Adrenocorticotrophic hormone stimulates phosphotyrosine phosphatase SHP2 in bovine adrenocortical cells: phosphorylation and activation by cAMP-dependent protein kinase

7During activation of adrenocortical cells by adrenocorticotrophic hormone (ACTH), tyrosine dephosphorylation of paxillin is stimulated and this corre lates with protrusion of filopodial structures and a decreased number of fo cal adhesions. These effects are inhibited by Na3VO4, a phosphotyrosine pho sphatase inhibitor [Vilgrain, Chinn, Gaillard, Chambaz and Feige (1998) Bio chem. J. 332, 533-540]. However, the tyrosine phosphatases involved in thes e processes remain to be identified. In this study, we provide evidence tha t the Src homology domain (SH)2-containing phosphotyrosine phosphatase (SHP )2, but not SHP1, is expressed in adrenocortical cells and is phosphorylate d upon ACTH challenge. ACTH (10(-8) M) treatment of P-32-labelled adrenocor tical cells resulted in an increase in phosphorylated SHP2, By probing SHP2 -containing immunoprecipitates with an antibody to phosphoserine we found t hat SHP2 was phosphorylated on serine in ACTH-treated cells in a dose- and time-dependent manner. Furthermore, using an in vitro kinase assay, we show ed that SHP2 was a target for cAMP-dependent protein kinase (PKA). Serine w as identified as the only target amino acid phosphorylated in SHP2. Phospho rylation of SHP2 by PKA resulted in a dramatic stimulation of phosphatase a ctivity measured either with insulin receptor substrate-1 or with the synth etic peptide [P-32]poly(Glu/Tyr) as substrate. In an in-gel assay of SHP2-c ontaining immunoprecipitates, phosphorylated in vitro by PKA or isolated fr om adrenocortical cells treated with 10 nM ACTH, a pronounced activation of SHP2 activity was shown. These observations clearly support the idea that a PKA-mediated signal transduction pathway contributes to SHP2 regulation i n adrenocortical cells and point to SHP2 as a possible mediator of the effe cts of ACTH.