title: RAFTK/Pyk2 mediates LPA-induced PC12 cell migration.
abstract: The phospholipid lysophosphatidic acid (LPA) is a normal constituent of serum that functions as a lipid growth factor and intracellular signaling molecule. In this report, we have investigated the signaling mechanism and function of the tyrosine kinase RAFTK/Pyk2 in LPA-induced cell migration. Analysis of tyrosine phosphorylation upon LPA stimulation in neuroendocrine PC12 cells revealed 6 major tyrosine-phosphorylated proteins with estimated sizes of 180, 120, 115, 68, 44, and 42 kDa. These proteins were identified as epidermal growth factor receptor (EGFR), focal adhesion kinase, RAFTK/Pyk2, paxillin, Erk 1, and Erk 2, respectively. Using specific pharmacological inhibitors, we found that the tyrosine phosphorylation of RAFTK/Pyk2 was intracellular Ca2+-dependent, but not EGFR-dependent, during LPA stimulation of these cells. Moreover, the cytoskeletal and signal scaffolding protein, paxillin, associated with and was regulated by RAFTK/Pyk2 in a Ca2+-dependent manner. Characterization of LPA receptors showed that LPA1 (Edg2) and LPA2 (Edg4) are major receptors for LPA, while LPA3 receptor (Edg7) expression was limited. Upon using the LPA1/LPA3 receptor-specific antagonist VPC 32179, we observed that inhibition of the LPA1/LPA3 receptors had no effect on the LPA-induced phosphorylation of RAFTK, strongly suggesting that the LPA2 receptor is a key mediator of RAFTK phosphorylation. Furthermore, LPA induced PC12 cell migration, which was subsequently blocked by the dominant-negative form of FAK, FRNK. Expression of a dominant-negative form of the small GTPase Ras also blocked LPA-induced cell migration and RAFTK phosphorylation. Taken together, these results indicate that RAFTK is a key signaling molecule that mediates LPA-induced PC12 cell migration in a Ras-dependent manner.
authors.affiliation: Division of Experimental Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115, United States.
authors.forename: Shin-Young
authors.initials: SY
authors.lastname: Park
authors.forename: Karin A
authors.initials: KA
authors.lastname: Schinkmann
authors.forename: Shalom
authors.initials: S
authors.lastname: Avraham
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002465:Cell Movement; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D051418:Focal Adhesion Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D008246:Lysophospholipids / Q000494:pharmacology / Q000502:physiology*; D016716:PC12 Cells; D051419:Paxillin / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors / Q000502:physiology; D051381:Rats; D049368:Receptors, Lysophosphatidic Acid / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D008246:Lysophospholipids; D051419:Paxillin; D047428:Protein Kinase Inhibitors; D049368:Receptors, Lysophosphatidic Acid; D014443:Tyrosine; D066246:ErbB Receptors; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; C496052:Ptk2 protein, rat; D048049:Extracellular Signal-Regulated MAP Kinases; D016283:Proto-Oncogene Proteins p21(ras); C032881:lysophosphatidic acid; D002118:Calcium