c-Jun Amino-terminal Kinase Is Regulated by Gα12/Gα13 and Obligate for Differentiation of P19 Embryonal Carcinoma Cells by Retinoic Acid

Abstract

Retinoic acid induces P19 mouse embryonal carcinoma cells to differentiate to endoderm and increases expression of the heterotrimeric G-protein subunits G␣ 12 and G␣ 13 . Retinoic acid was found to induce differentiation and sustained activation of c-Jun amino-terminal kinase, but not of ERK1,2 or of p38 mitogen-activated protein kinases. Much like retinoic acid, expression of constitutively active forms of G␣ 12 and G␣ 13 induced differentiation and constitutive activation of c-Jun amino-terminal kinase. Expression of the dominant negative form of c-Jun amino-terminal kinase 1 blocked both the activation of c-Jun amino-terminal kinase and the induction of endodermal differentiation in the presence of retinoic acid. These data implicate c-Jun amino-terminal kinase as a downstream element of activation of G␣ 12 or G␣ 13 obligate for retinoic acid-induced differentiation.The role of heterotrimeric guanine nucleotide binding proteins (G-proteins) 1 in cell differentiation and development has been shown in several systems (1-4). Adipogenesis of NIH 3T3-L1 cells in response to inducers such as insulin or dexamethasone plus methylisobutylxanthine is accompanied by a sharp decline in G␣ s subunit (5). Both suppression of G␣ s by antisense oligodeoxynucleotides and overexpression of the constitutively active mutant form of G␣ i2 promote adipogenesis in the absence of classical inducers (5). Differentiation of F9 embryonic stem cells to endodermal cells provokes a sharp reduction of G␣ i2 (2), a de-repression of phospholipase C, and activation of protein kinase C and mitogen-activated protein (MAP) kinase, especially via ERK1,2 (6). The expression of constitutively active forms of G␣ q or G␣ 16 can induce neuronal differentiation of PC 12 cells (7), via activation of c-Jun aminoterminal kinase but not via ERK1,2.The extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK/JNK), and mammalian homolog of the yeast-osmosensing ERK HOG1 (p38 MAPK) are conserved members of a MAP kinase cascade for regulation of targets such as transcription factors in response to growth factors or environmental stresses, such as ultraviolet light, and protein synthesis inhibitors (8 -16). ERK appears to play a major role in provoking cell proliferation and differentiation (17-19), whereas JNK mediates stress responses and some forms of apoptosis (20 -23). JNK has been implicated also in the induction of differentiation (7, 24) and oncogenesis (25). The ability of overexpression of c-Jun, which is a target for JNK, to stimulate differentiation of P19 embryonal carcinoma cells to a mixed endoderm/mesoderm population supports a role of JNK in differentiation (26). P19 embryonal carcinoma (P19) cells have been used as a model system for murine pre-implantation development (27,28). These pluripotent cells have the ability to differentiate into derivatives of three germ layers, endoderm, mesoderm, and ectoderm, upon different inducer stimulation (29,30). Monolayer cultures of P19 cells challenged with 50 nM retinoic acid (...

Ajuba, a Cytosolic LIM Protein, Shuttles into the Nucleus and Affects Embryonal Cell Proliferation and Fate Decisions

“…It has been reported that the endodermal differentiation in P19 cells induced by retinoic acid is also dependent on activation of a JNK1-dependent pathway (Jho et al, 1997). Increased JNK activity correlated with the ability of specific Ajuba isoforms to induce endodermal differentiation.…”

“…Previous reports have demonstrated that atRA-induced endodermal differentiation of P19 cells requires activation of JNK (Jho et al, 1997). Because Ajuba is a potent modulator of ERK activity in fibroblasts, we determined whether overexpression of the various isoforms of Ajuba in P19 cells affected the activity of the MAPK family of proteins (e.g., ERK, JNK, and p38) in these cells.…”

Scaffold proteins of MAP-kinase modules

“…Overexpressing this Ga 12/13 -interacting domain, it has been established that JLP is required for the activation of JNK by Ga 12/13 coupled LPA receptors. Likewise, a critical role for JLP has been established in retinoic acid-mediated endodermal differentiation of P19 embryonic carcinoma cells (Kashef et al, 2006), which requires the stimulation JNK module by Ga 13 (Jho et al, 1997). It is significant to note here that JLP is the first mammalian scaffold protein that has been demonstrated to link a heterotrimeric G protein to a MAPK module.…”

Selective Uncoupling of Gα12 from Rho-mediated Signaling

“…For binding assays utilizing untagged 35 S-labeled variants of G␣ 12 QL , the proteins were produced using a TNT in vitro coupled transcription/ translation system (Promega) according to the manufacturer's instructions. Reactions were diluted into reduced detergent lysis buffer (see above) and incubated with GST fusion proteins as described above, and then proteins were separated by SDS-PAGE and gels fixed in 10% acetic acid, 1% glycerol, dried under vacuum, and analyzed by autoradiography.…”