Orchestrating Intracellular Calcium Signaling Cascades by Phosphosite-Centric Regulatory Network: A Comprehensive Analysis on Kinases CAMKK1 and CAMKK2

Intracellular calcium signaling is a cornerstone in cell biology and a key molecular target for human health and disease. Calcium/calmodulin dependent protein kinase kinases, CAMKK1 and CAMKK2 are serine/threonine kinases that contribute to the regulation of intracellular calcium signals in response to diverse stimuli. CAMKK1 generally has stable dynamics, whereas CAMKK2 dysregulation triggers oncogenicity and neurological disorders. To differentiate the phosphosignaling hierarchy associated with predominant phosphosites of CAMKK1 and CAMKK2, we assembled and analyzed the global cellular phosphoproteome datasets. We found that predominant phosphosites in CAMKK1 and CAMKK2 are located outside the kinase domain, and their phosphomotifs are highly homologous. Further, we employed a coregulation analysis approach to these predominant phosphosites, to infer the co-occurrence patterns of phosphorylations within CAMKKs and the coregulation patterns of other protein phosphosites with CAMKK sites. We report herein that independent phosphorylations at CAMKK2 S100 and S511 increase their enzymatic activity in the presence of calcium/calmodulin. In addition, the study unveils kinase-substrate associations such as RPS6KB1 as a novel high-confidence upstream kinase of both CAMKK1 S74 and CAMKK2 S100. Further, CAMKK2 was identified as a primary orchestrator in mediating intracellular calcium signaling cascades compared to CAMKK1 based on coregulation patterns of phosphosites from proteins involved in the calcium signaling pathway. These molecular details shed promising insights into the pathophysiology of several diseases such as cancers and psychiatric disorders associated with kinase activity dysregulations of CAMKK2 and further open the avenue for novel PTM-directed therapeutic strategies to regulate CAMKK2.