Tyrosine phosphorylation is involved in reorganization of the actin cytoskeleton in response to serum or LPA stimulation.
Tyrosine phosphorylation is known to regulate the formation of focal adhesions in cells adhering to extracellular matrix (ECM). We have investigated the possible involvement of tyrosine phosphorylation and the focal adhesion kinase (FAK) in the cytoskeletal changes induced by serum or lysophosphatidic acid (LPA) in quiescent Swiss 3T3 fibroblasts. As shown previously by others, quiescent cells stimulated with serum or LPA reveal a rapid reappearance of focal adhesions and stress fibers. Here we show that this is accompanied by an increase in phosphotyrosine in focal adhesions and specifically an increase in the tyrosine phosphorylation of FAK. The LPA-stimulated reappearance of focal adhesions and stress fibers is blocked by inhibitors of phospholipase C but not by pertussis toxin (PTX), indicating that this LPA signaling pathway is mediated by phospholipase C activation and does not involve PTX-sensitive G proteins. In the absence of serum or LPA, these cytoskeletal effects and the tyrosine phosphorylation of FAK can be mimicked by sodium orthovanadate in conjunction with hydrogen peroxide, agents that inhibit protein tyrosine phosphatases and thereby elevate levels of phosphotyrosine. Two tyrosine kinase inhibitors, erbstatin and genistein block both the serum-induced tyrosine phosphorylation of FAK and the assembly of focal adhesions and stress fibers. Two other tyrosine kinase inhibitors, tyrphostins 47 and 25, previously shown to inhibit FAK, failed to prevent FAK phosphorylation or the reassembly of focal adhesions and stress fibers in response to serum. However, these inhibitors did prevent FAK phosphorylation and cytoskeletal assembly in response to lysophosphatidic acid (LPA), one component of serum previously shown to stimulate assembly of focal adhesions and stress fibers. Our findings suggest that the response to serum is complex and that although FAK phosphorylation is important, other tyrosine kinases may also be involved.
Chrzanowska-Wodnicka
M
M
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599-7090.
Burridge
K
K
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001790:Blood Physiological Phenomena; D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015151:Immunoblotting; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D037342:Pertussis Toxin; D010766:Phosphorylation; D011233:Precipitin Tests; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014443:Tyrosine / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000199:Actins; D015815:Cell Adhesion Molecules; D008246:Lysophospholipids; D010566:Virulence Factors, Bordetella; D014443:Tyrosine; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D017027:Protein Tyrosine Phosphatases; D010738:Type C Phospholipases