Phosphorylation of neurofibromin by PKC is a possible molecular switch in EGF receptor signaling in neural cells

Children with neurofibromatosis (NF1) typically develop central nervous system (CNS) abnormalities, including aberrant proliferation of astrocytes and formation of benign astrocytomas. The NF1 gene encodes neurofibromin, a Ras-GAP, highly expressed in developing neural cells; the mechanism of regulation of neurofibromin as a Ras-GAP, remains however unknown. We now show that, in response to EGF, neurofibromin is in vivo phosphorylated on serine residues by PKC-alpha, in human, rat, and avian CNS cells and cell lines. EGF-induced PKC phosphorylation was prominent in the cysteine/serine-rich domain (CSRD) of neurofibromin, which lies in the N-terminus and upstream of the Ras-GAP domain (GRD), and this modification significantly increased the association of neurofibromin with actin in co-immunoprecipitations. In addition, we show that Ras activation in response to EGF was significantly lowered when C62B cells overexpressed a construct encoding both CSRD+GRD. Moreover, when PKC-alpha was downregulated, the Ras-GAP activity of CSRD+GRD was significantly diminished, whereas overexpressed GRD alone acted as a weaker GAP and in a PKC-independent manner. Most importantly, functional Ras inhibition and EGF signaling shifts were established at the single cell level in C6-derived cell lines stably overexpressing CSRD+GRD, when transient co-overexpression of Ras and PKC-depletion prior to stimulation with EGF-induced mitosis. Taken together, these data provide the first evidence of a functional, allosteric regulation of GRD by CSRD, which requires neurofibromin phosphorylation by PKC and association with the actin cytoskeleton. Our data may suggest a novel mechanism for regulating biological responses to EGF and provide a new aspect for the understanding of the aberrant proliferation seen in the CNS of children with NF1.