Clustering of cell surface (beta)1,4-galactosyltransferase I induces transient tyrosine phosphorylation of focal adhesion kinase and loss of stress fibers.
It is well appreciated that clustering of receptors for the extracellular matrix, most notably the integrins, elicits intracellular signal cascades. One of the first indications that integrin-dependent signaling has occurred is by the activation of focal adhesion kinase (FAK). Another, although less well understood, receptor for the extracellular matrix is (beta)1, 4-galactosyltransferase I (GalT). GalT participates during lamellipodia formation and cell migration by recognizing terminal N-acetylglucosamine residues on basal lamina glycosides. In this study, we investigated whether GalT is also capable of eliciting intracellular signal cascades, specifically FAK activation, in response to ligand binding and/or aggregation. 3T3 fibroblasts were treated with two different reagents capable of aggregating GalT, either antibodies raised against recombinant GalT or multivalent polymers of N-acetylglucosamine, and the effects on tyrosine phosphorylation were analyzed. Both reagents induced an initial tyrosine phosphorylation (1-2 minutes) and subsequent dephosphorylation (5-10 minutes) of proteins with molecular mass 67 and 125 kDa. These proteins were identified as paxillin and FAK, respectively, by immunoprecipitation with anti-paxillin and anti-FAK antibodies. Preimmune IgG, anti-GalT Fab fragments, irrelevant polymers and monomeric N-acetylglucosamine had no effect. The ability of GalT aggregation to induce transient tyrosine phosphorylation was dependent upon cell density. In addition, FAK dephosphorylation was found to be sensitive to the phosphatase inhibitor, sodium pervanadate. Similar to the integrins, GalT requires association with the cytoskeleton in order to function as a matrix receptor. To determine if the transient tyrosine phosphorylation of FAK was dependent upon GalT binding to the cytoskeleton, stably transfected fibroblasts expressing different amounts of GalT were treated with polymeric N-acetylglucosamine. Cells expressing increased levels of GalT associated with the cytoskeleton showed increased levels of FAK tyrosine phosphorylation and prolonged dephosphorylation, relative to control cells. In contrast, cells in which a dominant negative form of GalT prevents association with the cytoskeleton showed no or weak response to polymeric N-acetylglucosamine. Concomitant with the GalT-stimulated dephosphorylation of FAK, cells treated with anti-GalT antibodies or polymeric N-acetylglucosamine showed a loss of actin stress fibers and focal adhesions. Pervanadate treatment inhibited the GalT-dependent loss of actin stress fibers. To confirm the requirement of GalT in transient FAK phosphorylation and stress fiber reorganization in this system, we created cells homozygous null for the GalT isoform that functions as a matrix receptor. These cells were incapable of phosphorylating FAK in response to GalT agonists and, interestingly, showed a lack of lamellar stress fibers when cultured on basal lamina matrices. These data suggest that GalT function as a basal lamina receptor involves transient activation of FAK and an associated reorganization of stress fibers.
Wassler
M J
MJ
Department of Cell Biology, Emory University School of Medicine, Room 100, Atlanta, GA 30322, USA.
Shur
B D
BD
D016475:3T3 Cells; D000117:Acetylglucosamine / Q000494:pharmacology; D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000201:enzymology; D005109:Extracellular Matrix / Q000187:drug effects / Q000201:enzymology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D007797:Laminin / Q000494:pharmacology; D051379:Mice; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D007797:Laminin; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; C113831:poly-N-acetyl glucosamine; D014443:Tyrosine; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D010744:Phosphoric Monoester Hydrolases; D000117:Acetylglucosamine