Abstract
RasGRF1 is a neuron-specific guanine nucleotide exchange factor for the small GTPases Ras and Rac. It is implicated in the regulation of memory formation and in the development of tolerance to drug abuse, although the mechanisms have been elucidated only in part. Here we report the isolation, by the yeast two-hybrid screen, of the microtubule-destabilizing factor SCLIP (SCG10-like protein) as a novel RasGRF1-interacting protein. This interaction requires the region spanning the Dbl-homology domain of RasGRF1, endowed with catalytic activity on Rac. In search for a possible function we found by biochemical means that SCLIP influences the signaling properties of RasGRF1, greatly reducing its ability to activate the Rac/p38 MAPK pathway, while the Ras/Erk one remains unaffected. Moreover, a potential role is suggested by transfection studies in neuronal PC12 cells in which RasGRF1 induces neurite outgrowth, and coexpression of SCLIP counteracts this effect, causing a dramatic decrease in the percentage of cells bearing neurites, which also appear significantly shortened. This study unveils a physical and functional interaction between RasGRF1 and SCLIP. We suggest that this novel interplay may have possible implications in mechanisms that regulate neuronal morphology and structural plasticity.
Highlights
RasGRF1 is a bifunctional guanine nucleotide exchange factor (GEF)3 that catalyzes the activation of the small GTP-binding proteins Ras and Rac, by facilitating the release of GDP favoring the loading of GTP
The involvement of RasGRF1 and its closely related, widely distributed homologue RasGRF2 in intracellular signaling is developmentally regulated, because recent findings demonstrated that the components of signaling cascades from both types of ionotropic glutamate receptors, N-methyl-D-aspartate (NMDA) receptor and ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, to mitogen-activated protein kinases (MAPKs)/extracellular signal-regulated protein kinases (Erks) switch during development and that RasGRFs play a major role in these signaling events only in mature neurons [8, 9]
Regarding the signaling from NMDA receptor, recent experiments on single and double knock-out mice revealed that RasGRF1 and -2 respond to diverse subclasses of NMDA receptors and contribute to different forms of synaptic plasticity, because RasGRF1 mediates mainly long term depression by regulating Rac and its effector p38 MAPK, whereas RasGRF2 predominantly signals to Ras/Erk cascade and mediates long term potentiation [10]
Summary
Yeast Two-hybrid Screen and Constructs—The yeast two-hybrid screen of an adult mouse brain cDNA library (BALB/c males, ages 9 –12 weeks) constructed in the GAL4 activation domain plasmid pACT2 was carried out using the DH PH region of mouse RasGRF1 (aa 249 –592) as bait. For expression of the myc-tagged DH PH tandem of RasGRF1 in mammalian cells the DNA fragment corresponding to aa 238 –595 of mouse sequence was amplified by PCR with primers designed to introduce proper restriction sites and subcloned into pcDNA3 downstream to a previously introduced myc epitope. After rocking for 2 h at 4 °C, the beads were washed with lysis buffer and incubated with 1 mg of total protein extract for each sample from HEK293 cells transfected with RasGRF1 full-length or myc-DH PH, overnight at 4 °C, and processed as above. Lysates from HEK293 cells transfected with the indicated plasmids (1 mg for each sample) were incubated with anti-FLAG M2 monoclonal antibodies (5 g, Sigma) overnight at 4 °C. Student’s t test was applied for statistical analysis between two treatments
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