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

**Background:** p27 is a multifunctional protein that regulates cell cycle progression, differentiation, and migration. Its canonical degradation involves K48-linked polyubiquitination by the E2 Ubc3 and E3 SCF^SKP2^, leading to proteasomal degradation. However, cytoplasmic p27 can be stabilized by non-K48 ubiquitin chains, particularly K29 and K63 linkages, which are not associated with proteasomal degradation. The E2 enzyme UbcH7 promotes p27 stabilization and delays S phase entry, but the cooperating E3 ligase was unknown. Since UbcH7 only functions with HECT and RBR class E3s, the authors aimed to identify the specific E3 that works with UbcH7 to ubiquitinate and stabilize p27, and to explore the functional consequences on cell migration.

**Methods:** The authors used a yeast deletion collection to screen HECT and RBR E3 ligases for their ability to support p27 ubiquitination in vitro. Yeast extracts from knockout strains (HEL1, HUL5, UFD4, ITT1, TOM1, and RSP5) were tested in ubiquitination assays with UbcH7 and His6-p27. p27 ubiquitination was assessed by Western blotting with anti-ubiquitin antibodies. For mammalian studies, HeLa and human lens epithelial cells (HLECs) were transfected with overexpression plasmids or RNAi for UbcH7 and SMURF1, and p27 levels were measured by Western blot. In vitro reconstituted ubiquitination assays used purified components (Ube1, UbcH7, SMURF1, p27, and ubiquitin variants) to determine chain linkage specificity. AlphaFold2 was used to predict protein-protein interactions. Cell migration was assessed by scratch wound healing assays, and colocalization was examined by immunofluorescence in HLECs.

**Key Results:** In yeast, deletion of RSP5 (the homolog of mammalian NEDD4 family) completely ablated p27 ubiquitination, while other E3 deletions did not. Purified wild-type Rsp5p rescued ubiquitination in a dose-dependent manner, but catalytically dead Rsp5p did not. In mammalian cells, overexpression of SMURF1 increased p27 levels, while RNAi knockdown of SMURF1 or UbcH7 decreased p27 levels. In contrast, SMURF2 overexpression decreased p27. Cycloheximide chase experiments showed that UbcH7 stabilizes p27, and this stabilization is blocked by K29R ubiquitin. In vitro reconstituted assays demonstrated that SMURF1 and UbcH7 together catalyze p27 ubiquitination with wild-type and K29-only ubiquitin, but not with K29R or K63-only ubiquitin. SMURF1 autoubiquitination was also observed, preferentially using K29 linkages. AlphaFold predicted interactions between UbcH7 and the HECT domain of SMURF1, and between p27 and the same HECT domain, but not a ternary complex. Pulldown experiments confirmed that full-length SMURF1 binds p27, but SMURF1 lacking the HECT domain does not. Scratch assays showed that RNAi knockdown of SMURF1 or UbcH7 significantly decreased cell migration speed, while overexpression increased migration. Immunofluorescence revealed colocalization of SMURF1 and p27, and UbcH7 and p27, in both the nucleus and cytoplasm at the leading edge of migrating HLECs.

**Clinical Implications:** This study identifies a novel non-degradative ubiquitination pathway for p27 that promotes cell migration. The SMURF1-UbcH7-p27 axis may be relevant to diseases where p27 accumulation or cell migration is dysregulated, such as cancer metastasis and cataract formation. The authors note that multiple congenital cataract models show p27 stabilization and delays in lens fiber cell denucleation, a process required for lens clarity. Understanding how K29-linked ubiquitination of p27 regulates its stability and function could provide new therapeutic targets for conditions involving aberrant cell migration or lens development.