Fig. 5.

Mutations at S551 alter the interaction of SIK3 with PKA and 14-3-3. (A) Expression plasmids for HA-Sik3 (WT), HA-S551A, HA-S551D, and HA-Sleepy were transfected to HEK293 cells, cell lysates immunoprecipitated with anti-HA antibodies, and precipitates were probed with anti-SIK3 (Ex13) antibody, anti-pPKA substrate antibody, anti–14-3-3 (pan) antibody, and anti-HA antibody. Control: no plasmid transfected. (B) The schematic diagram of molecular mechanisms for the sleep/wake phenotypes of WT, Sleepy (ΔEx13), S551A, and S551D mutants. A deletion/mutation at S551 of SIK3 result in decreased recognition from PKA and abolished binding with 14-3-3. This may alter the substrate selectivity or subcellular localization of SIK3, which leads to the increased NREMS and sleep need in Sleepy, S551A, and S551D mutant mice.