Protein Kinase A‐mediated Regulation of SLC5A8 Surface Expression

SLC5A8 (solute carrier gene family 5A, member 8) encodes the sodium monocarboxylate transporter, SMCT1. SMCT1 mediates cotransport of sodium and monocarboxylates, such as short chain fatty acids, in many tissues, including thyroid, kidney, and colon. Inspection of SMCT1’s primary amino acid sequence identifies not only a carboxyl terminus post‐synaptic density/discs large/zona occuldens 1 (PDZ) binding motif postulated to be important in macromolecular transport protein complexing, but also consensus sites for phosphorylation by multiple classes of kinases. Notably, SMCT1 contains a single protein kinase A (PKA) consensus site: RRMT (amino acids 50–53), with T53 potentially functioning as the site of phosphorylation. The present study considers the hypothesis that PKA regulates SMCT1 surface expression, as reported for the sodium‐glucose cotransporter (SGLT1, the product of the SLC5A1 gene). The following experiments test whether elimination of the phosphorylation site disrupts PKA‐mediated regulation of surface expression. Using site‐directed mutagenesis, we disrupted the consensus motif and concomitantly disabled phosphorylation by replacing T53 with an alanine residue (T53A). To facilitate downstream assays, we generated T53A on the background of a human SMCT1 expression construct containing an amino‐terminus, extracellular hemagglutinin (HA) tag. In transiently expressing COS‐7 cells, surface luminescence measurements confirmed T53A resides in the cell surface at levels similar to wild‐type SMCT1, and immunoblot measurements indicate similar total expression levels. Wild‐type SMCT1‐expressing cells displayed increased luminescence signal (~20%) subsequent to forskolin (10 μM) treatment to stimulate adenylate cyclase and activate PKA, consistent with augmented surface expression. In contrast, cells expressing T53A were indifferent to forskolin treatment. As an independent measure of surface expression, plans are underway to perform cell‐surface immunoprecipitation (CS‐IP) experiments. Taken together, these data suggest that, although constitutive plasma membrane residency does not depend on an intact PKA consensus site, forskolin‐activated, PKA‐dependent phosphorylation can increase SMCT1 levels in the plasma membrane, and thereby augment potential transport capacity. Support or Funding Information NIH/NIGMS Award 1R15GM101674‐01A1 (to PF)