Identification of neurofibromatosis type I gene product as an insoluble GTPase-activating protein toward ras p21.

The neurofibromatosis type I (NF1) gene was cloned as a gene whose structural change is closely related to the onset of a neurocutaneous disorder, neurofibromatosis type I. The predicted amino acid sequence of the NF1 gene product shows significant similarity to the catalytic domain of GTPase-activating protein (GAP) for ras p21. This GAP-related domain has been shown to have GAP activity when expressed by recombinant DNA technique, however little is known about the NF1 gene product expressed in tissues. Antibodies against the carboxy terminus and the GAP-related domain of the NF1 gene product were made to identify and characterize NF1 protein expressed in vivo. Both antibodies specifically reacted with a polypeptide with apparent molecular weight of 235 kDa in immunoblot and immunoprecipitation analyses. This 235 kDa protein is detected in brain but not in spleen, thymus, kidney, liver, lung and heart. The subcellular distribution of NF1 and GAP was studied using rat brain extract and was identified by the activity which was inhibited by antibodies against GAP-related domain of NF1 and anti-GAP respectively. The results show that NF1 protein mainly resides in the particulate fraction, in contrast to GAP, which is a soluble protein. We also found that a GAP activity which is not inhibited by anti-NF1 and anti-GAP exists in both the soluble and particulate fractions. The result suggests that there may be another GAP molecule.