Emergence of glycogen synthase kinase-3 interaction domain enhances phosphorylation of SARS-CoV-2 nucleocapsid protein

• Published in: bioRxiv 2022-01
• Main Authors: Yun, Jun Seop, Kim, Nam Hee, Song, Hyeeun, So Young Cha, Kyu Ho Hwang, Lee, Jae Eun, Cheol-Hee Jeong, Song, Sang Hyun, Kim, Seonghun, Eunae Sandra Cho, Kim, Hyun Sil, Jong In Yook
• Format: Article
• Language: English
• Subjects: Coronaviridae; Coronaviruses; Glycogen; Glycogen synthase kinase 3; Kinases; Mutants; N protein; Nucleocapsids; Phosphorylation; Protein structure; Proteins; Replication; Severe acute respiratory syndrome coronavirus 2; Wnt protein
• DOI: 10.1101/2022.01.24.477037

A structural protein of SARS-CoV-2, nucleocapsid (N) protein is abundantly expressed during viral replication. The N protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3β constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), commonly found in many endogenous GSK-3β binding proteins, such as Axins, FRATs, WWOX and GSKIP. Indeed, N interacts with GSK-3β similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. Unlike with endogenous GID proteins, the N interaction neither disturbs endogenous GSK-3 activity nor regulates subsequent canonical Wnt activity and the Snail-EMT program. Notably, N abundance in SARS-CoV-2 is incomparably high compared to other coronaviruses, such as 229E, OC43 and HKU1. Compared to other coronaviruses, N harbors a CDK1 primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3β. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allows increased abundance and hyper-phosphorylation of N. Our observations suggest that the emergence of GID and mutations for increased phosphorylation in N may have contributed to the emergence of SARS-CoV-2 and evolution of variants, respectively. Further study, especially in a BSL3-equipped facility, is required to elucidate the functional importance of GID and N phosphorylation in SARS-CoV-2 and variants. Competing Interest Statement The authors have declared no competing interest.