The Neuron-Specific Rai (ShcC) Adaptor Protein Inhibits Apoptosis by Coupling Ret to the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

Abstract

Rai is a recently identified member of the family of Shc-like proteins, which are cytoplasmic signal transducers characterized by the unique PTB-CH1-SH2 modular organization. Rai expression is restricted to neuronal cells and regulates in vivo the number of postmitotic sympathetic neurons. We report here that Rai is not a common substrate of receptor tyrosine kinases under physiological conditions and that among the analyzed receptors (Ret, epidermal growth factor receptor, and TrkA) it is activated specifically by Ret. Overexpression of Rai in neuronal cell lines promoted survival by reducing apoptosis both under conditions of limited availability of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) and in the absence of Ret activation. Overexpressed Rai resulted in the potentiation of the Ret-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt. Notably, increased Akt phosphorylation and PI3K activity were also found under basal conditions, e.g., in serum-starved neuronal cells. Phosphorylated and hypophosphorylated Rai proteins form a constitutive complex with the p85 subunit of PI3K: upon Ret triggering, the Rai-PI3K complex is recruited to the tyrosine-phosphorylated Ret receptor through the binding of the Rai PTB domain to tyrosine 1062 of Ret. In neurons treated with low concentrations of GDNF, the prosurvival effect of Rai depends on Rai phosphorylation and Ret activation. In the absence of Ret activation, the prosurvival effect of Rai is, instead, phosphorylation independent. Finally, we showed that overexpression of Rai, at variance with Shc, had no effects on the early peak of mitogen-activated protein kinase (MAPK) activation, whereas it increased its activation at later time points. Phosphorylated Rai, however, was not found in complexes with Grb2. We propose that Rai potentiates the MAPK and PI3K signaling pathways and regulates Ret-dependent and -independent survival signals.Shc-like gene sequences are present in all multicellular organisms analyzed to date, from nematodes to Homo sapiens (18). Three mammalian Shc genes have been identified, termed Shc (ShcA), Sli (ShcB/SCK), and Rai (ShcC/N-Shc) (26-28). Due to alternative initiation codon usage and splicing patterns, these three genes encode at least six proteins (p66Shc, p52Shc, p46Shc, p64Rai, p52Rai, and p68Sli), all of which contain a unique PTB-CH1-SH2 modular organization. PTB and SH2 are phosphotyrosine-binding domains that are found in hundreds of different proteins. The concomitant presence of the PTB and SH2 domains (in the N-PTB-SH2-C order) is unique to the proteins of the Shc family (18). The CH1 region contains tyrosine phosphorylation sites and several putative SH3-binding sites that might be important for the signaling function(s) of the Shc-like proteins.The modular organization of the proteins of the Shc family suggests that they function as adaptors within cytoplasmic signaling pathways. Indeed, several lines of evidence support a general role for the p52/46Shc proteins in the transduction of si...

Domain-specific function of ShcC docking protein in neuroblastoma cells

“…Cells expressing 3YF-ShcC become more susceptible to RA-induced apoptosis presumably due to inhibition of the Akt pathway. It has recently been shown that ShcC is physiologically involved in the regulation of the PI3K/Akt pathway as a downstream effecter of the ligand-stimulated Ret receptor in neuroblastoma cells (Pelicci et al, 2002). This study confirms that the survival of NB-39-nu cells is regulated by the signals downstream of ShcC.…”

Biological Role of Anaplastic Lymphoma Kinase in Neuroblastoma

“…28 A recent study also showed that ShcC is a physiological substrate of Ret kinase and that it exerts a prosurvival function in neuronal cells. 40 Although high levels of TrkA expression correlate with a favorable outcome of neuroblastoma patients, 20 TrkA expression was significantly high in NB-39-nu and Nagai, which derive from tumors with a poor prognosis. This discrepancy may also be explained by the overwhelming control of cell survival by ALK kinase in these cell lines.…”

Neural stem and progenitor cells: choosing the right Shc

“…It was also found that ShcC elicits these effects through a different kinetic of activation of downstream effector molecules with respect to ShcA. Indeed, ShcC elicits neuronal differentiation via prolonged stimulation of the MAPK (Conti et al, 2001;Pelicci et al, 2002). This behavior is reminiscent of that described in PC12 cells exposed to NGF, where persistent activation of MAPK is required for neuronal differentiation.…”

“…This behavior is reminiscent of that described in PC12 cells exposed to NGF, where persistent activation of MAPK is required for neuronal differentiation. On the contrary, ShcC-driven prosurvival effect occurs via recruitment of the PI3K-Akt pathway (Conti et al, 2001;Pelicci et al, 2002), as demonstrated by the fact that its pharmacological or molecular inhibition markedly abolishes this effect. To this respect, ShcC-induced Akt activation was found to cause phosphorylation (with inhibition) of Bad, a proapoptotic member of the Bcl2 family (Conti et al, 2001).…”