Expression of Galpha 13 (Q226L) induces P19 stem cells to primitive endoderm via MEKK1, 2, or 4.
Galpha13 mediates the ability of the morphogen retinoic acid to promote primitive endoderm formation from mouse P19 embryonal carcinoma stem cells, a process that includes the obligate activation of Jun N-terminal kinase. Expression of the constitutively activated (Q226L) GTPase-deficient form of Galpha13 mimics retinoic acid and was used to investigate the signaling upstream of primitive endoderm formation. Jun N-terminal kinase 1 activity, MEK1,2, MKK4, and MEKK1 were constitutively activated in clones stably transfected to express Q226L Galpha13. Dominant negative forms of MEKK1 and MEKK4 were expressed stably in the clones harboring Q226L Galpha13. Expression of dominant negative versions of either MEKK1 or MEKK4 effectively blocks both the activation of Jun N-terminal kinase as well as the formation of primitive endoderm. Depletion of MEKK1, -2, or -4 by antisense oligodeoxynucleotides suppressed signaling from Q226L Galpha13 to JNK1 and primitive endoderm formation. We demonstrate that the signal linkage map from Galpha13 activation to primitive endoderm formation in these stem cells requires activation at three levels of the mitogen-activated protein kinase cascade: MEKK1, -2, or -4 for MAP kinase kinase kinase; MKK4 and/or MEK1 for MAP kinase kinase; and JNK1 for MAP kinase.
Wang
Hsien-yu
HY
Department of Physiology & Biophysics, University Medical Center, State University of New York, Stony Brook, New York 11794-8661, USA. wangh@pharm.sunysb.edu
Kanungo
Jyotshnabala
J
Malbon
Craig C
CC
D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D048031:JNK Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; D048730:MAP Kinase Kinase Kinase 2; D048768:MAP Kinase Kinase Kinase 4; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*; D013724:Teratoma; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; D048730:MAP Kinase Kinase Kinase 2; D048768:MAP Kinase Kinase Kinase 4; D020930:MAP Kinase Kinase Kinases; C482578:Map3k1 protein, mouse; C482619:Map3k2 protein, mouse; C482780:Map3k4 protein, mouse; D044365:GTP-Binding Protein alpha Subunits, G12-G13