Integrin-mediated tyrosine phosphorylation and redistribution of paxillin during neuronal adhesion.
Integrins are important receptors for neuronal adhesion to laminin, which is one of the best promoters of neurite outgrowth. The present study was carried out to understand some of the intracellular mechanisms which allow integrin-mediated neurite extension on laminin. In chicken retinal neurons, integrin-mediated adhesion to laminin and antibody-induced integrin clustering caused an increase in tyrosine phosphorylation of paxillin and focal adhesion kinase. The kinetics of phosphorylation and dephosphorylation of these proteins were different in neurons plated on laminin, compared to neurons in which the receptors were clustered with anti-integrin antibodies. Analysis of sucrose velocity gradients could not show any association of paxillin and focal adhesion kinase with the integrin receptors. On the other hand, by using digitonin and milder extraction conditions, we found an enrichment of the tyrosine-phosphorylated polypeptides in the cytoskeletal, digitonin-insoluble fraction. Furthermore, neuronal adhesion induced a dramatic increase in the fraction of tyrosine-phosphorylated paxillin recovered with the digitonin-insoluble fraction, suggesting redistribution of this protein following adhesion of neurons to laminin. Localization studies on the detergent-insoluble fraction showed codistribution of both paxillin and focal adhesion kinase with integrins. We also found that paxillin tyrosine phosphorylation, but not paxillin expression, is developmentally regulated in the retina. Our results show that integrin-mediated neuronal adhesion leads to the accumulation of a pool of highly phosphorylated proteins at adhesion sites. There they may be responsible for the reorganization of the cytoskeleton, which underlies the process of neurite extension.
de Curtis
I
I
Department of Biological and Technological Research (DIBIT), S. Raffaele Scientific Institute, Milano, Italy. decurti@dibit.hsr.it
Malanchini
B
B
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002457:Cell Extracts; D002478:Cells, Cultured; D002642:Chick Embryo; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D019012:Integrin beta1 / Q000378:metabolism; D016023:Integrins; D007797:Laminin / Q000494:pharmacology; D016501:Neurites; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017474:Receptors, Laminin / Q000378:metabolism; D012160:Retina / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D012503:Saponins / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism*
D000906:Antibodies; D015815:Cell Adhesion Molecules; D002457:Cell Extracts; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D019012:Integrin beta1; D016023:Integrins; D007797:Laminin; D051419:Paxillin; D010750:Phosphoproteins; D017474:Receptors, Laminin; D012503:Saponins; D014443:Tyrosine; D004072:Digitonin