Dynamics of Scaffold Protein Tethered Signal Transduction

The basis of intracellular communication and information processing lies in highly connected and complex networks of signal transduction that can produce diverse responses to stimuli. Scaffold proteins bind two or more signaling proteins and play a key role in spatially and temporally organizing these networks of signal transduction. By tethering multiple signaling proteins in close proximity, scaffolds have been hypothesized to be an important determinant of signaling specificity and efficiency. While scaffolds have been observed to amplify and accelerate signaling dynamics, an open challenge remains to identify a mechanistic explanation for these scaffold-derived emergent phenomena. Additionally, the coordination of several interacting proteins can obscure the functional role of a given scaffold protein. Through the combination of computational modeling and live-cell imaging, this dissertation aims to investigate the effects of scaffold proteins on signaling dynamics, and the mechanism that underlies them.

To address the amplification and acceleration of scaffold tethered signaling, we propose the novel “scaffold state-switching” mechanism, where the enzyme-substrate-scaffold complex can stochastically switch between active and inactive intermediate states before the enzyme completes catalysis. We developed a computational model of this mechanism showing that scaffold proteins can amplify and accelerate tethered signal transduction by increasing the rate of enzyme-substrate interaction. To validate these predictions we exploited a direct interaction between Protein Kinase C (PKC) and AKAP7-alpha and found that both the strength and speed of substrate phosphorylation were enhanced in agreement with the computational model. Additionally, extension of this model to study the effects of scaffold proteins on inhibitors led to the prediction and subsequent validation that scaffold proteins can insulate tethered enzymes from substrate- and ATP-competitive inhibitors but not activation-competitive inhibitors. Together, these data provide theoretical and experimental evidence that scaffold proteins can amplify, accelerate and insulate signal transduction through the scaffold state-switching mechanism.

To investigate the coordination of multiple signaling pathways by scaffold proteins, we studied the role of AKAP5 in the coordination of crosstalk between oscillatory Protein Kinase A (PKA) and calcium signaling in MIN6 beta-cells. Using FRET biosensors, we show that Protein Kinase A activity at the plasma membrane oscillates out-of-phase with calcium whereas AKAP5-anchored PKA oscillates in-phase with calcium. The mechanism of these unique dynamics were studied through the development of computational models testing different hypotheses for the role of AKAP5. These models predicted that by regulating the activation of two distinct pools of adenylyl cyclase, AKAP5 simultaneously coordinates both the in- and out-of-phase PKA activity. Extension of this model also identified that the AKAP5 coordination of the positive feedback of PKA onto the Cav1.2 calcium channel is necessary for the development of calcium oscillations. Through the application of these computational models, these data show that the coordination of a specific network of signaling proteins by AKAP5 enable the development of unique signaling dynamics and make AKAP5 essential for the oscillatory dynamics in these cells.

Together, this body of work provides insight into both the mechanisms of scaffold tethered signal transduction and the effects that can arise from this tethering. In addition to improving our understanding of the very basis of cell signaling, this work provides a quantitative framework with which to analyze the effects of all scaffold proteins. This framework will be instrumental in evaluating the disruption of scaffold interactions as a therapeutic strategy and identifying which interactions to target.