| Summary: | Wnt signaling is dependent on dishevelled proteins (DVL1-3), which assemble an
intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by
multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTBKelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the
molecular mechanisms determining its substrate interactions have remained unknown. Here, we
mapped the interaction of DVL1-3 to a ‘PGXPP’ motif that is conserved in other known partners
and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the
binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in
complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate
adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades
of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the
binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a
degron motif for KLHL12 recruitment. These results define the molecular mechanisms
determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new
protein interaction module for a novel proline-rich motif.
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