P85Interacts With Spred2to Modulate The Activation Of ERK/MAPK Pathway

Sprouty-related proteins with an EVH1domain (Spreds) are a new protein family with an N-terminal Enabled/VASP homology1domain (EVH1), a central c-Kit binding domain (KBD) and a C-terminal Sprouty-related domain (SPR). Spreds, like Sproutys, modulate growth factor receptor signaling by inhibiting the Ras/ERK pathway. Importantly, the SPR (Sprouty-related) domains of Spred-2is essential for Spred-2-mediated inhibitory effect, but the molecular mechanism is largely unknown. In this study, we show that the p85subunit of phosphatidylinositol3-kinase (PI3K) was a new binding partner of Spred-2via the SPR domain and tyrosines303/343/353within this domain of Spred-2were essential for its interation with p85and inhibitory effect on Ras/ERK activation. Mutation of these three tyrosines not only abolished EGF-induced association of Spred-2with p85but also reduced the inhibitory effect on Ras/ERK activation by Spred-2. Consequently, proliferation of Hela cells and neurite outgrowth in PCI2cells were increased. Together, our findings indicate that p85binding to Spred-2enhances the Spred-2-mediated inhibitory effect via increased Ras binding to Spred2and decreased Spred-2ubiquitination. The main results are summarized as follows:1. Endogenous p85subunit of PI3K was immunoprecipitated with Myc-Spred-2in either EGF or FGF-stimulated human embryonic kidney293T cells. We found that p85regulatory subunit of PI3K is a novel binding partner of Spred-2. Furthermore, the SH2domains of p85and the SPR domains of Spred2were essential for the association of Spred-2with p85.2. In order to define the binding site for p85, tyrosines303,343,353at SPR domain of Spred-2were mutated to phenylalanine and a Spred2mutant with three tyrosine residues (Y303, Y343and Y353) mutated to phenylalanine was constructed. These data indicated that tyrosines Y303/Y343/Y353at the SPR domain of Spred-2are required for its interaction with p85and tyrosine phosphorylation of Spred2upon EGF treatment. 3. p85binding to Spred2enhanced Spred-2-mediated inhibitory effect via increased Ras binding to Spred-2and decreased Spred-2ubiquitination and EGF-induced activation of ERK1/2was further attenuated in cells co-transfecting p85with Spred-2compared to cells transfecting with Spred2or p85alone.4. Adenoviruses were constructed to express wild type Spred-2and mutants, then Hela cells and PC12cells were infected with adenoviruses at a multiplicity of infection of125. Our findings indicate that mutations of p85binding sites at Spred2promote Hela cell survival and PC12cell differentiation compared to wild type Spred-2.In summary, we presented evidence that p85is recruited to Spred-2upon EGF stimulation. Furthermore, tyrosines303,343and353within the SPR domain of Spred-2were essential for its interaction with p85and inhibitory effect on Ras/ERK activation. In addition, p85binding to Spred-2enhanced Spred-2-mediated inhibitory effect via increased Ras binding to Spred-2and decreased Spred-2ubiquitination.