Serine/Threonine Phosphatases in Neurological Diseases

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ABSTRACT. Phosphorylation is one of the most ubiquitous, reversible posttranslational modifications in cells, and is a critical component of most signaling cascades. Strict temporal and spatial control are essential for the fidelity of this process, as derailed signaling cascades lead to disease. Here, we continue our long-standing effort to investigate signaling in neurons. If neuronal signaling goes awry, the most prominent results are well known diseases, such as Alzheimer's disease and stroke. Recently, we successfully determined how the most ubiquitous neuronal ser/thr protein phosphatase (PPP) calcineurin (CN) recruits its substrates. Namely, CN binds regulators and substrates via CN-specific recruitment motifs (PxIxIT and LxVP). Further, we also discovered that CN uses an active site recognition sequence (TxxP) to target substrate phosphosites, which, in turn, drives vital biological functions. This is the first defined active site recognition sequence for any PPP, transforming our ability to identify novel CN-specific phosphosites. Here, we will leverage our newly established tools and discoveries to achieve three aims. First, we will establish the CN interactome and substratome in distinct neuronal populations. This will enable us to demonstrate the diversity of CN functions in neurons, define if they differ in response to stimuli as well as identify the molecular substrates that are necessary for these changes to occur. Building further on the success of the previous funding period, we will also advance our molecular understanding of CN substrate recruitment by studying two critical CN substrate signaling platforms: CN-AKAP5 and CN-TAK1. Specifically, we will show that the these signaling platforms utilize multiple, competing PxIxIT/LxVP motifs to recruit CN via different proteins and show how these distinct mechanisms define CN substrate dephosphorylation efficacy. Critically, our preliminary data suggest that posttranslational modification of AKAP5 modulates CN control of PKA activity and ultimately receptor regulation. Finally, we have also recently confirmed our prediction that the transforming growth factor-β activated kinase 1 (TAK1) binds directly to CN. However, unexpectedly the TAK1 regulator TAB2 also binds directly to CN via different LxVP and PxIxIT motifs. We will investigate the mechanisms and consequences of this interaction on CN recruitment and TAK1 function. The modes of action and regulation of CN in the AKAP5 and TAK1 signaling platforms are unexpected and highlight the broad variety of mechanisms used to regulate CN activity. Taken together, the proposed studies leverage a powerful integrated approach that combines atomic resolution techniques with biochemical and cell biology experiments to obtain novel insights into the molecular mechanisms used to direct CN activity. Because CN has critical roles in human diseases generally, and in the brain specifically, and because CN is the only successfully therapeutically targeted PPP (CN is the target of the blockbuster immunosuppressants cyclosporin A and FK-506), our proposed work will provide a critically needed molecular and cellular understanding of CN activity and regulation in neuronal function.