Expression of Gα13 (Q226L) induces P19 stem cells to primitive endoderm via MEKK1, 2, or 4

Gα13 mediates the ability of the morphogen retinoic acid to promote primitive endoderm formation from minoma stem cells, a process that includes the obligate activation of Jun N-terminal kinase. Expression of the constitutively activated (Q226L) GTPase-deficient form of Gα13 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 Gα13. Dominant negative forms of MEKK1 and MEKK4 were expressed stably in the clones harboring Q226L Gα13. 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 Gα13 to JNK1 and primitive endoderm formation. We demonstrate that the signal linkage map from Gα13 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.