The activity and function of many if not all proteins are regulated by chemical perturbations, such as phosphorylation, acetylation, and methylation. With around one third of the cell proteome being phosphorylated, phosphorylation in particular is of paramount importance to cell biology and signaling. This chemical modification is catalyzed by two very large families of similar enzymes: protein kinases and phosphatases. These enzymes are involved in almost all pathways, from cell division to cell death, and their aberrant function is implicated in diseases such as cancer, inflammation and neurological disorders. Thus, understanding their function, by turning them on or off, is important for understanding their biology and for designing new therapies.<br/>The structural similarity of kinases and phosphatases makes it impossible to selectively turn-on or turn-off desired kinases using inhibitors or activators. To address this problem, we first developed a sequence dissimilarity based approach to identify sites in kinases and phosphatases tolerant to a 25-residue loop insertion. Subsequently, we split the kinase or phosphatase at these sites into two inactive fragments and attached to two proteins, FKBP and FRB. Upon addition of a small molecule chemical inducer of dimerization (CID), rapamycin, that induces the dimerization of FKBP/FRB, we could turn-on the activity of the enzyme. We have successfully demonstrated the feasibility of this approach with members of the tyrosine kinase group, such as Lyn, Src, Fyn, Fak, Hck, Lck and Abl, implicated in cell-proliferation and cancer, as well as with the PTP1B, PTPH1 and SHP1 phosphatases. We are currently developing methods to selectively turn-on desired kinases and phosphatases in live cells. We further demonstrated that the CIDs abscisic acid and gibberellic acid can also be used to control the activity of split-kinases and split-phosphatases. Thus together, we provide a methodology that allows for posttranslational orthogonal small molecule control over the activity of user defined split-PTKs and split-PTPs. This methodology has the long term potential for both interrogating and redesigning signaling pathways.

Controlling the phosphoproteome: Ligand-gated split-kinases and split-phosphatases

249th National Meeting (2015)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.