Renata Zippel

Cloning and characterization of mouse UBPy, a deubiquitinating enzyme that interacts with the ras guanine nucleotide exchange factor CDC25(Mm)/Ras-GRF1.

We used yeast “two-hybrid” screening to isolate cDNA-encoding proteins interacting with the N-terminal domain of the Ras nucleotide exchange factor CDC25Mm. Three independent overlapping clones were isolated from a mouse embryo cDNA library. The full-length cDNA was cloned by RACE-polymerase chain reaction. It encodes a large protein (1080 amino acids) highly homologous to the human deubiquitinating enzyme hUBPy and contains a well conserved domain typical of ubiquitin isopeptidases. Therefore we called this new protein mouse UBPy (mUBPy). Northern blot analysis revealed a 4-kilobase mRNA present in several mouse tissues and highly expressed in testis; a good level of expression was also found in brain, where CDC25Mm is exclusively expressed. Using a glutathione S-transferase fusion protein, we demonstrated an “in vitro” interaction between mUBPy and the N-terminal half (amino acids 1–625) of CDC25Mm. In addition “in vivo” interaction was demonstrated after cotransfection in mammalian cells. We also showed that CDC25Mm, expressed in HEK293 cells, is ubiquitinated and that the coexpression of mUBPy decreases its ubiquitination. In addition the half-life of CDC25Mm protein was considerably increased in the presence of mUBPy. The specific function of the human homolog hUBPy is not defined, although its expression was correlated with cell proliferation. Our results suggest that mUBPy may play a role in controlling degradation of CDC25Mm, thus regulating the level of this Ras-guanine nucleotide exchange factor.

Modulation of extracellular signal-regulated kinases cascade by chronic Δ9-tetrahydrocannabinol treatment

Acute Delta(9)-tetrahydrocannabinol (THC) injection increased ERK pathway (ERK, pCREB, and c-fos) mostly in the caudate putamen and cerebellum. This effect underwent to homeostatic adaptation after chronic treatment. Moreover, chronic THC exposure induced increases in the ERK cascade (ERK, pCREB, and Fos B) in the prefrontal cortex and hippocampus, suggesting that different neuronal circuits seem to be involved in the early phase and late phase of exposure. The involvement of ERK pathway in cannabinoid chronic exposure was also confirmed in Ras-GRF1 knock out mice, a useful model where cannabinoid-induced ERK activation is lost. In fact, Ras-GRF1 ko mice did not develop tolerance to THC analgesic and hypolocomotor effect. Our data suggest that ERK cascade could play a pivotal role in the induction of synaptic plasticity due to cannabinoid chronic exposure.

CAMP cascade leads to Ras activation in cortical neurons

Monoaminergic G protein-coupled receptors (GPCRs) are highly expressed in the CNS at the cerebrocortical level, where they support a variety of behavioural responses. To elucidate possible intracellular signalling pathways coupled to these receptors, we have studied their ability to activate extracellular signal-regulated kinases (ERKs) in cultured cortical neurons. An increase in ERK activity was observed after stimulation of neurons with dopamine or serotonin, and with agonists selective for various GPCRs. In addition, ERK activation was also observed following treatment with phorbol dibutyrate (PdBu) and forskolin, activators of protein kinase C (PKC) and protein kinase A (PKA), respectively. Concomitant with ERK activation, all the monoaminergic agonists tested also increased the level of active Ras (Ras-GTP). Surprisingly, Ras activation was also observed after activation of cAMP pathway, and this effect was at least in part mediated by PKA. Ras activation by cAMP was unique for neurons, since in PC12 cells forskolin caused activation of ERK but did not increase Ras-GTP level. These results highlight the relevance of Ras as a target for multiple signalling cascades leading to activation of the ERK pathway in neurons.

Ras/ERK signalling in cannabinoid tolerance: from behaviour to cellular aspects

We investigated the role of the Ras/extracellular‐regulated kinase (ERK) pathway in the development of tolerance to Δ9‐tetrahydrocannabinol (THC)‐induced reduction in spontaneous locomotor activity by a genetic (Ras‐specific guanine nucleotide exchange factor (Ras‐GRF1) knock‐out mice) and pharmacological approach. Pre‐treatment of wild‐type mice with SL327 (50 mg/kg i.p.), a specific inhibitor of mitogen‐activated protein kinase kinase (MEK), the upstream kinase of ERK, fully prevented the development of tolerance to THC‐induced hypolocomotion. We investigated the impact of the inhibition of ERK activation on the biological processes involved in cannabinoid tolerance (receptor down‐regulation and desensitization), by autoradiographic cannabinoid CB1 receptor and cannabinoid‐stimulated [35S]GTPγS binding studies in subchronically treated mice (THC, 10 mg/kg s.c., twice a day for 5 days). In the caudate putamen and cerebellum of Ras‐GRF1 knock‐out mice and SL327 pre‐treated wild‐type mice, CB1 receptor down‐regulation and desensitization did not occur, suggesting that ERK activation might account for CB1 receptor plasticity involved in the development of tolerance to THC hypolocomotor effect. In contrast, the hippocampus and prefrontal cortex showed CB1 receptor adaptations regardless of the genetic or pharmacological inhibition of the ERK pathway, suggesting regional variability in the cellular events underlying the altered CB1 receptor function. These findings suggest that at least in the caudate putamen and cerebellum, the Ras/ERK pathway is essential for triggering the alteration in CB1 receptor function responsible for tolerance to THC‐induced hypomotility.

Changes in the expression of G protein-coupled receptor kinases and β-arrestins in mouse brain during cannabinoid tolerance : A role for Ras-ERK cascade

The focus of our study was to determine the role of G protein-coupled receptor kinases (GRKs) and β-arrestins in agonist-induced CB1 receptor modulation during cannabinoid tolerance and their dependence from the extracellular signal-regulated kinase (ERK) cascade. In wild-type mice, chronic Δ9-tetrahydrocannabinol (THC) exposure significantly activated specific GRK and β-arrestin subunits in all the considered brain areas (striatum, cerebellum, hippocampus, and prefrontal cortex), suggesting their involvement in the adaptive processes underlying CB1 receptor downregulation and desensitization. These events were ERK-dependent in the striatum and cerebellum, because they were prevented in the genetic (Ras-GRF1 knockout mice) and pharmacological (SL327-pretreated mice) models of ERK activation inhibition, whereas in the hippocampus and prefrontal cortex, they appeared to be mostly ERK-independent. In the latter areas, ERK activation after chronic THC increased the transcription factors cyclic adenosine monophosphate response element-binding protein and Fos B as well as a downstream protein known as brain-derived neurotrophic factor. As a whole, our data suggest that in the striatum and cerebellum, THC-induced ERK activation could represent a key signaling event to initiate homologous desensitization of CB1 receptor, accounting for the development of tolerance to THC-induced hypolocomotion. In the prefrontal cortex and hippocampus, THC-induced alteration in GRKs and β-arrestins primarily depends on other kinases, whereas ERK activation could be part of the molecular adaptations that underlie the complex behavioral phenotype that defines the addicted state.

CDC25MM/RAS-GRF1 REGULATES BOTH RAS AND RAC SIGNALING PATHWAYS

The Ras‐GRF1 exchange factor molecule contains in addition to the catalytic domain two pleckstrin homology (PH1 and PH2), one IQ and one Dbl homology (DH) domains. In this study we investigated the role of such additional domains. We found that a Ras‐GRF1 mutant lacking PH1 and IQ domains is sufficient to activate c‐fos promoter in response to lysophosphatidic acid (LPA). The same mutant did not increase external stimuli‐regulated kinase (ERK) activity, suggesting an additional mechanism for the induction of gene transcription. Isolated DH‐PH2 module activates c‐Jun NH2‐terminal kinase and the c‐fos promoter in response to LPA, providing the basis for an ERK‐independent mechanism. These results provide evidence that Ras‐GRF1 acts as a bifunctional molecule on both ERK‐dependent and independent pathways.

ERK-Dependent Modulation of Cerebellar Synaptic Plasticity after Chronic Δ9-Tetrahydrocannabinol Exposure

Chronic exposure to Δ9-tetrahydrocannabinol (THC) induces tolerance to cannabinoid-induced locomotor effects, which are mediated by cannabinoid receptors (CB1Rs) located in motor control regions, including the cerebellum. There is substantial evidence of cerebellar CB1R molecular adaptation and modifications in receptor signaling after prolonged cannabinoid exposure. However, very little is known about the effects of chronic cannabinoid administration on cerebellar synaptic plasticity, which may contribute to the development of cannabinoid behavioral tolerance. In the cerebellar cortex, activation of CB1R inhibits excitatory synaptic transmission at parallel fiber (PF)–Purkinje cell (PC) synapses by decreasing neurotransmitter release. Our study aimed to investigate the neurophysiological adaptive responses occurring at cerebellar PF-PC cell synapses after repeated THC exposure. In THC-tolerant mice, an increase of the basal release probability was found at PF-PC synapses, in parallel with a facilitation of slow mGluR1 (metabotropic glutamate receptor type 1)-mediated excitatory postsynaptic currents and a reduced sensitivity to the inhibitory effects of the CB1R agonist CP55,940 [(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol]. Additionally, after repeated THC exposures, presynaptic PF-PC long-term potentiation was blocked by A1R (adenosine receptor-1) activation. Inhibition of the extracellular signal regulated kinase (ERK) pathway prevented these alterations of cerebellar synaptic transmission and plasticity. In summary, we provide evidence for ERK-dependent modulatory mechanisms at PF-PC synapses after chronic THC administration. This contributes to generation of forms of pathological synaptic plasticity that might play a role in cannabinoid dependence.

A proteomic study using zebra mussels (D. polymorpha) exposed to benzo(α)pyrene: the role of gender and exposure concentrations.

It has recently been established that the use of proteomics can be a useful tool in the field of ecotoxicology. Despite the fact that the mussel Dreissena polymorpha is a valuable bioindicator for freshwater ecosystems, the application of a proteomic approach with this organism has not been deeply investigated. To this end, several zebra mussel specimens were subjected to a 7-day exposure of two different concentrations (0.1 and 2 μg L⁻¹) of the model pollutant benzo[α]pyrene (B[α]P). Changes in protein expression profiles were investigated in gill cytosolic fractions from control/exposed male and female mussels using 2-DE electrophoresis. B[α]P bioaccumulation in mussel soft tissue was also assessed to validate exposure to the selected chemical. We evaluated overall changes in expression profiles for 28 proteins in exposed mussels, 16 and 12 of which were, respectively, over- and under-expressed. Surprisingly, the comparative analysis of protein data sets showed no proteins that varied commonly between the two different B[α]P concentrations. Spots of interest were manually excised and analysed by MALDI-TOF/TOF mass spectrometry. The most significant proteins that were identified as altered were related to oxidative stress, signal transduction, cellular structure and metabolism. This preliminary study indicates the feasibility of a proteomic approach with the freshwater mussel D. polymorpha and provides a starting point for similar investigations. Our results confirm the need to increase the number of invertebrate proteomic studies in order to increase the following: their representation in databases and the successful identification of their most relevant proteins. Finally, additional studies investigating the role of gender and protein modulation are warranted.

PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse Swiss 3T3 and human skin fibroblasts

Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts we demonstrate that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA. Feed-back inhibition of surface receptors by protein kinase C-mediated phosphorylation is therefore not general, and cannot be the only process responsible for the attenuation of receptor-mediated responses in eukaryotic cells.

In vivo phosphorylation and dephosphorylation of the platelet-derived growth factor receptor studied by immunoblot analysis with phosphotyrosine antibodies

Antibodies against the synthetic hapten azobenzyl phosphonate which specifically crossreact with phosphotyrosine have been produced and used to detect the proteins phosphorylated in tyrosine following exposure of intact quiescent Swiss 3T3 fibroblasts to the platelet-derived growth factor (PDGF). Western blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated proteins followed by decoration with phosphotyrosine antibodies and 125I-labeled protein A have been used. The major tyrosine-phosphorylated component was a 170 kDa protein. The following lines of evidence suggest that this protein is the PDGF receptor in its tyrosine-phosphorylated form: (a) both proteins have the same (170 kDa) molecular weight; (b) the phosphorylated 170 kDa protein was detectable only in cell lines bearing the PDGF receptor; (c) the phosphorylation of the 170 kDa protein required PDGF and was dose-dependent. Kinetic studies showed that the phosphorylation of the receptor was maximal after 5-10 min at 37 degrees C and was followed by a rapid decrement of the band. The loss of the 170 kDa component was not prevented by inhibitors of membrane internalization and of lysosomal proteinases, while it was inhibited by lowering the temperature to 5 degrees C. In PDGF-stimulated cells, phosphotyrosine antibodies detected also a minor 36 kDa component phosphorylated at tyrosine.