Raf, but not MEK or ERK, is sufficient for differentiation of hippocampal neuronal cells.
To elucidate signal transduction pathways leading to neuronal differentiation, we have investigated a conditionally immortalized cell line from rat hippocampal neurons (H19-7) that express a temperature sensitive simian virus 40 large T antigen. Treatment of H19-7 cells with the differentiating agent basic fibroblast growth factor at 39 degrees C, the nonpermissive temperature for T function, resulted in the activation of c-Raf-1, MEK, and mitogen-activated protein (MAP) kinases (ERK1 and -2). To evaluate the role of Raf-1 in neuronal cell differentiation, we stably transfected H19-7 cells with v-raf or an oncogenic human Raf-1-estrogen receptor fusion gene (deltaRaf-1:ER). deltaRaf-1:ER transfectants in the presence of estradiol for 1 to 2 days expressed a differentiation phenotype only at the nonpermissive temperature. However, extended exposure of the deltaRaf-1:ER transfectants to estradiol or stable expression of the v-raf construct yielded cells that extended processes at the permissive as well as the nonpermissive temperature, suggesting that cells expressing the large T antigen are capable of responding to the Raf differentiation signal. deltaRaf-1:ER, MEK, and MAP kinase activities in the deltaRaf-1:ER cells were elevated constitutively for up to 36 h of estradiol treatment at the permissive temperature. At the nonpermissive temperature, MEK and ERKs were activated to a significantly lesser extent, suggesting that prolonged MAP kinase activation may not be sufficient for differentiation. To test this possibility, H19-7 cells were transfected or microinjected with constitutively activated MEK. The results indicate that prolonged activation of MEK or MAP kinases (ERK1 and -2) is not sufficient for differentiation of H19-7 neuronal cells and raise the possibility that an alternative signaling pathway is required for differentiation of H19-7 cells by Raf.
Kuo
W L
WL
Ben May Institute, University of Chicago, Ilinois 60637, USA.
Abe
M
M
Rhee
J
J
Eves
E M
EM
McCarthy
S A
SA
Yan
M
M
Templeton
D J
DJ
McMahon
M
M
Rosner
M R
MR
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D004958:Estradiol / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D048728:MAP Kinase Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009474:Neurons / Q000166:cytology*; D027384:Oncogene Proteins v-raf; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011960:Receptors, Estrogen / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013696:Temperature; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D016222:Fibroblast Growth Factor 2; D004958:Estradiol; D027384:Oncogene Proteins v-raf; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human