Phosphorylation is a versatile and reversible posttranslational modication that aects the subcellular location, catalytic activity, and interaction of proteins. e importance of phosphorylation is evident not only in its pervasive inuence over approximately one third of the human proteome but also in the nodal functions served by many phosphorylated proteins. e protein kinases that confer phosphorylation are thus pivotal components in many signaling cascades. Commensurate with this central role, kinase signaling is often nely regulated through mechanisms such as compartmentalization and feedback interactions. All of these processes are important pieces in the kinase-signaling puzzle, yet it is often dicult to assess their impact in a live cell context using traditional biochemical approaches. However, the recent development of uorescent reporters, along with advances in optical microscopy, has enabled us to monitor the spatiotemporal dynamics of signaling molecules such as kinases in real time in living cells, thus greatly complementing traditional biochemistry. e simplest optical readout that can be elicited from a uorescence-based

9.1 Introduction 247 9.2 Kinase Activation Sensors 249 9.3 Kinase Activity Biosensors 253 9.4 Outlook and Perspective 260 References 263

kinase reporter is the subcellular location of the kinase of interest; however, when the signal from a biosensor changes in parallel with the activity of a kinase, microscopy can further reveal the spatiotemporal dynamics of the kinase and allow a more meaningful examination of signaling. Such signaling dynamics are best captured by sensors with high dynamic ranges, high specicity, and rapid reversibility. e purpose of this chapter is to showcase prototypical kinase sensor designs and review the considerations that go into making optimal sensors that satisfy the aforementioned criteria. We introduce the general principles that underlie the design of these biosensors and highlight how such biosensors have aided the dissection of signaling pathways.