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

<jats:p>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.</jats:p>