title: Involvement of phosphorylation of Tyr-31 and Tyr-118 of paxillin in MM1 cancer cell migration.
abstract: We demonstrated previously that rat ascites hepatoma MM1 cells require both lysophosphatidic acid (LPA) and fibronectin (FN) for phagokinetic motility and transcellular migration and that these events are regulated through the RhoA-ROCK pathway. It remains to be elucidated, however, how the signals from both LPA and FN are integrated into cell migration. To examine this, total cellular lysates after stimulation with LPA or FN were subjected to time-course immunoblot analysis with anti-phosphotyrosine antibodies (Abs). Consequently, tyrosine-phosphorylation of paxillin was obviously persistent after stimulation with FN + LPA as compared to after stimulation with either alone. Tyrosine-phosphorylated paxillin comprised 2 components; slowly and fast migrating ones. Immunoblotting of anti-paxillin immunoprecipitates with phosphorylation site-specific Abs revealed the following: tyrosine-phosphorylation was enhanced preferentially on a slowly migrating component after stimulation with FN + LPA; this component contained phosphorylation at both tyrosine residue (Y) 31 and Y118; and phosphorylation of paxillin at Y181 was constitutive and not augmented by stimulation with either FN or LPA. Amiloride, an inhibitor of the Na+/H+ antiporter downstream of ROCK, suppressed cell motility and correspondingly paxillin tyrosine-phosphorylation at both Y31 and Y118. Paxillin phosphorylation weakly induced by FN alone, insufficient for cell migration, was not inhibited by amiloride. These results demonstrate that LPA collaborates with FN for persistent tyrosine phosphorylation of paxillin at both Y31 and Y118, regulated by the Na+/H+ antiporter downstream of ROCK and that this phosphorylated paxillin is essential for MM1 cancer cell migration.
authors.affiliation: First Department of Pathology, Hyogo College of Medicine, Hyogo, Japan.
authors.forename: Teruo
authors.initials: T
authors.lastname: Iwasaki
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nakata
authors.forename: Mutsuko
authors.initials: M
authors.lastname: Mukai
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Shinkai
authors.forename: Hajime
authors.initials: H
authors.lastname: Yano
authors.forename: Hisataka
authors.initials: H
authors.lastname: Sabe
authors.forename: Erik
authors.initials: E
authors.lastname: Schaefer
authors.forename: Masaharu
authors.initials: M
authors.lastname: Tatsuta
authors.forename: Tohru
authors.initials: T
authors.lastname: Tsujimura
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Terada
authors.forename: Eizo
authors.initials: E
authors.lastname: Kakishita
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Akedo
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005353:Fibronectins / Q000494:pharmacology; D015151:Immunoblotting; D008246:Lysophospholipids / Q000378:metabolism; D009361:Neoplasm Invasiveness; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D011554:Pseudopodia / Q000378:metabolism; D051381:Rats; D012697:Serine Endopeptidases / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*; D000099271:Prostasin
chemical_list: D000584:Amiloride; D003598:Cytoskeletal Proteins; D005353:Fibronectins; D008246:Lysophospholipids; D051419:Paxillin; D010750:Phosphoproteins; D019000:Phosphotyrosine; D012697:Serine Endopeptidases; D014443:Tyrosine; D000099271:Prostasin; C496063:Pxn protein, rat