A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases

Abstract

Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.

Chemical approaches towards unravelling kinase-mediated signalling pathways

“…[2][3][4] Other limitations, such as non-specific adsorption of proteins to chips or nitrocellulose membranes and incorrect folding of recombinant proteins also need to be considered. Furthermore, 50 for convenience, peptide analogues, rather than protein substrates, are often employed in in vitro studies, however, they often display K M values that are several orders of magnitude higher than their protein progenitors.…”

“…For this 50 reason, this residue, which is conserved in most protein kinases as a large or polar amino acid 22 , has been termed the 'gatekeeper' residue. Gatekeeper mutations decrease interactions between the adenine ring and the adenine-binding pocket of the protein kinase, and as a consequence decrease 55 the affinity of the kinase for ATP ~10 fold 23 The enlarged adenine binding site can be filled by providing an ATP analogue with a bulkier, hydrophobic adenine moiety.…”

“…For example, a series of enlarged adenine analogues (analogous to those outlined in Scheme 3) was found to have IC 50 values for WT v-Src or Fyn kinase of >300 M 24 , strongly suggesting that they had no 85 effect on other endogenous kinases. Mutation of the gatekeeper residue decreased IC 50 fold meaning that the as-kinase was selectively inhibited. 24 In summary, the biochemical data characterising the interactions of WT and as-kinases with ATP and adenine-enlarged ATP analogues 90 (Fig.…”

“…In the second, a triphosphate is chemically ligated to a modifying group to generate the triphosphate analogue. 45 Diphosphates can be accessed by several routes, however, two commonly used routes will be discussed in detail here: (i) Yoshikawa phosphorylation of the modified adenosine followed by introduction of the second phosphoryl group through the use of tri-or tetraalkylammonium salts of 50 inorganic phosphate, and (ii) preparation of a 5´-tosylate of the adenosine analogue and subsequent displacement of this leaving group using a tri-or tetraalkylammonium salt of the inorganic pyrophosphate ion (Scheme 9).…”

“…In a cellular context, subcellular co-localization of a 45 kinase and its substrate(s), and long-distance interactions between the kinase and its substrate(s) facilitate control over the selectivity of a particular protein kinase. Some approaches, notably the use of labelled ATP analogues, require cells to be lysed, because of the membrane 50 impermeability of these hydrophilic molecules. Thus, the spatial control over kinase-substrate pairs is lost.…”