Mecanismo de activación de la tirosina quinasa c-Src en células intestinales estimuladas con PTH

GENTILI CLAUDIA; MORELLI SUSANA; RUSSO DE BOLAND ANA

Buenos Aires

Congreso; XXI Reunión Anual de la Asociación Argentina de Osteología y Metabolismo Mineral (AAOMM); 2004

Asociación Argentina de Osteología y Metabolismo Mineral (AAOMM)

Parathyroid hormone (PTH) is a major mediator of bone remodeling and isan essential regulator of calcium homeostasis. PTH interacts in targettissues with a G protein-coupled receptor (GPCR) localized in the plasmamembrane. The heterotrimeric G proteins control diverse biologicalprocesses by conveying signals from cell-surface receptors to intracellulareffectors. Upon activation, the receptor facilitates the exchange of GDP forGTP in the Ga subunit. Ga is then thought to dissociate from the Ghgheterodimer allowing both complexes to play active roles in the signalingprocess through downstream regulation of effectors. Although activation ofGPCR can elicit rapid stimulation of cellular protein tyrosine phosphorylation,the mechanism by which G proteins activate protein-tyrosinekinases is not completely understood. In the present work we demonstratedthat PTH rapidly increases the activity of non-receptor tyrosine kinase c-Src in rat intestinal cells (enterocytes). The response is biphasic, the earlyphase is fast and transient, peaking at 30 sec (+120%) while the secondphase is maximal at 5 minutes of treatment with the hormone (+220%). Toevaluate whether G-proteins are required for PTH-induced c-Src activation,the enterocytes were treated with PTH (10-8 M, 30 sec-5 min) and celllysates were immunoprecipitated with a specific anti-cSrc antibody,resolved on SDS-PAGE, followed by Western blotting with anti-Gh,anti-Gas or anti-Gai antibodies. In basal condition the three subunits areassociated with c-Src and this association increases two-three fold, andwith different temporal profile, in cells treated with PTH. In addition, thehormone activates c-Src in intestinal cells through changes in the tyrosinephosphorylation of the enzyme. The response is very fast and biphasicwith a peak at 1 minute (+50%) and a second peak at 5 minute (+100%) ofPTH treatment. We demonstrate that the first event in the activation of c-Src is the dephosphorylation of tyrosine 527 (that happens after fewseconds of hormonal treatment), followed by a second event of activationwith phosphorylation at tyrosine 416.These results show that PTH activates c-Src in intestinal cells throughconformational changes via G proteins and modulates the phosphorylationof tyrosine residues of the enzyme.