Identification of novel RasGRF1 interacting partners by large-scale proteomic analysis.
The brain-specific Ras guanine nucleotide exchange factor RasGRF1 is a protein harbouring a complex array of structural motifs. It contains a pleckstrin homology (PH1) domain, a coiled coil region (CC) and an ilimaquinone (IQ) one in addition to the catalytic Ras and Rac exchange factor domains. In this study, we used the recombinant N-terminal PH1, CC and IQ region (PHCCIQ) fused to the chitin-binding domain to isolate interacting proteins from mouse brain extracts. The use of an advanced software tool, the Pep-Miner, allowed clustering similar spectra from multiple mass spectrometry analysis, simplifying and improving the analysis of the complex peptide mixture. The most representative classes of RasGRF1-interacting proteins were ribosomal and other RNA-binding proteins, cytoskeletal proteins and proteins involved in vesicular trafficking. We confirmed the interaction of some of the identified proteins using different experimental approaches. We also demonstrated an RNA-dependent association of the PHCCIQ moiety of RasGRF1 with ribosomal protein S6 and Ras-GTPase-activating protein SH3-domain binding protein 2. In addition, we found that purified total RNA binds to the PHCCIQ fusion protein and the recombinant protein associates with poly(A)-sepharose. These data indicate that RasGRF1 can interact with different protein categories and exhibits a potential RNA-binding property.
Lavagni
Paola
P
Department of Biomolecular Sciences and Biotechnology, University of Milan, Milan, Italy.
Indrigo
Marzia
M
Colombo
Graziano
G
Martegani
Enzo
E
Rosenblum
Kobi
K
Gnesutta
Nerina
N
Zippel
Renata
R
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D051379:Mice; D046228:Microarray Analysis; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020543:Proteome / Q000032:analysis*; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020666:ras-GRF1 / Q000235:genetics / Q000378:metabolism*
D020543:Proteome; D011993:Recombinant Fusion Proteins; D020666:ras-GRF1; D012313:RNA