Degradation of MyoD by the ubiquitin pathway: regulation by specific DNA-binding and identification of a novel site for ubiquitination.
MyoD is a tissue-specific transcriptional activator involvd in skeletal muscle differentiation. It is induced during transition from proliferating, non-differentiated myoblasts to the resting and well differentiated myotubes. Like many other transcriptional regulators, it is short-lived, however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved have remained obscure. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In cells, degradation is inhibited by lactacystin, a specific inhibitor of the 20S proteasome. Inhibition is accompanied by accumulation of MyoD-ubiquitin conjugates. In a cell free system, the proteolytic process requires both ATP and ubiquitin and is preceded by formation of MyoD-ubiquitin adducts. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds. Analysis of the ubiquitination site has revealed that the N-terminal residue of MyoD is sufficient and essential to promote conjugation and subsequent degradation of the protein: conjugation to internal Lys residues is not necessary. Substitution of all Lys residues did not affect significantly its degradation either in intact cells or in a reconstituted cell free system. Degradation was inhibited by specific proteasome inhibitors and was accompanied by accumulation of ubiquitinated species of the protein. We concluded that the first ubiquitin moiety is attached via its C-terminal Gly to the N-terminal residue of MyoD, and the polyubiquitin chain is then synthesized on Lys48 of this moiety.
Ciechanover
A
A
Department of Biochemistry, The Bruce Rappaport Faculty of Medicine and the Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa.
Breitschopf
K
K
Hatoum
O A
OA
Bengal
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D017570:MyoD Protein / Q000378:metabolism*; D011485:Protein Binding; D014452:Ubiquitins / Q000378:metabolism*
D017570:MyoD Protein; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D004247:DNA