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A Subtle Amino Acid Change Impacts Kinase Function in Dramatically Distinct Ways

Dysregulated protein kinase function is prevalent in cancer. Analyses of disease mutations reveal important insights into structure‐function relationships in kinases. Here we present how mutation of a conserved residue can have vastly different effects on kinase function among distinct kinases. A previous study by Weinreb et al. (Nat Genet. 2014) identified a point mutation within a conserved subdomain of PKD1 (E710D) in polymorphous low‐grade adenocarcinomas of the salivary gland. In their study, in vitro kinase assays revealed increased activity of this mutated PKD1 as well as a change in its subcellular localization. Through use of our D Kinase Activity Reporter (DKAR) to monitor kinase signaling, we confirm that E710D mutation exhibits constitutive activity and we observe that the kinase localization is significantly altered by this subtle amino acid change. Specifically, the mutant PKD1‐E710D protein localizes basally in the nucleus and at Golgi membranes compared to the solely cytoplasmic distribution of wild‐type PKD1. This Glu residue appears as Glu in nearly all of the CAMK family members as well as many members of the AGC family of kinases such as Akt and PKA; interestingly, most of the other members of the AGC family, such as PKC, possess Asp at this site. A search of other kinases mutated in cancer at this conserved residue revealed a Glu to Asp mutation in Akt3 in endometrial cancer. Introduction of this mutation within Akt3, however, does not result in a change in protein localization nor does it result in an active kinase when assessed using our Akt/B Kinase Activity Reporter (BKAR); surprisingly, Akt3‐E275D appears completely inactive. Thus, mutation from Glu to Asp in PKD results in a dramatic conformational alteration of the enzyme leading to constitutive activity, and mutation from Glu to Asp in Akt results in a complete loss of activity. These findings underscore how minute changes (Glu to Asp) can have dramatically different impacts on kinase structure and function, and how alterations of these components can have divergent effects on kinase biology. Further analyses of these mutations in the context of the kinase can yield insight into the role each kinase plays in tumor initiation, progression and metastasis.