Engineered orthogonal signaling systems for selective phosphorylation of protein kinase substrates

ABSTRACT Protein kinases function in cell signaling through regulated phosphorylation of specific substrates. Current methods allow one to comprehensively identify the substrates of a given kinase, yet we lack general methodology for determining which substrates are critical for specific functions of that kinase. Likewise, approaches for probing

the functional impact that phosphorylation has upon such key substrates are lacking. Here, we propose to develop technology allowing for directed phosphorylation of a single protein kinase substrate at defined sites. With this approach, we engineer a kinase mutant that can only phosphorylate a designer allele of one of its

substrates. To accomplish this goal we will leverage knowledge of kinase phosphorylation site specificity gained through our recent comprehensive analysis of the human serine-threonine kinome and consequent understanding of the structural determinants of kinase selectivity. We will establish the feasibility of this

technology using the tumor suppressor kinase LKB1 as a model system. LKB1 has a well-defined substrate repertoire, phosphorylating and activating a set of 14 downstream protein serine-threonine kinases exclusively on Thr residues. We will engineer LKB1 so that it instead phosphorylates Ser residues and does not act on its

endogenous substrates. We will then engineer compensating mutations in one of its key substrates, the AMP- activated protein kinase (AMPK), to restore phosphorylation and activation by mutant LKB1. Human cancer cell lines co-expressing these alleles will be analyzed for LKB1-dependent activation of AMPK to the exclusion of

other LKB1 substrates. To determine whether this system faithfully recapitulates endogenous signaling, we will examine phosphorylation of established substrates downstream of AMPK, and we will globally map changes to the phosphoproteome in response to AMPK activation. At the outcome of these studies, we will have established

a system in which a single substrate of a kinase is phosphorylated with identical dynamics as the native substrate. Future studies will expand this approach into other LKB1 substrates, facilitating studies of how LKB1 functions as a tumor suppressor. We will ultimately apply this technology generally to other protein kinases

implicated in cancer whose critical substrates are currently unknown.