Biology subjects

Church, T. W.

Publications and source records attributed to Church, T. W..

3 recordsLinked to original sources

bioRxiv2026

PKA can adopt structural or enzymatic roles depending on its anchoring site

Protein kinase A (PKA) is a central mediator of cAMP signaling whose spatial specificity is conferred by A-kinase anchoring proteins (AKAPs). However, functions of individual anchored PKA pools are difficult to resolve as current approaches cannot isolate anchoring at defined sites. Here, we develop a SpyCatcher-SpyTag-based replacement strategy enabling direct comparison of AKAP function with and without type II PKA regulatory (RII) subunit anchoring in intact signaling environments. Anchoring RII to AKAP79 or AKAP1 produced distinct phosphorylation-dependent effects, including catalytic subunit membrane recruitment and mitochondrial morphology regulation. In contrast, MAP2-anchored RII drove phosphorylation-independent microtubule straightening, increased formation of widely spaced bundles, and supported dendritic arborization. This structural function was recapitulated by native MAP2-RII binding and required an intact cAMP-binding pocket in the second cyclic nucleotide-binding domain. These findings establish that AKAP-bound PKA can function as either an enzymatic or structural component and provide a generalizable strategy to dissect compartmentalized kinase signaling.

molecular biology↗

bioRxiv2025

Widely used CaMKII regulatory segment mutations cause tight actinin binding and dendritic spine enlargement in unstimulated neurons

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is essential for long-term potentiation (LTP) of excitatory synapses that underlies learning. CaMKII responds to Ca2+ influx into postsynaptic spines by phosphorylating proteins and forming new protein interactions. The relative importance of these enzymatic and structural functions is debated. LTP induction triggers CaMKII docking to NMDA receptors, and recent evidence suggests that LTP can proceed without kinase activity after this event. Furthermore, CaMKII interaction with -actinin-2 is required for dendritic spine enlargement following LTP induction. CaMKII can auto-phosphorylate at T286, which enables autonomous activity after Ca2+/CaM dissociation. CaMKII also bears threonine at positions 305 and 306 in its regulatory segment. Experiments with CaMKII variants including a T305A/T306A ( AA) double substitution have led to a model whereby T305/T306 phosphorylation by autonomously active CaMKII prevents further Ca2+/CaM activation. However, this mechanism is not compatible with some existing data including CaMKII phospho-proteomics and measurements with reporters of CaMKII conformation. Furthermore, autonomous CaMKII activity is now thought to only endure for seconds after LTP induction. In this study, we show that the AA substitution has an unintended gain-of-function property - it enables tight binding to -actinin-2 in unstimulated neurons. In situ labelling shows that the AA substitution elevates CaMKII-actinin interactions in neurons to a level only normally observed after induction of LTP. The AA CaMKII variant also elevates the proportion of enlarged spines in unstimulated neurons without altering synaptic currents. Calorimetric measurements with purified protein confirm that -actinin-2 binds tightly to the AA variant of CaMKII with no requirement for kinase activation. Using x-ray crystallography, we show that the AA substitution enables -actinin-2 to adopt a different tighter binding mode. Our findings reinforce the notion that CaMKII primarily fulfils a structural role in LTP.

neuroscience↗

bioRxiv2021

AKAP79 enables calcineurin to directly suppress protein kinase A activity

Interplay between the second messengers cAMP and Ca2+ is a hallmark of dynamic cellular processes. A common motif is the opposition of the Ca2+-sensitive phosphatase calcineurin and the major cAMP receptor, protein kinase A (PKA). Calcineurin dephosphorylates sites primed by PKA to bring about changes including synaptic long-term depression (LTD). AKAP79 supports signaling of this type by anchoring PKA and calcineurin in tandem. In this study, we discovered that AKAP79 increases the rate of calcineurin dephosphorylation of type II PKA regulatory subunits by an order of magnitude. Fluorescent PKA activity reporter assays, supported by kinetic modeling, show how AKAP79-enhanced calcineurin activity enables suppression of PKA without altering cAMP levels by increasing PKA catalytic subunit capture rate. Experiments with hippocampal neurons indicate that this mechanism contributes towards LTD. This non- canonical mode of PKA regulation may underlie many other cellular processes.

biochemistry↗