Phosphorylation-dependent conformational change in the Keratin 19 head domain: Implications for filament assembly and binding to GSK3β and 14-3-3

Ji, Y.; Jeong, M.; Kim, J.; Yeom, J.; Han, M. A.; Park, C.; Lee, S.-H.; Hong, E.; Ko, Y. H.; Coulombe, P. A.; Kim, J. H.; Lee, C.-H.

doi:10.64898/2026.11.06.764774

bioRxiv · 10.64898/2026.11.06.764774

Phosphorylation-dependent conformational change in the Keratin 19 head domain: Implications for filament assembly and binding to GSK3β and 14-3-3

Ji, Y.·Jeong, M.·Kim, J.·Yeom, J.·Han, M. A.·Park, C.·Lee, S.-H.·Hong, E.·Ko, Y. H.·Coulombe, P. A.·Kim, J. H.·Lee, C.-H.

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Abstract

Keratins are intermediate filament (IF) proteins expressed in various cell types, playing a crucial role in mechanical support and signaling pathways. Keratin 19 (K19) is predominantly found in gut epithelia and early embryonic ectoderm. K19 interacts with signaling proteins such as GSK3{beta} and 14-3-3 to participate in cell signaling pathways. However, details on the binding sites of these proteins on K19, and specific molecular mechanisms governing these interactions, are lacking. Additionally, the N-terminal head domains of IFs including K19 are considered intrinsically disordered regions (IDRs), and their atomic-resolution structures are unavailable. Using NMR spectroscopy, we obtained the first solution structures of K19 head domain peptides. Structural models involving phosphomimetic peptides suggest a phosphorylation-dependent conformational change in the K19 head domain. The phosphomimetic S35E substitution alters the conformation of the head domain, affecting its association with keratin filaments and K19-binding partners. NMR spectral changes indicate that the S35E substitution affects the association of the K19 head domain to K8/K19 filaments and GSK3{beta}, whereas no significant binding changes were observed with 14-3-3{zeta} and 14-3-3{sigma}. Cell-based analyses further showed that the S35E mutation altered keratin filament organization and reduced the proximity between K19 and GSK3{beta} in 293T cells, whereas no significant change was observed in the proximity between K19 and 14-3-3{sigma}. Our findings provide insight into the structure-function relationship of the head domains in IF proteins with direct implications for the assembly of filaments and their role in signal modulation.

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Ji, Y., Jeong, M., Kim, J., Yeom, J., Han, M. A., Park, C., Lee, S.-H., Hong, E., Ko, Y. H., Coulombe, P. A., Kim, J. H., Lee, C.-H.. 2026-11-09. Phosphorylation-dependent conformational change in the Keratin 19 head domain: Implications for filament assembly and binding to GSK3β and 14-3-3. https://doi.org/10.64898/2026.11.06.764774

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