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.

Transporter regulator RS1 (RSC1A1) coats the trans-Golgi network and migrates into the nucleus.

The product of gene RSC1A1, named RS1, is involved in transcriptional and posttranscriptional regulation of sodium-d-glucose cotransporter SGLT1, and removal of RS1 in mice led to an increase of SGLT1 expression in small intestine and to obesity (Osswald C, Baumgarten K, Stümpel F, Gorboulev V, Akimjanova M, Knobeloch K-P, Horak I, Kluge R, Joost H-G, and Koepsell H. Mol Cell Biol 25: 78-87, 2005). Previous data showed that RS1 inhibits transcription of SGLT1 in LLC-PK1 cells derived from porcine kidney. A decrease of the intracellular amount of RS1 protein was observed during cell confluence, which was paralleled by transcriptional upregulation of SGLT1. In the present study, the subcellular distributions of endogenously expressed RS1 and SGLT1 were compared in LLC-PK1 cells and human embryonic kidney (HEK)-293 cells using immunofluorescence microscopy. RS1 was located at the plasma membrane, at the entire trans-Golgi network (TGN), and within the nucleus. Treatment of LLC-PK1 cells with brefeldin A induced rapid release of RS1 from the TGN, and confluence of LLC-PK1 cells was accompanied by reduction of nuclear location of RS1; 84-90% of subconfluent cells and 5-34% of confluent cells contained RS1 in the nuclei. This suggests that confluence-dependent transcriptional inhibition by RS1 is partially regulated by nuclear migration. Furthermore, we assigned SGLT1 to microtubule-associated tubulovesicular structures and dynamin-containing parts of the TGN. The data indicate that RS1 inhibits the dynamin-dependent release of SGLT1-containing vesicles from the TGN.