Biochemical and chemical biological approaches to mammalian sleep: roles of calcineurin in site-specific dephosphorylation and sleep regulation

Understanding of sleep mechanisms traditionally rely on electrophysiology and genetics but here we have initiated biochemical and chemical biological studies. Sleep was increased in mouse mutants with an alanine replacing threonine at residue 469 (T469A) of the salt inducible kinase 3 (SIK3). We searched for T469 phosphatases by classic purification with HEK293 cells and by a new photo-crosslinking method with mouse brains. Both led to PPP3CA, a catalytic subunit of calcium/calmodulin activated phosphatase (calcineurin). It dephosphorylated T469 and serine (S) 551 but not T221 in SIK3 in vitro. PPP3CA knockdown increased phosphorylation of T469 and S551 but not T221 in mouse brains. Knockdown of its regulatory subunit PPP3R1 significantly reduced daily sleep by more than 5 hours, exceeding other known mouse mutants. Our results have uncovered in vitro and in vivo evidence for site-specific SIK3 dephosphorylation by calcineurin, demonstrated a physiological role for calcineurin in sleep, and suggested sleep control by calcium dependent dephosphorylation.