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. 2024 Oct 22;12:RP88318. doi: 10.7554/eLife.88318

Figure 4. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) phosphorylates TSC2 at T1462 in vitro, and Ras Homolog Enriched in Brain (RHEB) overexpression rescues mTORC1 activity in cells.

(A) An in-vitro kinase assay was performed using DYRK1A and kinase-dead DYRK1A (K188R) that were purified from bacteria. Flag-TSC2 and HA3-TSC1 were co-expressed in HEK293 cells and purified using a combination of (1:1) of HA and Flag beads. Beads were equilibrated with kinase assay buffer before the reactions were initiated on beads. After incubation for 30 min at 30°C, reactions were stopped by the addition of SDS loading buffer. Since bacterially purified DYRK1A is autophosphorylated, it exhibits a fuzzier signal, whereas kinase-dead DYRK1A is incapable of phosphorylation and appears as a sharp signal. (B, C) RHEB overexpression partially rescues the size of HEK293 cells. HEK293 cells were first transduced with short hairpin RNA (shRNA) lentivirus targeting DYRK1A or control and selected with 1 ug/ml Puromycin for three days, after which they were re-transduced with lentivirus expressing Flag-RHEB. The concentration of Puromycin was raised to 2 ug/ml for the next 48 hr in order to select for the second round of transduction. (B) Panel shows knockdown efficiency of DYRK1A and overexpression of RHEB. (C) Lower panel shows cell size analysis. Data represent the mean ± SD (n=3 biological replicates). Student’s t-test was done to compare samples. Significant difference in p-value = *p<0.05.

Figure 4—source data 1. Uncropped blots for Figure 4A.
Figure 4—source data 2. Raw data showing in vitro kinase assay using recombinant DYRK1A and K188R for Figure 4A.
Figure 4—source data 3. Uncropped blots for Figure 4B.
Figure 4—source data 4. Raw data showing knockdown of DYRK1A and overexpression of Flag-RHEB for Figure 4B.

Figure 4.

Figure 4—figure supplement 1. mTORC1 inhibitors block the increase in cell size mediated by dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A).

Figure 4—figure supplement 1.

HEK293 cells expressing Flag-DYRK1A and the parental cells were treated with 40ng/ml Doxycycline. At 24hr mTOR inhibitors Torin1/Rapamycin were added and the cells were further incubated for 24hr. Data represent the mean ± SD (n=3 biological replicates).