Figure 3. SRPK2 but not Akt redistributes acinus in the nucleus.

(A). Acinus phosphorylation mimetic mutant S422D redistributes in the nucleus. Wild-type acinus and S422A mutants resided in the nuclear speckles, whereas S422D occurred in the whole nucleoplasm. Wild-type SRPK2 mainly localized in the cytoplasm, and a portion of it also distributed in the nucleus. SRPK2-KD exclusively localized in the cytoplasm. SRPK2 phosphorylation triggers acinus relocation from the nuclear speckle to the nucleoplasm. Wild-type acinus redistributed in the nucleoplasm when cotransfected with wild-type SRPK2, and it localized in the nuclear speckles when cotransfected with SRPK2-KD. S422D resided in the nucleoplasm regardless of SRPK2 wild-type or KD. (B). Akt can not relocate acinus from the nuclear speckles. All Akt proteins (wild-type, CA and KD) and acinus-S colocalized in the nuclear speckles of transfected cells. (C). S422A exhibited lower affinity to SRPK2. Myc-SRPK2 was cotransfected into HEK293 cells with various GST-tagged acinus constructs. Transfected acinus proteins were pulled down with glutathione beads and probed with anti-myc antibody. S422A mutants exhibited decreased binding activity to SRPK2 (top panel). The expression of transfected constructs was confirmed (middle and bottom panels). (D). Akt enhances the interaction between SRPK2 and acinus, when SRPK2 kinase activity is low. Acinus and SRPK2 were cotransfected into HEK293 cells, followed by knocking down of Akt with si-RNA. Wild-type and SRPK2-KD displayed the similar affinity to wild-type acinus and lower binding activity to S422A. Depletion of Akt slightly decreased the affinity of wild-type SRPK2 to acinus, while SRPK2 KD binding to acinus wild-type was evidently reduced and completely abolished to acinus S422A (top panel). The expression of transfected constructs and Akt protein level were confirmed (2^nd to bottom panels).