Engineering and Functional Analysis of Mitotic Kinases Through Chemical Genetics

Abstract

During mitosis, multiple protein kinases transform the cytoskeleton and chromosomes into new and highly dynamic structures that mediate the faithful transmission of genetic information and cell division. However, the large number and strong conservation of mammalian kinases in general pose significant obstacles to interrogating them with small molecules, due to the difficulty in identifying and validating those which are truly selective. To overcome this problem, a steric complementation strategy has been developed, in which a bulky "gatekeeper" residue within the active site of the kinase of interest is replaced with a smaller amino acid, such as glycine or alanine. The enlarged catalytic pocket can then be targeted in an allele-specific manner with bulky purine analogs. This strategy provides a general framework for dissecting kinase function with high selectivity, rapid kinetics, and reversibility. In this chapter we discuss the principles and techniques needed to implement this chemical genetic approach in mammalian cells.

Keywords: Analog-sensitive; Bump-hole; Chemical genetics; Genome editing; Kinase engineering.

Figure 1. Selective kinase inhibition through engineered steric complementarity

(A) The gatekeeper residue within your favorite kinase is mutated to a smaller residue to enlarge the catalytic pocket. (B) Selective inhibition is achieved amongst all other cellular kinases using the PP1 analogs 1-NA-PP1, 1-NM-PP1 or 3-MB-PP1. (C) Clustal alignment showing the hinge region and gatekeeper residue for Src and a selection of human and yeast mitotic kinases. (D) Steric complementarity visualized by overlaying the Hck kinase structure and the PP1 (brown), 1-NA-PP1(green), 1-NM-PP1(pink), and 3-MB-PP1 (yellow) inhibitors. The gatekeeper region is displayed in white. Adapted from (6).

Figure 2

Key steps for generating a chemical genetic system in human somatic cells.

Figure 3. Construction of analog-sensitive kinase alleles via Gibson assembly

(A) Overlapping N and C terminal kinase fragments are generated using PCR. The tails of the forward N-terminal primer and the reverse C-terminal primer need ~20 bp of overlap homology overlap with the expression vector. The N-terminal reverse primer and the C terminal forward primer also overlap and incorporate the desired gatekeeper mutation (_*). (B) The kinase fragments are combined with the linearized expression plasmid in the Gibson assembly reaction mix, which contains three enzymatic activities: (1) an exonuclease that generates single-stranded 3′ overhangs, allowing for strand annealing betweent the fragments; (2) a DNA polymerase that fills in any remaining gaps; and (3) a DNA ligase that seals nicks to create covalently closed circular DNA, generating the final expression construct.

Figure 4. Visualizing analog-dependent and allele-specific growth inhibition using crystal violet staining

Plk1^wt or Plk1^as cells were cultured in the presence of 1-NM-PP1 or 3-MB-PP1 (left to right: 0, 0.078 μM, 0.313 μM, 1.25 μM, 5.0 μM, and 20.0 μM) for 8 days. Adapted from (20).