DYRK1A regulates cell size.

shRNA-mediated knockdown of DYRK1A was performed in (A-B) HEK293 and (C-D) SH-SY5Y cells using lentivirus. Transduced cells were selected for four days before analysis. Western blot shows the efficiency of DYRK1A knockdown. (E-F) NIH3T3 cells were treated with Dyrk1a-targeting sgRNA expressing lentivirus and selected for four days before analysis. Western blot shows the efficiency of DYRK1A knockdown. (G-H) HEK293 cells expressing Flag-DYRK1A and the parental cells were treated with 40ng/ml Doxycycline for 48 hours and analyzed for cell size. (G) Overexpression was analyzed by qRT-PCR. GAPDH mRNA was used to normalize RNA in q-RT-PCR samples. Data represent the mean ± SD (n = 3 biological replicates).

DYRK1A interacts with the Tuberous Sclerosis Complex

(A) The MS/MS datasets previously acquired by MudPIT analyses of FLAG-DYRK1A affinity purifications and negative FLAG controls (18) were searched against the most recent releases of the human protein sequence databases (built by collating and removing redundant entries from NCBI Homo sapiens RefSeq GCF_000001405.40_GRCh38.p14 and GCF_009914755.1_T2T-CHM13v2.0). Highly enriched proteins include known and novel DYRK1A-interacting partners and are reported with their peptide counts and distributed Normalized Spectral Abundance Factor (dNSAF) values, which reflect their relative abundance in the samples (56). (B) Flag beads were used to pull-down Flag-DYRK1A from whole cell extracts of HEK293 transfected with Flag-DYRK1A, Protein A beads were used as a control. The blots were probed with TSC1 and TSC2 antibodies. Actin was used to normalize the lysates inputs. (C) endogenous DYRK1A was immunoprecipitated with DYRK1A antibody from HEK293 cytoplasmic fraction generated using the Dignam protocol (18) and probed with antibodies against endogenous DYRK1A, TSC1 and TSC2. Rabbit IgG was used as the IP control (D) Flag-DYRK1A and Flag-DYRK1A kinase domain constructs were affinity purified using Flag-beads from HEK293 cells co-transfected with HA3-TSC1, and probed with α-HA and α-Flag antibodies. Actin was used as loading control.

DYRK1A promotes the activation of mTORC1 pathway in human and mouse cells.

(A) HEK293 cells treated with DYRK1A shRNA or control shRNA were serum starved for 12 hours before being activated with serum for the indicated times. Cells were then harvested, lysates and probed with the indicated antibodies. Actin was used as loading control. (B - C) Quantification of proteins in A, levels of pS6K (T389), S6K, pS6 (pS235/236) and S6 were quantified using Image J software and the ratio of pS6K/S6K and pS6/S6 were plotted (n = 3 biological replicates). (D) NIH3T3 cells were treated with sgRNA-targeting Dyrk1a or non-targeting control and selected for four days with Puromycin before harvesting. Lysates were probed with indicated antibodies. (E - F) Quantification of proteins in D, levels of pS6K (T389), S6K, pS6 (pS235/236) and S6 were quantified (as described for B and C) and ratios were plotted (n = 3 biological replicates). Student’s t tests were done to compare samples. p value = * (p < 0.05).

DYRK1A phosphorylates TSC2 at T1462 in vitro, and 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 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 2ug/ml for the next 48 hours in order to select for the second round of transduction. (B) 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)

mnb mutant phenotype can be rescued by TOR activation in flies.

(A-F and J-L) 3rd instar larval NMJ (muscles 6/7) were stained using anti-HRP (Green) and anti-Dlg (Red). Muscles are stained with phalloidin (Blue, A-F). HRP (green) stains the entire neuron and Dlg (red) stains only boutons (Red+Green). (G-I, M) Quantification of bouton numbers, normalized to muscle area (Bouton-NMA). Error bars represent standard deviation. Statistical significance (p-values: *** < 0.001; ** < 0.01; * < 0.05) is calculated by unpaired student’s t-test. (A, B, G) mnb1 alleles show fewer boutons numbers as compared to wild-type (WT, Canton S) control (B). Data are quantified in G. (C, D, H) mnb overexpression (D42-Gal4>UAS-mnb, D) increases bouton numbers as compared with mCherry overexpression (D42-Gal4>UAS-mCherry, Control, C). D42-Gal4 is a motor-neuron specific driver. Data are quantified in H. (E, F, I) Rheb overexpression (D42-Gal4>UAS-Rheb, F) increases bouton numbers as compared with mCherry overexpression (D42-Gal4>UAS-mCherry, Control, E). Data are quantified in I. (J-M) Rheb overexpression in mnb mutant (mnb1/Y D42-Gal4>UAS-Rheb, L) suppressed bouton phenotype as compared to mnb mutant (mnb1/Y D42-Gal4/+, K). Wild type is heterozygous D42-Gal4 (+/Y; D42-Gal4/+, J). Data is quantified in M.