Fish CDK2 recruits Dtx4 to degrade TBK1 through ubiquitination in the antiviral response

Reviewer #1 (Public Review):

Summary:

The authors set out to evaluate the regulation of interferon (IFN) gene expression in fish, using mainly zebrafish as a model system. Similar to more widely characterized mammalian systems, fish IFN is induced during viral infection through the action of the transcription factor IRF3 which is activated by phosphorylation by the kinase TBK1. It has been previously shown in many systems that TBK1 is subjected to both positive and negative regulation to control IFN production. In this work, the authors find that the cell cycle kinase CDK2 functions as a TBK1 inhibitor by decreasing its abundance through the recruitment of the ubiquitinylation ligase, Dtx4, which has been similarly implicated in the regulation of mammalian TBK1. Experimental data are presented showing that CDK2 interacts with both TBK1 and Dtx4, leading to TBK1 K48 ubiquitinylation on K567 and its subsequent degradation by the proteasome.

Strengths:

The strengths of this manuscript are its novel demonstration of the involvement of CDK2 in a process in fish that is controlled by different factors in other vertebrates and its clear and supportive experimental data.

Weaknesses:

The weaknesses of the study include the following. 1) It remains unclear whether the function described for CDK2 is regulatory, that is, it affects TBK1 levels during physiological responses such as viral infection or cell cycle progression, or if it is homeostatic, governing the basal abundance of TBK1 but not responding to signaling. 2) The authors have not explored whether the catalytic activity of CDK2 is required for TBK1 ubiquitinylation and, if so, what its target specificity is. 3) Given the multitude of CDK isoforms in fish, it remains unexplored whether the identified fish CDK2 homolog is a requisite cell cycle regulator or if its action in the cell cycle is redundant with other CDKs.

Reviewer #2 (Public Review):

Summary:

In this paper, the authors describe a novel function involving the cell cycle protein kinase CDK2, which binds to TBK1 (an essential component of the innate immune response) leading to its degradation in a ubiquitin/proteasome-dependent manner. Moreover, the E3 ubiquitin ligase, Dtx4, is implicated in the process by which CDK2 increases the K48-linked ubiquitination of TBK1. This paper presents intriguing findings on the function of CDK2 in lower vertebrates, particularly its regulation of IFN expression and antiviral immunity.

Strengths:

(1) The research employs a variety of experimental approaches to address a single question. The data are largely convincing and appear to be well executed.

(2) The evidence is strong and includes a combination of in vivo and in vitro experiments, including knockout models, protein interaction studies, and ubiquitination analyses.

(3) This study significantly impacts the field of immunology and virology, particularly concerning the antiviral mechanisms in lower vertebrates. The findings provide new insights into the regulation of IFN expression and the broader role of CDK2 in immune responses. The methods and data presented in this paper are highly valuable for the scientific community, offering new avenues for research into antiviral strategies and the development of therapeutic interventions targeting CDK2 and its associated pathways.

Weaknesses:

(1) While the study focuses on fish, the broader implications for other lower vertebrates and higher vertebrates are not extensively discussed.

(2) The study heavily relies on specific fish models, which may limit the generalizability of the findings across different species.