title: The JNK/SAPK activator mixed lineage kinase 3 (MLK3) transforms NIH 3T3 cells in a MEK-dependent fashion.
abstract: Mixed lineage kinases (MLKs) form a family of serin/threonine protein kinases with multiple protein/protein interaction domains (SH3, Cdc42 Rac interactive binding sequence, leucine zipper, and proline rich region), the physiological roles of which are largely unknown. We show that overexpression of wild type MLK3 leads to morphological transformation of NIH 3T3 fibroblasts and growth in soft agar. Consistent with this transforming potential, we demonstrate that MLK3 strongly induces transcription from a reporter construct that is driven by a composite AP-1-/Ets-1-enhancer element in HEK 293 cells. In the same cell system, MLK3 preferentially activates the c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) mitogen-activated protein kinase cascade and to a lesser degree the extracellular signal-regulated kinase (ERK) pathway. Activation of the latter can be further enhanced by coexpression of wild type MEK1 and is blocked by the synthetic MEK inhibitor PD 098059 or a kinase-dead MEK1 mutant. Immunoprecipitated MLK3 catalyses the phosphorylation of MEK1 in vitro, but this phosphorylation leads only to a marginal activation. In support of these data, we also show that MEK1 is highly phosphorylated in vivo on Ser 217/221 in MLK3-transformed fibroblasts, whereas activating ERK phosphorylations are barely detectable. Nevertheless, MLK3-transformed NIH 3T3 fibroblasts are partially reverted when activation of MEK is specifically blocked with PD 098059. Our combined data show that although MLK3 is primarily an activator of the JNK/SAPK pathway, overexpression of the wild type protein leads to a transformed phenotype in NIH 3T3 cells that can be partially reversed by a synthetic MEK inhibitor. We conclude that the ERK pathway is necessary for MLK3-mediated transformation.
authors.affiliation: Institut für Medizinische Strahlenkunde und Zellforschung, Universität Würzburg, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Hartkamp
authors.forename: J
authors.initials: J
authors.lastname: Troppmair
authors.forename: U R
authors.initials: UR
authors.lastname: Rapp
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D017930:Genes, Reporter; D048369:MAP Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010641:Phenotype; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
chemical_list: D000952:Antigens, Polyomavirus Transforming; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D005419:Flavonoids; D048369:MAP Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; C482127:Map2k1 protein, mouse; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one