Identification of RPTPalpha as responsible for Fyn activation in PrPc signalling

Cellular prion protein (PrPc) is a ubiquitous glycoprotein which physiological role is poorly characterized. It has been suggested that PrPc participates in synaptic structure, neurite formation, copper metabolism, and signal transduction (1, 2). PrPc antibody cross-linking stimulates the caveolin-dependent activation of Fyn leading to a rapid and transient phosphorylation of Erk1/2 (2, 3). The activity of Src family tyrosine kinases (SFK) is regulated by tyrosine phosphorylation at two sites, but with opposing effects. Phosphorylation of a tyrosine residue in the activation loop of the kinase domain upregulates enzyme activity, whereas phosphorylation by Csk of a tyrosine residue in the carboxy-terminal tail renders the enzyme less active stabilizing a noncatalytic conformation. An increasing number of protein-tyrosine phosphatases (PTPs) are being implicated in controlling SFK phosphorylation and activity. Such SFK-activating PTPs include both non-receptor (e.g. PTP1B, SHP1, and SHP2) and receptor PTPs (RPTPs, such as CD45, RPTPα, RPTPβ, and LAR) (4). Following this lead, we investigated the possibility that one of these PTPs is involved in PrPc-dependent activation of Fyn. We peformed co-immunprecipitation experiments, PTPs in gel assay and immunoblotting assay with specific antibodies on isolated caveolae-like membrane domains. The analysis were conducted on 2D-electrophoresis gels. The results obtained with all these tests suggest RPTPα as major phosphatase involved in PrPc signalling. This hypothesis was confirmed by cell stimulation experiments after siRNA-mediated RPTPalpha knock down.