Abstract
Myf-5 and MyoD are members of a family of muscle-specific basic helix-loop-helix (bHLH) proteins that are fundamental for myogenic cell differentiation and transcriptional activation of muscle-specific genes. Here we report that elevated levels of the intracellular signaling molecule cAMP and overexpression of cAMP-dependent protein kinase (PKA) inhibit myogenic differentiation. PKA represses the transcriptional activation of muscle-specific genes by the myogenic regulators Myf-5 and MyoD. The repression is directed at the basic HLH domain and is mediated through the E-box DNA consensus motif to which these proteins bind. However, phosphorylation of Myf-5 and MyoD by PKA in vitro does not affect their ability to bind to DNA. PKA specifically inhibits the activity of myogenic bHLH proteins, but not of other HLH proteins, such as the ubiquitously expressed E2A gene products E12 and E47 (E2-5). Our results demonstrate that PKA mediates the cAMP-induced inhibition of muscle cell differentiation by repressing the activity of Myf-5 and MyoD. The inhibition by PKA occurs post-translationally and presumably affects the transactivation process at a step following DNA-binding. The regulation of Myf-5 and MyoD function by a cAMP-dependent pathway may partly explain how external signals generated by serum and certain peptide growth factors can be transduced to the nucleus and inhibit dominant-acting factors that are responsible for myoblast differentiation.
Highlights
Myf-5 andMyoD are members of a family of muscle- late cell differentiation
T o begin to dissect mechanisms whereby CAMP-dependent pathways may inhibit the muscle differentiation program, we investigated the role of PKAin the activation of musclespecific gene expression
To analyze the effect of elevated cAMP on the differentiation program of other myogenic cell lines, mouse buffer, and proteins were eluted with the samebuffer containing 0.8 C2 and rat .L6 myoblasts were cultured in medium which
Summary
Cell Cultures, Transfections, and Plasmid Constructs-C3H 10T1/ 2 fibroblasts and mouse C2 and rat L6 myoblasts were cultured in Dulbecco'smodified Eagle's medium supplemented with 10%fetal calf serum. Dibutyryl CAMPwas used at 3 pmol/ml final concentration.Transfections were performed using calcium phosphate precipitation as described previously (Braun et al, 1989~)I.n transient assays cells were shifted to differentiation medium 24 h after transfection and cultured for additional 48 h. DNA Binding Assays and in Vitro Phospho~~ation-DNAbinding of GST-Myf fusion proteins was measured by gel retardation assays as described (Braun et al, 1989c, 1991). In vitro phosphorylation of bacterially produced fusion proteins was done in 10 p1 of kinase reaction buffer containing 17.5 mM Tris-HC1, pH 7.4, 2,5 mM magnesium chloride, 40 p~ ATP plus 10 pCi of [Y-~'P]ATP(specific activity, 3000 Ci/mmol), and 500 ng of purified fusion protein. Prepuru~~on~ N u c ~ a r E ~ t ~ aTcitssu~erCoumlture Cells-Nuclear extracts from undifferentiated and different.iated C2C12 cells were prepared from 20 dishes containing approximately 3 X lo6cells each and processed as described previously (Braun et al, 1992).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
