title: Phosphorylation of neurofibromin by PKC is a possible molecular switch in EGF receptor signaling in neural cells.
abstract: 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.
authors.affiliation: Department of Pediatrics, The University of Chicago, Chicago, IL, USA. mangoura@bioacademy.gr and Neurosciences Division, Institute for Biomedical Research, Athens, Greece.
authors.forename: D
authors.initials: D
authors.lastname: Mangoura
authors.forename: Y
authors.initials: Y
authors.lastname: Sun
authors.forename: C
authors.initials: C
authors.lastname: Li
authors.forename: D
authors.initials: D
authors.lastname: Singh
authors.forename: D H
authors.initials: DH
authors.lastname: Gutmann
authors.forename: A
authors.initials: A
authors.lastname: Flores
authors.forename: M
authors.initials: M
authors.lastname: Ahmed
authors.forename: G
authors.initials: G
authors.lastname: Vallianatos
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D025542:Neurofibromin 1 / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D020703:ras GTPase-Activating Proteins / Q000378:metabolism
chemical_list: D017931:DNA Primers; D025542:Neurofibromin 1; D020703:ras GTPase-Activating Proteins; D066246:ErbB Receptors; D011493:Protein Kinase C