Members of the Myc family are known to function as transcription factors that regulate many aspects of cellular behavior and are implicated in many cancers. Cytoplasmic Myc and an interaction with tubulins (the proteins that form microtubules) have also been reported, but the physiological importance of these pools of Myc is unknown. Conacci-Sorrell et al. noticed a correlation between the decrease in abundance of full-length Myc and the appearance of a 42-kD cytoplasmic protein that was detectable with antibodies directed against the N-terminal region of Myc but not with those against the C-terminal region. This 42-kD protein, which they dubbed “Myc-nick,” was present in primary cell cultures, mouse tissues, and multiple cell lines. In vitro assays revealed that recombinant or purified Myc was cleaved by cytoplasmic extracts, not nuclear extracts. Through various inhibitor experiments in vitro and in cultured cells, calpain was determined to be the protease responsible for Myc-nick production, and siRNA knockdown of the regulatory subunit of calpain in cells reduced Myc-nick abundance and increased that of full-length Myc. Expression of a plasmid encoding only the Myc-nick sequence into Rat1a myc-null fibroblasts caused the cells to become spindle shaped with long protrusions and, in a scratch assay, caused the cells to align parallel to each other, extending long cellular protrusions into the open space. The elongated protrusions resembled structures formed by stabilized microtubules, and Myc-nick coimmunoprecipitated with α-tubulin and with the acetyltransferase GCN5. Acetylation stabilizes microtubules, and the Myc-nick–expressing cells exhibited staining of acetylated tubulin at the sites of the elongated cellular processes, cytoplasmic extracts supplemented with Myc and GCN5 exhibited the greatest amount of tubulin acetylation, and expression of Myc-nick with a mutation that blocked GCN5 binding prevented the morphological changes associated with stabilized microtubules. Immunoblotting of mouse tissues indicated that endogenous Myc-nick was most abundant in brain and mature skeletal muscle. When human primary myoblasts or mouse C2C12 myoblasts were stimulated to differentiate, the abundance of Myc-nick and the calpain activity increased relative to that in proliferating cultures, and the in vitro Myc cleavage activity in cytoplasmic extracts increased. Moreover, forced expression of Myc-nick enhanced muscle cell differentiation, tubulin acetylation, and production of muscle-specific proteins. Thus, Myc-nick appears to be an active calpain cleavage product of Myc that functions in muscle differentiation. It will be interesting to determine whether this cleavage-mediated activation of cytoplasmic Myc occurs in neurons, which also undergo substantial cytoskeletal reorganization during differentiation.
M. Conacci-Sorrell, C. Ngouenet, R. N. Eisenman, Myc-Nick: A cytoplasmic cleavage product of Myc that promotes α-tubulin acetylation and cell differentiation. Cell 142, 480–493 (2010). [PubMed]