Degradation of myogenic transcription factor MyoD by the ubiquitin pathway in vivo and in vitro: regulation by specific DNA binding.

MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway.

We have previously shown that the degradation of c-myc and N-myc in vitro is mediated by the ubiquitin system. However, the role of the system in targeting the myc proteins in vivo and the identity of the conjugating enzymes and possible ancillary proteins involved has remained obscure. Here we report that the degradation of the myc proteins in cells is inhibited by lactacystin and MG132, two inhibitors of the 20S proteasome. Inhibition is accompanied by accumulation of myc-ubiquitin conjugates. Dissection of the ancillary proteins involved revealed that the high-risk human papillomavirus oncoprotein E6-16 stimulates conjugation and subsequent degradation of the myc proteins in vitro. Expression of E6-16 in cells results in significant shortening of the t1/2 of the myc proteins with subsequent decrease in their cellular level. Analysis of the conjugating enzymes revealed that under basal conditions the proteins can be conjugated by two pairs of E2s and E3s-E2-14 kDa and E3alpha involved in the "N-end rule" pathway, and E2-F1 (UbcH7) and E3-Fos involved also in conjugation of c-Fos. In the presence of E6-16, a third pair, E2-F1 and E6-AP mediate conjugation of myc by means of a mechanism that appears to be similar to that involved in the targeting of p53, formation of a myc. E6.E6-AP targeting complex. It is possible that in certain cells E6-mediated targeting of myc prevents myc-induced apoptosis and thus ensures maintenance of viral infection.

Degradation of tyrosine aminotransferase (TAT) via the ubiquitin-proteasome pathway.

Most of the known cellular substrates of the ubiquitin system are short-lived growth regulators and transcriptional activators. Very few enzymes involved in intermediary metabolism have been shown to be targeted by the system. In a reconstituted cell-free system, we show that tyrosine aminotransferase (TAT), a key enzyme involved in amino acid metabolism, is conjugated and degraded in an ATP- and ubiquitin-dependent manner. Degradation of ubiquitin-TAT adducts requires, in addition to the 26S proteasome, a novel, yet unidentified, factor. TAT can be protected from degradation by association with its coenzyme pyridoxal phosphate. To examine the potential role of the ubiquitin system in regulating the stability of the enzyme in vivo, we show that cell extracts derived from livers of animals in which TAT was induced, display a corollary increase in the formation of specific TAT-ubiquitin adducts.

Ubiquitin-mediated degradation of tyrosine aminotransferase (TAT) in vitro and in vivo.

Degradation of a protein via the ubiquitin proteolytic pathway involves two successive steps. Covalent attachment of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Most native cellular proteins that are targeted by the ubiquitin system are short-lived transcriptional activators and growth and cell cycle regulators, as well as unstable membrane proteins. In the present study we demonstrate the involvement of the system in the degradation of tyrosine aminotransferase (TAT), a key enzyme in intermediary metabolism. In vitro, we have shown that the native enzyme is conjugated and degraded in a system that requires ATP and ubiquitin. Degradation was monitored by following the decrease of catalytic activity as well as disappearance of the protein molecule. The enzyme could be protected from degradation by association with its specific cofactor, pyridoxal phosphate (PLP). In vivo, we prepared cell extracts from livers of animals in which TAT was induced by starvation and corticosteroid administration. The dramatic increase in the level of the enzyme was accompanied by a concomitant increase in the level of specific TAT-ubiquitin adducts.