Galpha12 and Galpha13 stimulate Rho-dependent tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130 Crk-associated substrate.
We examined whether constitutively active mutants of the Galpha proteins Galpha12 and Galpha13, which together comprise the G12 subfamily of Galpha proteins, induce Rho-dependent tyrosine phosphorylation of the focal adhesion proteins p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate. We report that transient expression of the constitutively active mutants of Galpha12 or of Galpha13 in human embryonic kidney 293 cells stimulates tyrosine phosphorylation of a set of proteins of Mr of 110,000-130,000, 97,000, and 60,000-70,000. We identified p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate as prominent tyrosine-phosphorylated proteins in human embryonic kidney 293 cells expressing constitutively active Galpha12 and Galpha13. In common with the increased tyrosine phosphorylation of these proteins mediated by mitogens acting through heptahelical receptors, the Galpha12- and Galpha13-mediated increase in tyrosine phosphorylation is blocked by cytochalasin D, which specifically disrupts the actin cytoskeleton, and by the Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho. Our results support the hypothesis that Galpha12 and Galpha13 activate Rho and suggest that Galpha12 and Galpha13 may mediate the tyrosine phosphorylation of p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate.
Needham
L K
LK
Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.
Rozengurt
E
E
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000199:Actins / Q000502:physiology; D001905:Botulinum Toxins; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D050719:Crk-Associated Substrate Protein; D015638:Cytochalasin D / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000502:physiology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000196:embryology; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction / Q000502:physiology; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection / Q000235:genetics; D054460:rho-Associated Kinases
D000199:Actins; C493841:BCAR1 protein, human; D015815:Cell Adhesion Molecules; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D019000:Phosphotyrosine; D015638:Cytochalasin D; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D013917:Thrombin; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins