Figure 3. CDK5-dependent phosphorylation of PRMT1 at S307 promotes its cytoplasmic localization and mTORC1 signaling.

(A) IF analysis of PRMT1 localization in PRMT1^FLAG knockin HEK293T cells depleted of CDK5. Cells were starved of aa for 50 min or starved for 50 min and then restimulated with aa. for 10 min. Scale bar, 10 μm. Similar results were obtained in three independent experiments.

(B) IB analysis of WCLs derived from Huh7 cells depleted of CDK5. Similar results were obtained in three independent experiments.

(C) IB analysis of WCLs and anti-hemagglutinin (HA) immunoprecipitates (IPs) derived from HEK293T cells stably expressing HA-PRMT1. Similar results were obtained in three independent experiments.

(D) In vitro kinase assays using recombinant GST-PRMT1 protein as the substrate. HA-CDK5-WT and HA-CDK5-D144N were immunopurified from HEK293T cells. The reaction products were subjected to IB analysis. Similar results were obtained in three independent experiments.

(E) IB analysis of WCLs and anti-HA IPs derived from HEK293T cells depleted of CDK5 and transfected with HA-PRMT1. Similar results were obtained in three independent experiments.

(F) A schematic presentation of the evolutionarily conserved S307 of PRMT1.

(G) In vitro kinase assays using recombinant GST-PRMT1-WT or GST-PRMT1-S307A proteins as substrate. HA-CDK5 protein purified from HEK293T cells was used as the kinase source. The reaction products were subjected to IB analysis. Similar results were obtained in three independent experiments.

(H) IF analysis of PRMT1 localization in PRMT1^WT and PRMT1^S307A cells. Cells were starved of aa for 50 min or starved for 50 min then restimulated with aa for 10 min. Scale bar, 10 μm. Similar results were obtained in three independent experiments.

(I) In vitro arginine methylation assays using H4 protein as the substrate. PRMT1 protein immunopurified from PRMT1^WT and PRMT1^S307A cells was used as the methyltransferase. The reaction products were subjected to IB analysis. Similar results were obtained in three independent experiments.

(J) IB analysis of WCL derived from PRMT1^WT or PRMT1^S307A cells. Cells were starved of aa for 50 min or starved for 50 min then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.

(K) A model depicting CDK5-mediated phosphorylation of PRMT1 at S307 controls as PRMT1 subcellular localization and mTORC1 activation.

See also Figure S3 and Table S1.