Neurofibromin interacts with CRMP-2 and CRMP-4 in rat brain

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Abstract

Neurofibromin, encoded by the neurofibromatosis type 1 (NF1) gene, regulates the Ras and cAMP pathways and plays a role in proliferation and neuronal morphogenesis. The details of the molecular mechanism of neurofibromin action in these processes are still unclear. In this study, immunoprecipitation and proteomics were used to identify novel proteins from rat brain that interact with neurofibromin. Mass spectrometry analysis showed that two proteins, the collapsin response mediator protein-2 (CRMP-2) and propionyl-CoA carboxylase alpha chain (PCCA), associated with neurofibromin. Immunoprecipitation–immunoblotting analysis confirmed the interactions between neurofibromin and CRMP-2 and CRMP-4, but not CRMP-1, in rat brain. CDK5, a kinase that regulates CRMP-2 in axonal outgrowth, was required for the interaction between neurofibromin and CRMP-2. Since both neurofibromin and CRMP proteins are involved in proliferation and axonal morphogenesis, these results suggest that the interaction with CRMPs contributes to the function of neurofibromin in tumorigenesis and neuronal morphogenesis.

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Materials and methods

Antibodies. Neurofibromin antibodies NF1(N) and NF1(D) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Myc antibody 9B11 and CRMP-4 antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA) and BD Biosciences (San Jose, CA), respectively. Rabbit polyclonal pan-CRMP antibodies were generated using a GST-tagged full length CRMP-2 recombinant protein, and the resulting pan-CRMP antibodies were purified by His-tagged-CRMP-2 conjugated to an AminoLink® Plus column

Results and discussion

To identify neurofibromin-associated proteins, proteins from homogenized rat brains were co-immunoprecipitated using the neurofibromin NF1(D) antibody and separated by 2D electrophoresis. Two sets of phosphorylated proteins, around 60 and 70 kDa, were precipitated by the neurofibromin antibody but not by control IgG (Fig. 1A). Mass spectrometry analysis identified the phosphorylated proteins at ∼60 kDa as collapsin response mediator protein-2 (CRMP-2) (Fig. 1B and C) and the phosphorylated

Acknowledgments

We thank Dr. Li-Huei Tsai, Massachusetts Institute of Technology, USA, for the CDK5 and p35 constructs, and the staff at the Proteomic Core Facility at the Institute of Biological Chemistry, Academia Sinica for technical assistance. This work was supported by grants from Academia Sinica, the National Science Council (NSC 96-2321-B-001-005 to Y.-P. H.), and the National Health Research Institute (NHRI-EX96-9403NI to Y.-P. H.).

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