Involvement of c-Src tyrosine kinase in SHP-1 phosphatase activation by Ang II AT2 receptors in rat fetal tissues

Abstract

Angiotensin II (Ang II) AT2 receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10-7 M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT2 antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT2 receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT2 or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT2 or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT2 receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT2 receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT2 subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.