2.3. NF1 protein

The NF1 protein (neurofibromin) is relatively large, containing 2818 amino acids with a calculated molecular mass of 327kDa (Fig. 1) (Marchuk et al. 1991). However, the molecular mass identified in electrophoretic analysis is 220-280kDa (DeClue et al. 1991, Gutmann et al. 1991, Daston et al. 1992, Hirvonen et al. 1998). This is most likely due to the protein folding during migration through denaturing polyacrylamide gels because there is no evidence of glycosylation or processing of the full-length protein (Gutmann & Collins 1993). The NF1 protein has a GAP-related (GRD) domain, which is about 300-400 amino acids long, in the middle of the protein product (Gutmann & Collins 1993).

The NF1 protein is expressed in a variety of cell types in adults and during embryonic development (Daston & Ratner 1992, Daston et al. 1992, Huynh et al. 1994). The expression is most abundant in the nervous system (Daston et al. 1992). Subcellular localization studies have shown the NF1 protein to occur in the particulate and soluble fractions of the cytoplasm (DeClue et al. 1991, Golubic et al. 1992, Hattori et al. 1992). The NF1 protein has been shown to associate with microtubules, actin filaments, mitochondria, smooth endoplasmic reticulum, the nuclear/perinuclear area or heparan sulphate proteoglycans (Gregory et al. 1993, Nordlund et al. 1993, Roudebush et al. 1997, Hsueh et al. 2001, Li et al. 2001). The NF1 protein has also been shown to be constitutively phosphorylated at the cysteine/serine-rich domain of the N-terminus and the C-terminal region (Izawa et al. 1996). The half-life of the NF1 protein is regulated by a post-translational mechanism, which is likely to depend on protein phosphorylation. Its half-life is increased in melanocytes by the addition of growth factors, such as the basic fibroblast growth factor (bFGF) or phorbol myristate acetate (PMA) (Griesser et al. 1997, Kaufmann et al. 1999b). A recent study has shown that the NF1 protein is rapidly degraded in response to various growth factors, and the protein levels are re-elevated shortly afterwards in NIH3T3 fibroblasts, primary IMR90 cells and RT4 Schwannoma cells (Cichowski et al. 2003).

The best-known function of the NF1 protein is to act as a Ras-GAP. The Ras-GAP proteins stimulate the intrinsic Ras-GTPase to hydrolyze Ras attached GTP to GDP and inactivate the Ras by doing this (Fig. 2). The Ras-GAP function of the NF1 protein was first suggested after gene cloning and the identification of a region homologous to other Ras-GAP proteins (Ballester et al. 1990, Xu et al. 1990b). p120GAP and GAPIII are the closest relatives, sharing ~30% identity with the GAP region of the NF1 protein. The NF1 protein has been shown to interact with Ras-GTP and to stimulate its GTPase activity (Martin et al. 1990, Xu et al. 1990a, Bollag & McCormick 1991). The affinity of the NF1 protein to Ras-GTP is higher than that of p120GAP, but the stimulation of the intrinsic GTPase activity of Ras is less marked compared to p120GAP (Martin et al. 1990, Bollag & McCormick 1991). The NF1 protein also binds to oncogenic Ras, but is not able to stimulate its intrinsic GTPase activity (Xu et al. 1990a, Bollag & McCormick 1991). Furthermore, certain lipids (e.g., arachidonic acid, phosphatidic acid, stearic acid, oleic acid, phosphatidylinositol-4,5-bisphosphate, some n-6 and n-3 polyunsaturated fatty acids) decrease the GAP activity of the NF1 protein (Bollag & McCormick 1991, Golubic et al. 1991).

Elevated Ras-GTP levels have been demonstrated in some cells and tissues obtained from NF1 patients. Elevated Ras-GTP levels have been demonstrated in Schwann cells, primary leukemias, malignant schwannomas, neurogenic sarcomas and dermal neurofibromas (Basu et al. 1992, Yan et al. 1995, Bollag et al. 1996, Guha et al. 1996, Sherman et al. 2000). However, low levels of NF1 protein have not been shown to lead to increased Ras-GTP levels in various cell types, including melanomas, neuroblastomas, melanocytes and fibroblasts (Boddrich et al. 1995, Griesser et al. 1995, Sherman et al. 2000). Transfection studies have shown that overexpression of NF1 protein may lead to suppression of cell growth, transformed phenotype and decreased tumorigenicity without any change in Ras-GTP levels, suggesting that the NF1 gene can act independently of GAP activity (Johnson et al. 1994, Li & White 1996). Furthermore, in Drosophila, the NF1 protein seems to be a regulator of the cAMP-PKA-dependent signaling pathway instead of the Ras pathway (Guo et al. 1997, The et al. 1997, Tong et al. 2002). Defective calcium signaling has also been demonstrated in keratinocytes cultured from NF1 patients (Korkiamaki et al. 2002).

Taken together, the NF1 protein seems to be involved in the regulation of the Ras-MAPK pathway. However, the GAP region of the NF1 protein is only a fraction of the length of the total protein product. Consequently, it is likely that the NF1 protein has functions other than acting as a Ras-GAP. These other functions could involve regulation of the cAMP/PKA pathway or calcium-related signaling. The complexity of the regulation of the NF1 protein is shown by studies demonstrating various subcellular localizations for it.