The dissertation is devoted to the study of a novel post-translational modification of ribosomal protein kinase S6 (S6K1) — CoAlation and its impact on the functional activity of this kinase. S6K1 is one of the key components of the mTOR/Akt1/S6K-dependent signaling pathway, which regulates cell growth, proliferation, metabolism, and differentiation. Dysregulation of this pathway is associated with the development of various metabolic diseases, making the study of mechanisms controlling p70S6K1 activity of significant scientific and practical interest. p70S6K1 undergoes several post-translational modifications, including CoAlation, which can affect its functionality and stability. In the course of the study, monoclonal antibodies against CoA were characterized. These antibodies demonstrated high specificity toward CoAlated proteins and can serve as powerful tools for detecting these modifications using various immunological methods. Using LC-MS analysis, a peptide sequence LTDFGLC*K was identified among the general pool of CoAlated proteins, which belongs to the S6K family, with cysteine at position 217 as the only modified amino acid residue, located in the kinase’s activation loop. In cellular studies, the CoAlation of immunoprecipitated overexpressed p70S6K1 confirmed that oxidative stress significantly increases the level of kinase CoAlation compared to cells cultured under normal conditions. Using immunofluorescence and PLA methods, it was shown that the CoAlation of endogenous p70S6K1 occurs in response to various metabolic and oxidative stresses. This suggests that CoAlation plays a protective role, shielding S6K1 from excessive oxidation and subsequent inactivation or degradation in environments with elevated ROS levels. The existence of an additional Cys231 in the catalytic center of S6K1 suggests the potential for forming a bond between Cys231 and Cys217, which could hinder CoA attachment to Cys217. To investigate this, site-directed mutagenesis was used to study changes in S6K1 CoAlation levels depending on the mutation of one of the cysteines in the activation loop. Based on the experimental results of expressing mutant forms of S6K1 containing C217A and C231A substitutions, it was demonstrated that the absence of the Cys217 CoAlation site reduces the kinase’s CoAlation level by 30% compared to the wild-type p70S6K1. In contrast, the Cys231 mutation increases the CoAlation level of p70S6K1 by 40%, likely due to the availability of the Cys217 -SH group for CoA attachment, if there is an intramolecular bond between Cys217 and Cys231. These findings confirm the CoAlation of Cys217 in S6K1 and indicate a possible regulatory role for Cys231 in this process. Using the Bac-to-Bac expression system, a recombinant constitutively active form of p70S6K1 was generated and characterized. A “dual” vector was employed, allowing infected cells to express p70S6K1 in its active form through co-expression with its activator PDK1. It was discovered that the degree of CoAlation of the recombinant protein is dose- and time-dependent. Since it is known that post-translational modifications of S6K1 are crucial in regulating kinase activity, the potential influence of the CoAlation process on the activity of recombinant S6K1 kinase was investigated in vitro, and it was demonstrated that CoAlation reduces the enzyme’s activity by 40%.
Molecular docking results confirm the possibility of forming a disulfide bond between the –SH groups of CoA and Cys217 of p70S6K1. The ADP moiety of CoA is stabilized through hydrogen, multiple hydrophobic, and ionic interactions within the ATP-binding pocket. As a result of this formation, competition occurs between CoA and ATP for the enzyme’s active site, leading to kinase inhibition. Molecular dynamics simulations established that the CoA complex with the activated form of the kinase is more stable compared to the complex with the non-phosphorylated enzyme. These data indicate that phosphorylation is critically important for stabilizing the interaction between p70S6K1 and CoA, highlighting the importance of CoAlation in regulating the activated form of the kinase. The results obtained from this study indicate that CoAlation of S6K1 is an important mechanism for protecting the protein from excessive oxidation. Additionally, CoAlation of S6K1 at Cys217 has the potential to inhibit S6K1 activity. This regulatory mechanism is associated with the type of interaction between the CoA molecule and S6K1 during the CoAlation process, in addition to forming a disulfide bond between the –SH groups of the CoA tail and the kinase cysteine, CoA also stabilizes its ADP moiety in the catalytic center, preventing ATP binding and further functional activity of the enzyme. These results lay the foundation for further exploration of the role of CoAlation in the regulation of other proteins and open new opportunities for developing effective therapeutic strategies for correcting related pathologies.
