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-a, 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 Nterminus 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 þ GRD. Moreover, when PKC-a was downregulated, the Ras-GAP activity of CSRD þ 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 þ 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.

Differential Regulation of RasGAPs in Cancer

“…72 In addition, protein kinase C (PKC) has also been shown to phosphorylate NF1. 73,74 In response to EGF, PKCα-mediated phosphorylation of NF1 increases its RasGAP activity and also its interaction with actin. 73 Altogether, this might point at NF1 targeting K-Ras at the plasma membrane, which requires an intact actin cytoskeleton for signaling.…”

“…73,74 In response to EGF, PKCα-mediated phosphorylation of NF1 increases its RasGAP activity and also its interaction with actin. 73 Altogether, this might point at NF1 targeting K-Ras at the plasma membrane, which requires an intact actin cytoskeleton for signaling. 75 Further implicating protein kinases in NF1 localization and activity, NF1 can be degraded via the proteosome with growth factors that stimulate both G protein-coupled receptors and receptor tyrosine kinases.…”

Interplay Between Protein Kinase C Isoforms Alpha and Epsilon, Neurofibromin, and the Ras/MAPK Pathway in Neuroblastoma Differentiation

“…Neurofibromin is phosphorylated by PKCs in vitro, particularly at the C-terminal domain (Izawa et al, 1996). Evidence for direct PKC and PKC -dependent neurofibromin phosphorylation was www.intechopen.com documented later in cultured neurons, and neuroblastoma and glioma cell lines (Mangoura et al, 2006a). Moreover, phosphorylation of neurofibromin results in both its increased association with actin and enhancement of its GAP activity (Mangoura et al, 2006a).…”

“…Activation of PKCs and the ensuing Ras/ERK signalling cascade have been highlighted as central modulators of NB differentiation, with novel (PKC ) and conventional (PKC ) PKC isoforms critically controlling the signalling output and dynamics of MAPKs (Griner & Kazanietz, 2007). The importance of the PKC/Ras/ERK pathway is further emphasized by recent studies showing that neurofibromin, a prominent tumor suppressor and a neuronal RasGAP protein (a) is a PKC and PKC substrate (Mangoura et al, 2006a) actively phosphorylated during phorbol ester-induced differentiation (Leondaritis et al, 2009) and (b) provides responsiveness to retinoic acid (Holzel et al, 2010). In these studies, the role of PKC and PKC may be viewed as differential and even opposing, with PKC emerging as a crucial, neuronal differentiationspecific PKC isoform.…”

“…Mechanistically, PKC activation is necessary for the formation of Ras and activated Raf-1 complexes Hamilton et al, 2001). PKCs may directly phosphorylate Raf (Ueda et al, 1996), RasGEFs (Ebinu et al 1998;Roose et al, 2005;Zheng et al, 2005), and RasGAPs (Izawa et al, 1996;Mangoura et al, 2006a;Leondaritis et al, 2009), hence regulating the output of Ras/ERK signalling at multiple levels. PKCs on GEFs: Members of the GRP family of RasGEFs possess C1-domains that "recognize" DAG produced by phospholipases in the membrane upon membrane receptor stimulation, and through these interactions translocate to the membranes too (Ebinu et al 1998).…”