The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration

Authors: WEINBERG, JASPER - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; WHITCOMB, ELIZABETH - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; BOHM, ANDREW - Tufts University; CHEKKILLA, UDAY KUMAR - Harvard University; TAYLOR, ALLEN - Tufts University

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2024
Publication Date: 2/21/2024
Citation: Weinberg, J., Whitcomb, E., Bohm, A., Chekkilla, U., Taylor, A. 2024. The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration. Journal of Biological Chemistry. https://doi.org/10.1016/j.jbc.2024.105693.
DOI: https://doi.org/10.1016/j.jbc.2024.105693

Interpretive Summary: During the development of the eye lens, lens fiber cells get rid of their nuclei, a process that uses many of the same components as cell division. One such cell division protein is p27, an important regulator of the cell cycle. However, little is known how the process works. Through a series of cell studies, we identified an enzyme that is involved in the p27 pathway and may promote its cell-cycle independent of its function of regulating cell migration.

Technical Abstract: Ubiquitination is a key regulator of protein stability and function. The multifunctional protein p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293 and HELA cells. But the ubiquitin ligase associated with this stabilization of p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1, but not its close homolog SMURF2, stabilizes p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of p27, we also found that SMURF1, UbcH7, and p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated colocalization of SMURF1/p27 and UbcH7/p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.