Fluoroaluminate stimulates phosphorylation of p130 Cas and Fak and increases attachment and spreading of preosteoblastic MC3T3-E1 cells.
Fluoroaluminate is a G-protein activator, it stimulates osteoblastic cells in culture, and is a bone-forming agent in vivo. To elucidate the mechanisms of G-protein-mediated action of fluoroaluminate in osteoblasts, we studied protein tyrosine phosphorylation in the preosteoblastic cell line MC3T3-E1. Fluoroaluminate, lysophosphatidic acid (LPA; an agonist for G-protein-coupled receptor), or adhesion to type I collagen all stimulated phosphorylation of a similar set of proteins, including p130, p120, p110 (previously identified as proline-rich tyrosine kinase 2, Pyk2), and p70. The phosphorylation of these proteins was sensitive to an Src inhibitor, but not to a Gi-protein inactivator, pertussis toxin. By purification/mass spectrometry and by immunodepletion, p130 protein was identified as p130 Cas (Crk-associated protein), a Src substrate and a protein involved in signaling by cell-adhesion receptors, integrins. Phosphorylation of immunoprecipitated p130 Cas increased upon stimulation with fluoroaluminate and with agonists of G-protein-coupled receptors, but not with growth factors. By immunodepletion, the p120 protein was identified as focal adhesion kinase, Fak. The addition of fluoroaluminate during cell attachment to type I collagen further stimulated phosphorylation of p130 Cas and of Fak. Simultaneously, fluoroaluminate increased the number of attached MC3T3-E1 cells and their spreading. These novel aspects of fluoroaluminate action in cell culture may be important for the bone-forming action of fluoroaluminate in vivo.
Freitas
F
F
Research Bone Metabolism Novartis Pharma AG, Basel, Switzerland.
Jeschke
M
M
Majstorovic
I
I
Mueller
D R
DR
Schindler
P
P
Voshol
H
H
Van Oostrum
J
J
Susa
M
M
D000535:Aluminum / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D050719:Crk-Associated Substrate Protein; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005461:Fluorine / Q000494:pharmacology*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D037342:Pertussis Toxin; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins; D011743:Pyrimidines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; D007328:Insulin; D008246:Lysophospholipids; D010750:Phosphoproteins; D011506:Proteins; D011743:Pyrimidines; D011758:Pyrroles; D050717:Retinoblastoma-Like Protein p130; D010566:Virulence Factors, Bordetella; C053629:fluoroaluminum; D005461:Fluorine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D003094:Collagen; D000535:Aluminum; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D019061:src-Family Kinases; D019204:GTP-Binding Proteins; C119957:CGP 77675