biostudies-literatureKravec MAgence Nationale de la Recherche (ANR)Institut de convergence Q-life grantCzech Academy of Sciences grant RVOFunded by the European Union - Next Generation EU - National Institute for Cancer ResearchAgence Nationale de la RechercheEuropean Regional Development Fund-ProjectLabEx Cell&Scale grantMEYS CRCharles University Grant AgencyLabEx Cell&Scale grante-Infrastruktura CZCzech Science Foundation5635-5666https://www.ebi.ac.uk/biostudies/studies/S-EPMC11574253biostudies-literatureUnknown43(22)Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal rThe EMBO journalCarboxy-terminal polyglutamylation regulates signaling and phase separation of the Dishevelled protein.PMC11574253ANR-17-CONV-0005INFRA LM201814086652036LX22NPO5102ANR-10-IDEX-0001-02LM2018127ANR-12-BSV2-0007LM2018129ANR-17-CE13-0021CZ.02.1.01/0.0/0.0/18_046/0015974GA22-06405SGA22-25365S1414120ANR-11-LBX-0038GA23-07149SGX19-28347XNedvedova JJanke CBryja VTripsianes KGomoryova KSedo OZdrahal ZBologna SSulcova MKravec MZezula NHarnos JCervenka IMicka MBarinka CPotesil DSri Ganji RfalseCarboxy-terminal polyglutamylation regulates signaling and phase separation of the Dishevelled protein.Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal r2024-01-01T00:00:00Z2024 Nov2026-06-01T20:29:40.092Z2025-04-04T11:42:48.896ZS-EPMC115742533934984610.1038/s44318-024-00254-7