308
specific kinase remains challenging. Though powerful, genetic
methods such as siRNA-based genetic knockdown and CRISPRCas gene editing, which provide insight regarding the function
of a specific protein kinase, presently lack spatial and temporal
control and often fall prey to compensatory cellular mechanisms.
Toward the goal of establishing temporal control of a specific
protein kinase, three elegant posttranslational control methods
have been developed that build on structural studies [15–17]
and seek to either turn a specific kinase on or off.
Shokat and coworkers pioneered a pharmacological knockdown
approach by designing inhibitor-sensitive kinase alleles, where
mutation of the gatekeeper residue adjacent to the kinase ATPbinding cleft allowed for inhibition with uniquely complementary
inhibitors [18–20]. A more recent protein engineering approach
described by Hahn and coworkers provides allosteric control over
kinase activity by using a modified FK506 binding protein (FKBP)
as an insertion motif in a highly conserved region of the catalytic
domain of a protein kinase, which renders the kinase inactive. The
subsequent addition of the small molecule rapamycin as well as the
FKBP12 − rapamycin binding protein (FRB), likely restores catalytic activity through the reestablishment of the native tertiary
structure of the disrupted catalytic domain [21, 22]. A third
approach, pioneered by Chin and coworkers, incorporated a genetically encoded nonnatural photocaged lysine implicated in catalysis
that renders the kinase inactive [23]. Irradiation of cells expressing
the mutant kinase uncages lysine, leading to a gain of kinase activity and downstream signaling. Each of these innovative approaches,
focusing on rendering a specific kinase inactive or active, has merits
for interrogating kinase-dependent biological pathways. However,
these approaches do not currently allow for the simultaneous control and study of multiple kinases.
The approach discussed in this chapter, split protein kinases
(split kinases), complements the three previously described methods, providing ligand-induced gating of the activity of not only
one, but multiple kinases, which can allow for understanding and
engineering of more complex signal transduction pathways [24–26].
A split kinase consists of a protein kinase dissected into two fragments, each of which is attached to an interacting protein pair from
a system commonly called chemical inducers of dimerization (CID)
(Fig. 1). A CID system is composed of two proteins that do not
interact with each other until an input, typically a small molecule,
is added, which causes the proteins to interact or dimerize [27].
Thus in our design, each fragment of a split kinase attached to
ligand responsive proteins is inactive until reassembled through
the addition of the appropriate chemical input with attendant
restoration of catalytic activity. Conceptually, the control of multiple kinases is possible by using orthogonal sets of CIDs. We have
Javier Castillo-Montoya and Indraneel Ghosh
specific kinase remains challenging. Though powerful, genetic
methods such as siRNA-based genetic knockdown and CRISPRCas gene editing, which provide insight regarding the function
of a specific protein kinase, presently lack spatial and temporal
control and often fall prey to compensatory cellular mechanisms.
Toward the goal of establishing temporal control of a specific
protein kinase, three elegant posttranslational control methods
have been developed that build on structural studies [15–17]
and seek to either turn a specific kinase on or off.
Shokat and coworkers pioneered a pharmacological knockdown
approach by designing inhibitor-sensitive kinase alleles, where
mutation of the gatekeeper residue adjacent to the kinase ATPbinding cleft allowed for inhibition with uniquely complementary
inhibitors [18–20]. A more recent protein engineering approach
described by Hahn and coworkers provides allosteric control over
kinase activity by using a modified FK506 binding protein (FKBP)
as an insertion motif in a highly conserved region of the catalytic
domain of a protein kinase, which renders the kinase inactive. The
subsequent addition of the small molecule rapamycin as well as the
FKBP12 − rapamycin binding protein (FRB), likely restores catalytic activity through the reestablishment of the native tertiary
structure of the disrupted catalytic domain [21, 22]. A third
approach, pioneered by Chin and coworkers, incorporated a genetically encoded nonnatural photocaged lysine implicated in catalysis
that renders the kinase inactive [23]. Irradiation of cells expressing
the mutant kinase uncages lysine, leading to a gain of kinase activity and downstream signaling. Each of these innovative approaches,
focusing on rendering a specific kinase inactive or active, has merits
for interrogating kinase-dependent biological pathways. However,
these approaches do not currently allow for the simultaneous control and study of multiple kinases.
The approach discussed in this chapter, split protein kinases
(split kinases), complements the three previously described methods, providing ligand-induced gating of the activity of not only
one, but multiple kinases, which can allow for understanding and
engineering of more complex signal transduction pathways [24–26].
A split kinase consists of a protein kinase dissected into two fragments, each of which is attached to an interacting protein pair from
a system commonly called chemical inducers of dimerization (CID)
(Fig. 1). A CID system is composed of two proteins that do not
interact with each other until an input, typically a small molecule,
is added, which causes the proteins to interact or dimerize [27].
Thus in our design, each fragment of a split kinase attached to
ligand responsive proteins is inactive until reassembled through
the addition of the appropriate chemical input with attendant
restoration of catalytic activity. Conceptually, the control of multiple kinases is possible by using orthogonal sets of CIDs. We have
Javier Castillo-Montoya and Indraneel Ghosh