Novel shuttling domain in a regulator (RSC1A1) of transporter SGLT1 steers cell cycle-dependent nuclear location

The gene product of RSC1A1, RS1, participates in the regulation of the Na(+)-D-glucose cotransporter SGLT1. RS1 inhibits release of SGLT1 from the trans Golgi network. In subconfluent LLC-PK(1) cells, RS1 migrates into the nucleus and modulates transcription of SGLT1, whereas most confluent cells do not contain RS1 in the nuclei. We showed that confluence-dependent nuclear location of RS1 is because of different phases of the cell cycle and identified a RS1 nuclear shuttling domain (RNS) with an associated protein kinase C (PKC) phosphorylation site (RNS-PKC) that mediates cell cycle-dependent nuclear location. RNS-PKC contains a novel non-conventional nuclear localization signal interacting with importin beta1, a nuclear export signal mediating export via protein CRM1 and a Ca(2+)-dependent calmodulin binding site. PKC and calmodulin compete for binding to RNS-PKC. Mutagenesis experiments and analyses of the phosphorylation status suggest the following sequences of events. Subconfluent cells without and with synchronization to the G2/M phase contain non-phosphorylated RNS-PKC that mediates nuclear import of RS1 but not its export. During confluence or synchronization of subconfluent cells to the G2/M phase, phosphorylation of RNS-PKC mediates rapid nuclear export of RS1.