SCLIP, a Microtubule-destabilizing Factor, Interacts with RasGRF1 and Inhibits Its Ability to Promote Rac Activation and Neurite Outgrowth*
- ‡Department of Biomolecular Sciences and Biotechnology and the §Interdepartmental Center for Advanced Microscopy, University of Milan, Via Celoria 26, 20133 Milan, Italy
- 1 To whom correspondence should be addressed: Tel.: 39-02-5031-4914; Fax: 39-02-5031-4912; E-mail: simona.baldassa{at}unimi.it.
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 Dblhomology 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.
Footnotes
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↵3 The abbreviations used are: GEF, guanine nucleotide exchange factor; DH, Dbl homology domain; PH, pleckstrin homology domain; SLD, stathmin-like domain; MAPKs, mitogen-activated protein kinases; Erks, extracellular signal-regulated protein kinases; MTs, microtubules; NGF, nerve growth factor; MBP, maltose-binding protein; GST, glutathione S-transferase; HA, hemagglutinin epitope; GFP, green fluorescent protein; Trk, tropomycin-related kinase; NMDA, N-methyl-d-aspartate; AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; aa, amino acid(s); BSA, bovine serum albumin; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; DIV, days in vitro; JNK1, c-Jun N-terminal kinase 1; SCLIP, SCG10-like protein; SCG10, superior cervical ganglion-10 protein.
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↵4 A. Colombatti, personal communication.
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↵* This work was supported in part by grants from MIUR (PRIN 2005) and by FIRST 2004-5. Confocal facilities were supported by the Centro Interdisciplinare di Materiali e Interfacce Nanostrutturali-Università di Milano. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵2 Supported by E. C. Marie Curie IRG (Grant MIRG-CT-2004-003902).
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- Received May 10, 2006.
- Revision received November 22, 2006.
- The American Society for Biochemistry and Molecular Biology, Inc.
