| The studies presented in this thesis examined the role of the Raf signaling pathway in the regulation of myogenic differentiation. 23A2RafER DD myoblasts, a stable myogenic cell line expressing an inducible form of activated Raf kinase, were created to study the effects of Raf signal intensity on myocyte formation. In the absence of Raf activity, the cells readily differentiate into large multinucleated myofibers comparable to parental 23A2 myocytes. Unexpectedly, low level induction of Raf activity stimulates myocyte formation and enhances contractile and regulatory protein expression. High level Raf activation causes the predicted reduction in both myofiber number and muscle reporter gene expression. Treatment of myoblasts with spent culture medium from Raf-expressing myoblasts indicates that a TGF-beta-like factor is involved in the repression of myofiber formation. Closer analysis of the Raf-repressive myoblasts revealed an increase in TGF-beta1 expression and Smad transcriptional activity. However, neutralizing the biological activity of TGF-beta1 failed to reinstate the myogenic program, suggesting that the autocrine TGF-beta1 loop is not entirely responsible for the differentiation-defective phenotype imposed by Raf signaling.; An additional aim of this thesis focused on the role of MEK-independent signaling in response to activated Raf. The existence of MEK-independent Raf signaling was proposed because Raf-repressive myoblasts remain refractile in the presence of MEK inhibition. RafBXB-T481A, a Raf kinase that fails to interact with MEK and activate ERK, can sufficiently inhibit muscle gene transcription. MRF and MEF2 transcriptional activities are unaffected by RafBXB-T481A signaling. To identify additional Raf effector, a yeast two-hybrid screen of human skeletal muscle cDNA library was performed with RafBXB-T481A as bait, resulting in the isolation of the Ran binding protein RanBPM as a Raf-interacting protein. RanBPM localized within both cytoplasm and nucleus, and is known to shuttle between these compartments as part of its function. However, RanBPM does not affect MyoD function. Moreover, RanBPM and activated Raf kinase appear to synergistically inhibit myogenic differentiation, and RanBPM might be a direct target of Raf phosphorylation. (Abstract shortened by UMI.)... |
