Adapter protein CRKII signaling is involved in the rat pancreatic acini response to reactive oxygen species.
Recent studies demonstrate that reactive oxygen species (ROS) are important mediators of acute pancreatitis, whether induced experimentally or in necrotizing pancreatitis in humans; however, the cellular processes involved remain unclear. Adapter protein CrkII, plays a central role for convergence of cellular signals from different stimuli. Cholecystokinin (CCK), which induces pancreatitis, stimulates CrkII tyrosine phosphorylation and CrkII protein complexes, raising the possibility it can be important in the acinar cell responses to ROS. Therefore, our aim was to investigate whether CrkII signaling is involved in the biological response of rat pancreatic acini to H2O2 and the intracellular mediators implicated. Treatment of isolated rat pancreatic acini with H2O2 rapidly stimulates CrkII phosphorylation, measured as electrophoretic mobility shift and by using a phosphospecific antibody (pTyr221). Tyrosine kinase blocker B44 inhibits the higher phosphorylation state, demonstrating that it occurs mainly in tyrosine residues. H2O2-induced CrkII phosphorylation is time- and concentration-dependent, showing maximal effect with 3 mM H2O2 at 5 min. The intracellular pathways induced by H2O2 leading to CrkII tyrosine phosphorylation do not involve PKC, intracellular calcium, PI3-K or the actin cytoskeleton integrity. ROS generation clearly promotes the formation of protein complex CrkII-PYK2. In conclusion, ROS clearly affect the key adapter protein CrkII signaling by two ways: stimulation of CkII phosphorylation and a functional consequence: formation of CrkII-protein complexes. Because of its central role in activating more distal pathways, CrkII might likely play an important role in the ability of ROS to induce pancreatic cellular injury and pancreatitis.
Andreolotti
Alberto G
AG
Department of Physiology, University of Extremadura, Cáceres 10071, Spain.
Bragado
María J
MJ
Tapia
José A
JA
Jensen
Robert T
RT
Garcia-Marin
Luis J
LJ
D000199:Actins; D000818:Animals; D002118:Calcium / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008297:Male; D010179:Pancreas / Q000378:metabolism*; D046790:Pancreas, Exocrine / Q000187:drug effects / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology; D050737:Proto-Oncogene Proteins c-crk / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000494:pharmacology*; D015398:Signal Transduction; D013997:Time Factors
D000199:Actins; D050737:Proto-Oncogene Proteins c-crk; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D011494:Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D051418:Focal Adhesion Kinase 2; D002118:Calcium