P2Y12 receptor signalling towards PKB proceeds through IGF-I receptor cross-talk and requires activation of Src, Pyk2 and Rap1.
Previously it was shown that stimulation of the P2Y12 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 Gbetagamma-scavenger beta-ARK1/GRK2 or Rap1GAPII, stimulation with 2MeSADP failed to enhance PKB phosphorylation demonstrating that the signalling proceeds through Gbetagamma-subunits and Rap1. Moreover, Rap1-GTP pull-down assays revealed that P2Y12 receptor stimulation induced a rapid activation of Rap1. Treatment of cells with the Ca2+ chelator BAPTA-AM and inhibition of Src and PLD2 with PP2 or 1-butanol, respectively, abrogated P2Y12 receptor-mediated activation of Rap1 and PKB. In addition inhibition of PKCzeta 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 Ca2+ chelation, inhibition of PLD2 and IGF-I receptor tyrosine kinase activity. In conclusion, the data reveal a novel type of cross-talk between P2Y12 and IGF-I receptors that proceeds through Gbetagamma-, Ca2+-and PLD2-dependent activation of the Pyk2/Src pathway resulting in GTP-loading of Rap1 required for an increased PKB phosphorylation.
Van Kolen
Kristof
K
Laboratory of Cellular Biochemistry, Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk-Antwerpen, Belgium.
Gilany
Kambiz
K
Moens
Luc
L
Esmans
Eddy L
EL
Slegers
Herman
H
D000818:Animals; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D010739:Phospholipase D / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058925:Receptors, Purinergic P2Y12; D015398:Signal Transduction; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D008565:Membrane Proteins; C553058:P2ry12 protein, rat; D020033:Protein Isoforms; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D017526:Receptor, IGF Type 1; D051418:Focal Adhesion Kinase 2; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; C105606:phospholipase D2; D010739:Phospholipase D; D020962:Heterotrimeric GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins; D002118:Calcium