Regulation of brain-specific kinases 1 and 2 (BRSK1/2) by Ca2+/calmodulin

• BRSK1 and BRSK2 are identified as novel Ca 2+ /CaM-interacting kinases • Ca 2+ /CaM inhibits LKB1-catalyzed phosphorylation and activation of BRSK1/2 • Ca 2+ /CaM interacts with BRSK1 kinase domain to inhibit Thr189 phosphorylation • Ca 2+ /CaM interacts with phosphorylated BRSK1 without inhibiting its kinase activity We conducted a genome-wide calmodulin (CaM) interaction screening of 462 GST-fused human protein kinases to identify novel CaM-dependent protein kinases (CaMKs). In addition to known CaMKs, including myosin light chain kinases, CaMK2γ, and death-associated kinase 2, we identified the brain-specific protein kinase 2 (BRSK2, also known as SAD-A) as a novel CaM interactant. Proximity biotinylation and CaM–sepharose chromatography assays revealed that rat BRSK isoforms (BRSK1/2) interact with CaM in a Ca 2+ -dependent manner in vitro . We found that CaM suppresses the activation-loop phosphorylation of BRSK1 (at Thr189) and BRSK2 (at Thr175) by liver kinase B1 (LKB1), an activating kinase, in a Ca 2+ -dependent manner (IC 50 of ∼7 µM), thereby inhibiting BRSK activation. LKB1-catalyzed phosphorylation of the catalytic domain mutant of BRSK1 (residues 1–294) at Thr189 was suppressed by the addition of Ca 2+ /CaM, consistent with direct CaM binding of the kinase domain, as well as wild-type BRSK1. We confirmed that the LKB1 activity was not directly suppressed by Ca 2+ /CaM, supporting the hypothesis that the direct interaction of Ca 2+ /CaM with the kinase domain blocks the phosphorylation/activation of BRSK1/2 by LKB1. The kinase activity and PP2Cα-catalyzed dephosphorylation of LKB1-phosphorylated BRSK1 were not altered by Ca 2+ /CaM, although it was demonstrated to bind to Ca 2+ /CaM like that of unphosphorylated BRSK1. This unrecognized mechanism of BRSK1/2 regulation, involving the direct role of Ca 2+ /CaM binding, which inhibits phosphorylation/activation by LKB1, may open a new Ca 2+ signal transduction pathway in neurons.