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Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.

Functional interplay between ionotropic and metabotropic receptors frequently involves complex intracellular signaling cascades. The group I metabotropic glutamate receptor mGlu5a co-clusters with the ionotropic N-methyl-d-aspartate (NMDA) receptor in hippocampal neurons. In this study, we report that a more direct cross-talk can exist between these types of receptors. Using bioluminescence resonance energy transfer in living HEK293 cells, we demonstrate that mGlu5a and NMDA receptor clustering reflects the existence of direct physical interactions. Consequently, the mGlu5a receptor decreased NMDA receptor current, and reciprocally, the NMDA receptor strongly reduced the ability of the mGlu5a receptor to release intracellular calcium. We show that deletion of the C terminus of the mGlu5a receptor abolished both its interaction with the NMDA receptor and reciprocal inhibition of the receptors. This direct functional interaction implies a higher degree of target-effector specificity, timing, and subcellular localization of signaling than could ever be predicted with complex signaling pathways.

Calpains are Ca(2+)-dependent cysteine proteases known to be important for the regulation of cell functions and which aberrant activation causes cell death in a number of degenerative disorders. To provide a tool for monitoring the status of calpain activity in vivo under physiological and pathological conditions, we created a mouse model that expresses ubiquitously a fluorescent reporter consisting of eCFP and eYFP separated by a linker cleavable by the ubiquitous calpains. We named this mouse CAFI for calpain activity monitored by FRET imaging. Our validation studies demonstrated that the level of calpain activity correlates with a decrease in FRET (fluorescence resonance energy transfer) between the two fluorescent proteins. Using this model, we observed a small level of activity after denervation and fasting, a high level of activity during muscle regeneration and ischemia, and local activity in damaged myofibers after exercise. Finally, we crossed the CAFI mouse with the alpha-sarcoglycan-deficient model, demonstrating an increase of calpain activity at the steady state. Altogether, our results present evidence that CAFI mice could be a valuable tool in which to follow calpain activity at physiological levels and in disease states.

The GTPase-activating protein RasGAP functions as both a negative regulator and an effector of Ras proteins. In tumor cells, RasGAP is no longer able to deactivate oncogenic Ras proteins, and its effector function becomes predominant. As RasGAP itself has no obvious enzymatic function that may explain this effector function, we looked for downstream RasGAP effectors that could fulfill this role. We looked for the existence of RasGAP Src homology 3 (SH3) domain partners as this domain is involved in the regulation of cell proliferation and has an anti-apoptotic effect. We report here the identification of a new RasGAP SH3 domain-binding protein, named Aurora. This Drosophila melanogaster Ser/Thr kinase has three human orthologs called Aurora/Ipl1-related kinase or HsAIRK-1, -2, and -3. Coimmunoprecipitation experiments in COS cells confirmed that HsAIRK-1 and HsAIRK-2 both interact with RasGAP. RasGAP pull-down experiments showed that it interacts with HsAIRK-1 in G(2)/M HeLa cells. We also demonstrated that RasGAP binds to the kinase domain of Aurora and that this interaction inhibits the kinase activity of HsAIRK-1 and HsAIRK-2. Finally we showed that RasGAP forms a ternary complex with HsAIRK and survivin. This complex may be involved in the regulation of the balance between cell division and apoptosis.

Oxygen free radicals may act as second messengers in signal transduction pathways and contribute to inflammatory diseases. We studied the action in vitro of radiolytically generated hydroxyl radicals (.OH) and superoxide radicals (O-2) on the cAMP-dependent protein kinases, I and II (PKAI and -II, respectively). The effects of the gasses O2 and N2O used to produce O-2 or .OH radicals by gamma-radiolysis of the water were also studied. PKAI is more sensitive than PKAII to oxygen gas (10 mM sodium formate) and to hydroxyl and superoxide radicals. Hydroxyl radicals decreased the kinase phosphotransferase activities stimulated either by cAMP or its site-specific analogs for both PKAI and PKAII; however, PKAI was more affected. The binding of [3H]cAMP and of 8-N3-[32P]cAMP to RI regulatory subunits was decreased. .OH caused a loss of tryptophan 260 fluorescence at site A of PKAI and of bityrosine production. Superoxide radicals affected only PKAI. O-2 modified both cAMP-binding sites A and B of the regulatory subunit but had a smaller effect on the catalytic subunit. The catalytic subunit was more sensitive to radicals when free than when part of the holoenzymes during exposure to the oxygen free radicals. These results suggest that oxygen free radicals alter the structure of PKA enzymes. Thus, oxidative modifications may alter key enzymes, including cAMP-dependent protein kinases, in certain pathological states.