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Fyn-Dependent PI 3K Activation in the Regulation of Beta2 Integrin- Supported Cell Migration in Response to N-Formyl Peptide

Posted on: Thursday, 17 June 2004, 06:00 CDT

Aim. The activation of LFA-1 on the cell membrane is tightly regulated by the association of cytoplasmic molecules to the cytoplasmic domain of the integrin during leukocyte interaction with endothelial cells. Although biochemical and pharmacological approaches have implicated phosphatidylinhosithol 3 kinase (PI 3K) in chemoattractant induction of LFA-1 activation, the sequence of events that allow this regulation is not well characterized. Methods. In order to identify signaling molecules involved in chemoattractant receptor-integrin crosstalk we analyzed LFA-1 association with members of different families of kinases by measuring both Src kinase and lipid kinase activities associated with LFA-1 immunoprecipitates from a lymphoid cell line stably transfected with the formyl-peptide receptor (FPR). LFA1-associated PI 3kinase activity was detected upon treatment of the cells with formyl-peptide (fMet-Leu-Phe). Results. The increase of PI 3K activity was G protein and Src kinase dependent as shown by impairment of LFA-1-associated PI 3K activity in cells treated with pertussis toxin and PP2 pharmacological inhibitors respectively. We were able to demonstrate increased association of lipid kinase activity to Fyn but not to Lck immunoprecipitates upon FPR triggering suggesting that Src-dependent activation of PI 3K may be specifically induced by Fyn kinase in these cells. In addition, Fyn protein was detected in LFA-1 immunoprecipitates of cells stimulated with fMetLeu-Phe but not in unstimulatecl cells, suggesting that the chemoattractant promotes the direct or indirect association of the kinase with LFA-1. We thus analyzed the relevance of Fyn in the regulation of LFA1-associated PI 3K activation and of LFA-1- dependent cell migration. We were able to observe an impairment of LFA-1 associated PI 3Kinase activation in cells transiently expressing kinase inactive and SH2 mutant forms of Fyn compared to control cells. We thus conclude that the increase of PI 3K activation in proximity of LFA-1 is at least in part mediated by Fyn. LFA-1-supported migration in response to fMet-Leu-Phe of cells expressing the mutant forms of Fyn was also significantly lower than migration of cells transfected with control vector. On the contraiy, ICAM-1-inclependent migration was not affected. Conclusion. Our observations indicate that chemoattractant receptor-triggering induces Fyn-clependent PI 3K activation in association with LFA-1 and that activation of Fyn is necessaiy to initiate and/or to regulate directional migration that depend upon chemoattractant- mediated LFA-1 activation.

G. Bernardini1, J.Y. Kim2, A. Gismondi1, E.C. Butcher2, A. Santoni1

1Department of Experimental Medicine and Pathology, Fondazione CenciBolognetti, La Sapienza University of Rome, Rome, Italy; 2Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

Copyright Edizioni Minerva Medica Mar 2004

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