P2Y 12 receptor signalling towards PKB proceeds through IGF-I receptor cross-talk and requires activation of Src, Pyk2 and Rap1

Abstract

Previously it was shown that stimulation of the P2Y 12 receptor activates PKB signalling in C6 glioma cells [K. Van Kolen and H. Slegers, J. Neurochem. 89, 442.]. In the present study, the mechanisms involved in this response were further elucidated. In cells transfected with the Gβγ-scavenger β-ARK1/GRK2 or Rap1GAPII, stimulation with 2MeSADP failed to enhance PKB phosphorylation demonstrating that the signalling proceeds through Gβγ-subunits and Rap1. Moreover, Rap1-GTP pull-down assays revealed that P2Y 12 receptor stimulation induced a rapid activation of Rap1. Treatment of cells with the Ca 2+ chelator BAPTA-AM and inhibition of Src and PLD2 with PP2 or 1-butanol, respectively, abrogated P2Y 12 receptor-mediated activation of Rap1 and PKB. In addition inhibition of PKCζ decreased basal and 2MeSADP-stimulated phosphorylation of PKB indicating a role for this PKC isoform in PKB signalling. Although the increased PKB phosphorylation was abolished in the presence of the IGF-I receptor tyrosine kinase inhibitor AG 1024, 2MeSADP did not significantly increase receptor phosphorylation. Nevertheless, phosphorylation of a 120 kDa IGF-I receptor-associated protein was observed. The latter protein was identified by MALDI-TOF/TOF-MS as the proline-rich tyrosine kinase 2 (Pyk2) that co-operates with Src in a PLD2-dependent manner. Consistent with the signalling towards Rap1 and PKB, activation of Pyk2 was abrogated by Ca 2+ chelation, inhibition of PLD2 and IGF-I receptor tyrosine kinase activity. In conclusion, the data reveal a novel type of cross-talk between P2Y 12 and IGF-I receptors that proceeds through Gβγ-, Ca 2+-and PLD2-dependent activation of the Pyk2/Src pathway resulting in GTP-loading of Rap1 required for an increased PKB phosphorylation.