Kristina Lorenz

Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2

Feedback inhibition is a fundamental principle in signal transduction allowing rapid adaptation to different stimuli. In mammalian cells, the major feedback inhibitor for G-protein-coupled receptors (GPCR) is G-protein-coupled receptor kinase 2 (GRK-2), which phosphorylates activated receptors, uncouples them from G proteins and initiates their internalization. The functions of GRK-2 are indispensable and need to be tightly controlled. Dysregulation promotes disorders such as hypertension or heart failure. In our search for a control mechanism for this vital kinase, here we show that the Raf kinase inhibitor protein (RKIP) is a physiological inhibitor of GRK-2. After stimulation of GPCR, RKIP dissociates from its known target, Raf-1 (refs 6–8), to associate with GRK-2 and block its activity. This switch is triggered by protein kinase C (PKC)-dependent phosphorylation of the RKIP on serine 153. The data delineate a new principle in signal transduction: by activating PKC, the incoming receptor signal is enhanced both by removing an inhibitor from Raf-1 and by blocking receptor internalization. A physiological role for this mechanism is shown in cardiomyocytes in which the downregulation of RKIP restrains β-adrenergic signalling and contractile activity.

Conformational cross-talk between |[alpha]|2A-adrenergic and |[mu]|-opioid receptors controls cell signaling

Morphine, a powerful analgesic, and norepinephrine, the principal neurotransmitter of sympathetic nerves, exert major inhibitory effects on both peripheral and brain neurons by activating distinct cell-surface G protein-coupled receptors-the mu-opioid receptor (MOR) and alpha2A-adrenergic receptor (alpha2A-AR), respectively. These receptors, either singly or as a heterodimer, activate common signal transduction pathways mediated through the inhibitory G proteins (G(i) and G(o)). Using fluorescence resonance energy transfer microscopy, we show that in the heterodimer, the MOR and alpha2A-AR communicate with each other through a cross-conformational switch that permits direct inhibition of one receptor by the other with subsecond kinetics. We discovered that morphine binding to the MOR triggers a conformational change in the norepinephrine-occupied alpha2A-AR that inhibits its signaling to G(i) and the downstream MAP kinase cascade. These data highlight a new mechanism in signal transduction whereby a G protein-coupled receptor heterodimer mediates conformational changes that propagate from one receptor to the other and cause the second receptors rapid inactivation.

A new type of ERK1/2 autophosphorylation causes cardiac hypertrophy

The extracellular-regulated kinases ERK1 and ERK2 (commonly referred to as ERK1/2) have a crucial role in cardiac hypertrophy. ERK1/2 is activated by mitogen-activated protein kinase kinase-1 (MEK1) and MEK2 (commonly referred to as MEK1/2)-dependent phosphorylation in the TEY motif of the activation loop, but how ERK1/2 is targeted toward specific substrates is not well understood. Here we show that autophosphorylation of ERK1/2 on Thr188 directs ERK1/2 to phosphorylate nuclear targets known to cause cardiac hypertrophy. Thr188 autophosphorylation requires the activation and assembly of the entire Raf-MEK-ERK kinase cascade, phosphorylation of the TEY motif, dimerization of ERK1/2 and binding to G protein βγ subunits released from activated Gq. Thr188 phosphorylation of ERK1/2 was observed in isolated cardiomyocytes induced to undergo hypertrophic growth, in mice upon stimulation of Gq-coupled receptors or after aortic banding and in failing human hearts. Experiments using transgenic mouse models carrying mutations at the Thr188 phosphorylation site of ERK2 suggested a causal relationship to cardiac hypertrophy. We propose that specific phosphorylation events on ERK1/2 integrate differing upstream signals (Raf1-MEK1/2 or G protein–coupled receptor–Gq) to induce cardiac hypertrophy.

The transcriptional repressor Nab1 is a specific regulator of pathological cardiac hypertrophy

Hypertrophy represents the major physiological response of the heart to adapt to chronically enhanced workload, but is also crucial in the development of heart failure. Although we know of numerous inducers of cardiac hypertrophy, little is known about mechanisms that limit cardiac hypertrophy. Here, we describe the transcriptional repressor NAB1 as an endogenous regulator of cardiac growth. We identified NAB1 as being upregulated in both mouse and human heart failure. Nab1 is highly expressed in mammalian cardiac myocytes and it inhibited cardiomyocyte hypertrophy through repression of its targets, transcription factor Egr. Transgenic mice with cardiac-specific overexpression of Nab1 showed that Nab1 is a potent inhibitor of cardiac growth in response to pathological stimuli in vivo. Nab1 overexpression suppressed adrenergically induced and pressure overload–induced hypertrophy, whereas physiological growth during development and in response to exercise was not affected. These findings implicate the Nab1-Egr1 axis as a crucial regulator of pathological cardiac growth.

Cardiac hypertrophy: Targeting Raf/MEK/ERK1 /2-signaling

Over the past two decades, basic research has revealed a complex network of regulatory mechanisms that control the ERK1/2-signaling cascade. ERK1/2 mediate cardiac hypertrophy, a major risk factor for the development of arrhythmias, heart failure and sudden death, but also beneficial effects, e.g. protection of the heart from cell death and ischemic injury. Selective targeting of these ambiguous ERK functions could provide a powerful tool in the treatment of cardiac disease. This short review will discuss new mechanistic insights into ERK1/2-dependent development of cardiac hypertrophy and the prospect to translate this knowledge into future therapeutic strategies.

FTY720 Ameliorates Acute Ischemic Stroke in Mice by Reducing Thrombo-Inflammation but Not by Direct Neuroprotection

Background and Purpose— Lymphocytes are important players in the pathophysiology of acute ischemic stroke. The interaction of lymphocytes with endothelial cells and platelets, termed thrombo-inflammation, fosters microvascular dysfunction and secondary infarct growth. FTY720, a sphingosine-1-phosphate receptor modulator, blocks the egress of lymphocytes from lymphoid organs and has been shown to reduce ischemic neurodegeneration; however, the underlying mechanisms are unclear. We investigated the mode of FTY720 action in models of cerebral ischemia. Methods— Transient middle cerebral artery occlusion (tMCAO) was induced in wild-type and lymphocyte-deficient Rag1−/− mice treated with FTY720 (1 mg/kg) or vehicle immediately before reperfusion. Stroke outcome was assessed 24 hours later. Immune cells in the blood and brain were counted by flow cytometry. The integrity of the blood–brain barrier was analyzed using Evans Blue dye. Thrombus formation was determined by immunohistochemistry and Western blot, and was correlated with cerebral perfusion. Results— FTY720 significantly reduced stroke size and improved functional outcome in wild-type mice on day 1 and day 3 after transient middle cerebral artery occlusion. This protective effect was lost in lymphocyte-deficient Rag1−/− mice and in cultured neurons subjected to hypoxia. Less lymphocytes were present in the cerebral vasculature of FTY720-treated wild-type mice, which in turn reduced thrombosis and increased cerebral perfusion. In contrast, FTY720 was unable to prevent blood–brain barrier breakdown and transendothelial immune cell trafficking after transient middle cerebral artery occlusion. Conclusions— Induction of lymphocytopenia and concomitant reduction of microvascular thrombosis are key modes of FTY720 action in stroke. In contrast, our findings in Rag1−/− mice and cultured neurons argue against direct neuroprotective effects of FTY720.

β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle

(β-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs). They bind to active, phosphorylated GPCRs and thereby shut off ‘classical’ signalling to G proteins, trigger internalization of GPCRs via interaction with the clathrin machinery and mediate signalling via ‘non-classical’ pathways. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) β-arrestin proteins (β-arrestin1 and β-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs). The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (β-)arrestins that have recently been solved by X-ray crystallography. Here we investigate both the interaction of β-arrestin with GPCRs, and the β-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based β-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in β-arrestin2 that occur rapidly after the receptor–β-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and β-arrestins. They further indicate that β-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signalling.

Blocking of α4 Integrin Does Not Protect From Acute Ischemic Stroke in Mice

Background and Purpose— T lymphocytes have recently been identified as key mediators of tissue damage in ischemic stroke. The interaction between very late antigen-4 (VLA-4) and vascular adhesion molecule-1 is crucial for the transvascular egress of T lymphocytes, and inhibition of this interaction by specific antibodies is a powerful strategy to combat autoimmune neuroinflammation. However, whether pharmacological blocking of T-lymphocyte trafficking is also protective during brain ischemia is still unclear. We investigated the efficacy of a monoclonal antibody directed against VLA-4 in mouse models of ischemic stroke. Methods— Transient and permanent middle cerebral artery occlusion was induced in male C57Bl/6 mice. Animals treated with a monoclonal anti-CD49d antibody (300 &mgr;g) 24 hours before or 3 hours after the onset of cerebral ischemia and stroke outcome, including infarct size, functional status, and mortality, were assessed between day 1 and day 7. The numbers of immune cells invading the ischemic brain were determined by immunocytochemistry and flow cytometry. Results— Blocking of VLA-4 significantly reduced the invasion of T lymphocytes and neutrophils on day 5 after middle cerebral artery occlusion and inhibited the upregulation of vascular adhesion molecule-1. However, the anti-CD49d antibody failed to influence stroke outcome positively irrespective of the model or the time point investigated. Conclusions— Pharmacological inhibition of the VLA-4/vascular adhesion molecule-1 axis in experimental stroke was ineffective in our hands. Our results cast doubt on the effectiveness of anti-CD49d as a stroke treatment. Further translational studies should be performed before testing anti–VLA-4 antibodies in patients with stroke.

Dual Role of the β2-Adrenergic Receptor C Terminus for the Binding of β-Arrestin and Receptor Internalization

Homologous desensitization of β2-adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind β-arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by β-arrestins, we have investigated β-arrestin interaction and internalization of a set of mutants of the human β2-adrenergic receptor. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor-β-arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of β-arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and β-arrestin2 translocation. A β2-adrenergic receptor truncated distal to residue 381 interacted normally with β-arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and β-arrestin, but its last eight amino acids facilitate receptor internalization in concert with β-arrestin2.

Raf kinase inhibitor protein (RKIP) dimer formation controls its target switch from Raf1 to G protein coupled receptor kinase (GRK) 2

Background: Raf kinase inhibitor protein (RKIP) is a regulator of several distinct kinases, including Raf1 and G protein-coupled receptor kinase 2 (GRK2). Results: Protein kinase C-mediated phosphorylation of RKIP triggers dimer formation of RKIP, which enables RKIP to switch specificity between Raf1 and GRK2. Conclusion: Phosphorylation-dependent dimerization of RKIP coordinates specific interactions with Raf1 and GRK2. Significance: Control switches in a kinase regulator permit specific control of multiple kinase signaling pathways and their downstream functions. Proteins controlling cellular networks have evolved distinct mechanisms to ensure specificity in protein-protein interactions. Raf kinase inhibitor protein (RKIP) is a multifaceted kinase modulator, but it is not well understood how this small protein (21 kDa) can coordinate its diverse signaling functions. Raf1 and G protein-coupled receptor kinase (GRK) 2 are direct interaction partners of RKIP and thus provide the possibility to untangle the mechanism of its target specificity. Here, we identify RKIP dimer formation as an important mechanistic feature in the target switch from Raf1 to GRK2. Co-immunoprecipitation and cross-linking experiments revealed RKIP dimerization upon phosphorylation of RKIP at serine 153 utilizing purified proteins as well as in cells overexpressing RKIP. A functional phosphomimetic RKIP mutant had a high propensity for dimerization and reproduced the switch from Raf1 to GRK2. RKIP dimerization and GRK2 binding, but not Raf1 interaction, were prevented by a peptide comprising amino acids 127–146 of RKIP, which suggests that this region is critical for dimer formation. Furthermore, a dimeric RKIP mutant displayed a higher affinity to GRK2, but a lower affinity to Raf1. Functional analyses of phosphomimetic as well as dimeric RKIP demonstrated that enhanced dimerization of RKIP translates into decreased Raf1 and increased GRK2 inhibition. The detection of RKIP dimers in a complex with GRK2 in murine hearts implies their physiological relevance. These findings represent a novel mechanistic feature how RKIP can discriminate between its different interaction partners and thus advances our understanding how specific inhibition of kinases can be achieved.