The Ser-Thr directed kinase CaMKKβ activates a variety of substrates in response to increases in intracellular calcium. CaMKKβ plays a critical role in regulating long-term potentiation¹ and neuronal morphology². Additionally, genome wide association studies have implicated CaMKKβ in the pathogenesis of schizophrenia and anxiety disorders³. The enzyme contains an N-terminal regulatory sequence (NTS), a kinase domain and an overlapping autoinhibitory domain/calmodulin-binding domain (AID/CaMBD). Binding of Ca²⁺/calmodulin (Ca²⁺/CaM) to the AID/CaMBD causes an unknown conformational change that relieves auto-inhibition and activates the enzyme. Ca²⁺/CaM independent activity is minimised by phosphorylation of S129, S133 and S137 in the NTS. The absence of phosphorylation of these sites leads to increased basal activity. These sites were determined to be sequentially phosphorylated by MS-TOF⁴, and inhibitor studies have revealed these sites to be phosphorylated by the protein kinases CKI and GSK3. A number of single nucleotide polymorphisms (SNPs); S137L (rs201145728), P138S (rs199839786), R139W (rs202048546) and R142W (rs201263450), have been reported in proximity to the S129/S133/S137 regulatory cluster. These mutants were expressed in HeLa cells and their effect on the Ca²⁺/CaM dependence of CaMKKβ were determined by kinase assays. The S137L and P138S mutants showed increased Ca²⁺/CaM independent activity, consistent with these mutations blocking the sequential phosphorylation of S129/S133/S137. Interestingly, the R139W and R142W mutants displayed heightened Ca²⁺/CaM dependence, suggesting that these residues play a role interacting with the CaMBD.