased when cellular GRK2 levels are enhanced or reduced, respectively. Changes in GRK2 levels and activity can modify p38-dependent processes such as differentiation of preadipocytic cells and LPS-induced cytokine release, enhanced in macrophages from GRK2(+/-) mice. Phosphorylation of p38 at a region key for its interaction with different partners uncovers a new mechanism for the regulation of this important family of kinases.

G protein‐coupled receptor kinase 2 (GRK2): mechanisms of regulation and physiological functions

G protein‐coupled receptor kinase 2 (GRK2) plays a key role in determining the rate and extent of G protein‐coupled receptor (GPCR) desensitization and resensitization. Recent data indicate that GRK2 activity, subcellular distribution and expression are tightly regulated. The important physiological function of GRK2 as a modulator of the efficacy of GPCR signal transduction systems is exemplified by its relevance in cardiovascular physiopathology as well as by its emerging role in the regulation of chemokine receptors.

High Affinity Binding of β-Adrenergic Receptor Kinase to Microsomal Membranes MODULATION OF THE ACTIVITY OF BOUND KINASE BY HETEROTRIMERIC G PROTEIN ACTIVATION

The β-adrenergic receptor kinase (βARK) modulates β-adrenergic and other G protein-coupled receptors by rapidly phosphorylating agonist-occupied receptors at the plasma membrane. We have recently shown that βARK also associates with intracellular microsomal membranes both “in vitro” and “in situ” (García-Higuera, I., Penela, P., Murga, C., Egea, G., Bonay, P., Benovic, J. L., and 5or, F., Jr. (1994) J. Biol. Chem. 269, 1348-1355), thus suggesting a complex modulation of the subcellular distribution of βARK. In this report, we used recombinant [35S]methionine-labeled βARK to show that this kinase interacts rapidly with a high affinity binding site (Kd of 20 ± 1 nM) present in salt-stripped rat liver microsomal membranes. Although βARK binding is not modulated by membrane preincubation with G protein activators, the activity of bound βARK toward rhodopsin or a synthetic peptide substrate was markedly enhanced upon stimulation of the endogenous heterotrimeric G proteins present in the microsomal membranes by AlF4− or mastoparan/guanosine 5′-(3-O-thio)triphosphate, thus strongly suggesting a functional link between these proteins and membrane-associated βARK. Interestingly, βARK association with microsomal membranes is not significantly affected by a fusion protein derived from the carboxyl terminus of βARK1 (the proposed location of the βγ subunit binding site), whereas it is markedly inhibited by fusion proteins corresponding to the amino-terminal region of the kinase. The main determinants of binding appear to be localized to an ∼60-amino acid residue stretch (residues 88 to 145). Our results further indicate a functional relationship between βARK and heterotrimeric G proteins in different intracellular organelles, and suggest that additional proteins may be involved in modulating the cellular localization of the kinase through a new targeting domain of βARK.

Phosphorylation of Phosducin and Phosducin-like Protein by G Protein-coupled Receptor Kinase 2

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gβγ subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 ± 0.1 and 0.83 ± 0.09 mol of Pi/mol of protein, respectively). The phosphorylation reactions exhibit apparent K m values in the range of 40–100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with Gβγ subunits. Phosphorylation of phosducin by GRK2 markedly reduces its Gβγ binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.

Role of G Protein–Coupled Receptor Kinase 2 and Arrestins in β-Adrenergic Receptor Internalization

G protein-coupled receptors (GPCRs) mediate the action of messengers that are key modulators of the function, growth, and differentiation of cardiac and vascular cells. A general feature of GPCRs is the existence of complex regulatory mechanisms that modulate receptor responsiveness and underlie important physiologic phenomena such as signal integration and desensitization. The molecular mechanisms of desensitization have been investigated with the beta2-adrenergic receptor (beta2AR) used as the main model system. Rapid regulation of betaAR and other GPCRs appears to involve agonist-promoted receptor phosphorylation by G protein-coupled receptor kinases (GRKs). This is followed by binding of uncoupling proteins termed arrestins and transient receptor internalization, which plays a key role in resensitizing GPCR by allowing its dephosphorylation and recycling. Recent data indicate that, besides the uncoupling function, GRK2 and beta-arrestin also directly participate in beta2AR sequestration, thus providing the trigger for its resensitization. A detailed knowledge of the role of GRKs and arrestins in betaAR internalization would make their physiologic role in the modulation of cellular responses to messengers better understood.

Polymorphism in genes involved in adrenergic signaling associated with Alzheimer's.

To investigate the potential involvement of adrenergic signaling in Alzheimers disease (AD) pathogenesis, we performed genetic and functional studies of genes initiating the cascade. We chose two functional single-nucleotide polymorphisms (SNPs) in the beta1-adrenergic receptor (ADRB1) and the G protein beta3 subunit (GNB3) genes, respectively, and analyzed their allelic frequencies in a case-control sample of AD. We found that the GNB3 T allele produces a significant risk for AD in individuals homozygous for the ADRB1 C allele, suggesting that the combined effect of both polymorphisms influences AD susceptibility. Interestingly, the co-expression of GNB3 T and ADRB1 C alleles, compared with GNB3 C and ADRB1 G, produced increased cAMP levels and MAPK activation following adrenergic stimulation of transfected human cell lines. Furthermore, the co-expression of these alleles also produced increases in APP expression. These data strongly indicate that the combination of GNB3 and ADRB1 polymorphisms produces AD susceptibility by changing the cell responsiveness to adrenergic stimulation, pointing to the modulation of brain adrenergic receptors as a potential target for novel AD therapeutic strategies.

Role of the Drosophila Non-Visual ß-Arrestin Kurtz in Hedgehog Signalling

The non-visual ß-arrestins are cytosolic proteins highly conserved across species that participate in a variety of signalling events, including plasma membrane receptor degradation, recycling, and signalling, and that can also act as scaffolding for kinases such as MAPK and Akt/PI3K. In Drosophila melanogaster, there is only a single non-visual ß-arrestin, encoded by kurtz, whose function is essential for neuronal activity. We have addressed the participation of Kurtz in signalling during the development of the imaginal discs, epithelial tissues requiring the activity of the Hedgehog, Wingless, EGFR, Notch, Insulin, and TGFβ pathways. Surprisingly, we found that the complete elimination of kurtz by genetic techniques has no major consequences in imaginal cells. In contrast, the over-expression of Kurtz in the wing disc causes a phenotype identical to the loss of Hedgehog signalling and prevents the expression of Hedgehog targets in the corresponding wing discs. The mechanism by which Kurtz antagonises Hedgehog signalling is to promote Smoothened internalization and degradation in a clathrin- and proteosomal-dependent manner. Intriguingly, the effects of Kurtz on Smoothened are independent of Gprk2 activity and of the activation state of the receptor. Our results suggest fundamental differences in the molecular mechanisms regulating receptor turnover and signalling in vertebrates and invertebrates, and they could provide important insights into divergent evolution of Hedgehog signalling in these organisms.

Skeletal muscle myogenesis is regulated by G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) is an important serine/threonine-kinase regulating different membrane receptors and intracellular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced a defective differentiation of somatic muscles, loss of fibers, and a flightless phenotype. In vertebrates, GRK2 hemizygous mice contained less but more hypertrophied skeletal muscle fibers than wild-type littermates. In C2C12 myoblasts, overexpression of a GRK2 kinase-deficient mutant (K220R) caused precocious differentiation of cells into immature myotubes, which were wider in size and contained more fused nuclei, while GRK2 overexpression blunted differentiation. Moreover, p38MAPK and Akt pathways were activated at an earlier stage and to a greater extent in K220R-expressing cells or upon kinase downregulation, while the activation of both kinases was impaired in GRK2-overexpressing cells. The impaired differentiation and fewer fusion events promoted by enhanced GRK2 levels were recapitulated by a p38MAPK mutant, which was able to mimic the inhibitory phosphorylation of p38MAPK by GRK2, whereas the blunted differentiation observed in GRK2-expressing clones was rescued in the presence of a constitutively active upstream stimulator of the p38MAPK pathway. These results suggest that balanced GRK2 function is necessary for a timely and complete myogenic process.

The basal subcellular distribution of β-adrenergic receptor kinase is independent of G-protein βγ subunits

β‐adrenergic receptor kinase (βARK‐1 or GRK2) is a key regulatory protein involved in the regulation of G‐protein‐coupled receptors which associates with microsomal and plasma membranes. βγ subunits of G‐proteins have been suggested to mediate agonist‐dependent membrane translocation of βARK, but their possible role in maintaining the complex subcellular distribution of the kinase is not known. In this study we show that lovastatin‐mediated inhibition of Gγ subunits isoprenylation in HEK‐293 cells stably transfected with βARK1 leads to a significant release of Gβ subunits to the cytosol without causing changes in total particulate βARK or in the association of this kinase to plasma or microsomal membrane fractions. In addition, transient overexpression of mutant forms of Gγ unable to become isoprenylated resulted in a marked sequestration of Gβ to the soluble compartment, but caused no rearrangement in the distribution of cotransfected βARK. These results indicate that anchoring of βARK to cellular membranes under basal conditions is independent of the availability of heterotrimeric G‐protein subunits.

Glycinergic ligands modulate the rate of phosphorylation of the glycine receptor by protein kinase C

The alpha subunit of the glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C (Ruiz-Gómez et al., (1991) J. Biol. Chem. 266, 559-566). We report here that the rate of phosphorylation of the glycine receptor by this kinase is higher in the presence of agonists (glycine, beta-alanine) than in the presence of antagonists (strychnine, RU-5135). These results suggest that activated glycine receptors would be a preferential target for functional regulation through phosphorylation mechanisms.