307
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_19, © Springer Science+Business Media LLC 2017
Chapter 19
Engineering Small Molecule Responsive Split Protein
Kinases
Javier Castillo-Montoya and Indraneel Ghosh
Abstract
The over 500 human protein kinases are estimated to phosphorylate at least one-third of the proteome.
This posttranslational modification is of paramount importance to intracellular signaling and its deregulation
is linked to numerous diseases. Deciphering the specific cellular role of a protein kinase of interest remains
challenging given their structural similarity and potentially overlapping activity. In order to exert control
over the activity of user-defined kinases and allow for understanding and engineering of complex signal
transduction pathways, we have designed ligand inducible split protein kinases. In this approach, protein
kinases are dissected into two fragments that cannot spontaneously assemble and are thus inactive. The two
kinase fragments are attached to chemical inducers of dimerization (CIDs) that allow for ligand induced
heterodimerization and concomitant activation of kinase activity.
Key words Protein kinase, Split kinase, Split protein, Ligand gating, Chemical inducer of dimerization
1 Introduction
The activity and function of many, if not all, proteins are regulated by a multitude of chemical perturbations, collectively
termed posttranslational modifications (PTMs) [1]. Of these,
protein phosphorylation may be the most common [2], where
some estimate that three-quarters of the proteome can be phosphorylated [3, 4]. Protein phosphorylation is catalyzed by a class
of enzymes known as protein kinases. Protein kinases are involved
in almost all pathways, from cell division to cell death [5], and
their aberrant function is implicated in a plethora of diseases
such as cancer [6], metabolic disorders [7], inflammation [8],
and neurological disorders [9]. This renders kinases very attractive targets for therapeutic intervention [10–12], and protein
kinase inhibitors are currently the second largest group of therapeutics. Kinase research has advanced tremendously since the
discovery of the first serine [13] and tyrosine kinases [14],
respectively. However, deciphering the specific cellular role of a
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_19, © Springer Science+Business Media LLC 2017
Chapter 19
Engineering Small Molecule Responsive Split Protein
Kinases
Javier Castillo-Montoya and Indraneel Ghosh
Abstract
The over 500 human protein kinases are estimated to phosphorylate at least one-third of the proteome.
This posttranslational modification is of paramount importance to intracellular signaling and its deregulation
is linked to numerous diseases. Deciphering the specific cellular role of a protein kinase of interest remains
challenging given their structural similarity and potentially overlapping activity. In order to exert control
over the activity of user-defined kinases and allow for understanding and engineering of complex signal
transduction pathways, we have designed ligand inducible split protein kinases. In this approach, protein
kinases are dissected into two fragments that cannot spontaneously assemble and are thus inactive. The two
kinase fragments are attached to chemical inducers of dimerization (CIDs) that allow for ligand induced
heterodimerization and concomitant activation of kinase activity.
Key words Protein kinase, Split kinase, Split protein, Ligand gating, Chemical inducer of dimerization
1 Introduction
The activity and function of many, if not all, proteins are regulated by a multitude of chemical perturbations, collectively
termed posttranslational modifications (PTMs) [1]. Of these,
protein phosphorylation may be the most common [2], where
some estimate that three-quarters of the proteome can be phosphorylated [3, 4]. Protein phosphorylation is catalyzed by a class
of enzymes known as protein kinases. Protein kinases are involved
in almost all pathways, from cell division to cell death [5], and
their aberrant function is implicated in a plethora of diseases
such as cancer [6], metabolic disorders [7], inflammation [8],
and neurological disorders [9]. This renders kinases very attractive targets for therapeutic intervention [10–12], and protein
kinase inhibitors are currently the second largest group of therapeutics. Kinase research has advanced tremendously since the
discovery of the first serine [13] and tyrosine kinases [14],
respectively. However, deciphering the specific cellular role of a