DYRK1A Interacts with the Tuberous Sclerosis Complex and Promotes mTORC1 Activity

Reviewer #1 (Public Review):

In this manuscript, Wang et al. demonstrate that knockdown of DYRK1A results in reduced cell size, which is mediated by mTORC1 activity. They found that DYRK1A interacts with TSC1/TSC2 proteins which leads to the phosphorylation of TSC2 at T1462. Phosphorylation of TSC2 at T1462 inhibits TSC2 activity leading to the activation of mTORC1. The authors complement their findings by demonstrating that overexpression of RHEB (positive regulator of mTORC1) rescues the phenotype of DYRK1A (mnb in flies) mutation in the NMJ.

The authors' findings on the regulation of cell size and mTORC1 activity by DYRK1A reflect the previous findings of Levy et al. (PMID: 33840455) that cortical deletion of Dyrk1a in mice causes decreased neuronal size associated with a decreased activity of mTORC1 that can be rescued by the inhibition of Pten or supplementation of IGF1.

The authors demonstrate that T1462 phospho-site at TSC2 is phosphorylated in response to the overexpression of WT but not kinase-dead DYRK1A. However, the authors do not provide any evidence that the regulation of mTORC1 is mediated via phosphorylation of this site. In addition, T1462 site is known to be phosphorylated by Akt. There is a possibility that Akt was co-purified with TSC1/TSC2 complex and DYRK1A promotes phosphorylation of TSC2 indirectly via the activation of AKT that can be tested by using AKT depleted cells.

RHEB is the most proximal regulator of mTORC1 and can activate mTORC1 even under amino acid starvation. The fact that RHEB overexpression rescues the cell size under DYRK1A depletion or mnb (DYRK1A in Drosophila) mutant phenotype does not prove that DYRK1A regulates the cell size via TSC1 as it would rescue any inhibitory effects upstream to mTORC1.

Reviewer #2 (Public Review):

This study aims to describe a physical interaction between the kinase DYRK1A and the Tuberous Sclerosis Complex proteins (TSC1, TSC2, TBC1D7). Furthermore, this study aims to demonstrate that DYRK1A, upon interaction with the TSC proteins regulates mTORC1 activity and cell size. Additionally, this study identifies T1462 on TSC2 as a phosphorylation target of DYRK1A. Finally, the authors demonstrate the role of DYRK1A on cell size using human, mouse, and Drosophila cells.

This study, as it stands, requires further experimentation to support the conclusions on the role of DYRK1A on TSC interaction and subsequently on mTORC1 regulation. Weaknesses include, 1) The lack of an additional assessment of cell growth/size (eg. protein content, proliferation), 2) the limited data on the requirement of DYRK1A for TSC complex stability and function, and 3) the limited perturbations on the mTORC1 pathway upon DYRK1A deletion/overexpression. Finally, this study would benefit from identifying under which nutrient conditions DYRK1A interacts with the TS complex to regulate mTORC1.

The interaction described here is highly impactful to the field of mTORC1-regulated cell growth and uncovers a previously unrecognized TSC-associated interacting protein. Further characterization of the role that DYRK1A plays in regulating mTORC1 activation and the upstream signals that stimulate this interaction will be extremely important for multiple diseases that exhibit mTORC1 hyper-activation.

Reviewer #3 (Public Review):

The manuscript describes a combination of in vitro and in vivo results implicating Dyrk1a in the regulation of mTORC. Particular strengths of the data are this combination of cell and whole animal (drosophila) based studies. However, most of the experiments seem to lack a key additional experimental condition that could increase confidence in the authors' conclusions. Overall some tantalizing data is presented. However, there are several issues that should be clarified or otherwise addressed with additional data.

1. In Figure 1G, why not test overexpression levels of Dyrk1a via western rather than only looking at the RNA levels?

2. In Figure 2, while there is clearly TSC1 protein in the Dyrk1a and FLAG-Dyrk1a IPs that supports an interaction between the proteins, it would be good to see the reciprocal IP experiment wherein TSC1 or TSC2 are pulled down and then the blot probed for Dyrk1a.

3. Figures 3 A and D tested the effects of Dyrk1a knockdown using different methods in different cell lines. This is a reasonable approach to ascertain the generalizability of findings. However, each experiment is performed differently. For example, in 3A, the authors found no difference in baseline pS6, so they did a time course of treatment to induce phosphorylation and found differences depending on Dyrk1a expression. In 3D, they only show baseline effects from the CRISPr knockdown. Why not do the time course as well for consistency? Also, why the inconsistency in approaches wherein one shows baseline effects and the other does not? The authors could also consider the pharmacologic inhibition of Dyrk1a activity as well.

4. In Figure 4, RHEB overexpression increases cell size in both Dyrk1a wt and Dyrk1a shRNA treated cells, although the magnitude of the effect appears reduced in Dyrk1a shRNA cells. However, there is the possibility here that RHEB acts independently of Dyrk1a. Why not also do the experiment of Figure 1 wherein Dyrk1a is overexpressed and then knockdown RHEB in that context? If the hypothesis is supported, then RHEB knockdown should eliminate the cell size effect of Dyrk1a overexpression.

5. The discussion should incorporate relevant findings from other models, such as Arabidopsis. Barrada et al., Development (2019), 146 (3).