# Lessons Learned from PKA: Assembly of Dynamic Macromolecular Switches

> **NIH NIH R35** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2024 · $632,000

## Abstract

ABSTRACT
My history with cAMP-dependent protein kinase (PKA) and NIGMS, from active site labeling to holoenzyme
structures and tissue imaging, has been long and productive. My career has been guided by the fundamental
principle that structure will reveal function with the ultimate goal being to elucidate how PKA signaling regulates
biology and how it is altered in disease. Our tools include biochemistry, biophysics, and molecular biology to
probe mechanisms as well as crystallography, cryoEM, molecular dynamics, and imaging to explore conforma-
tional space and localization in cells. A hallmark of my laboratory has been to build interdisciplinary teams that
reach across all of these scales. Although it has been over 30 years now since we solved that first protein
kinase structure of the PKA catalytic (C) subunit, which has served ever since as the prototypical protein
kinase, surprisingly we are still learning new things. PKA signaling in cells is mediated by full-length R2C2
holoenzymes that are targeted to discreet sites in the cell near dedicated substrates, and a major recent
achievement was our solving the cryoEM structure of the compact full-length RII holoenzyme in 2020 where
for the first time all of the domains could be visualized. During this next phase we will continue with our
characterization of holoenzyme complexes focusing, in particular, on RIIβ, which is enriched in neurons and
localizes to Golgi. In addition, however, we will build on two new discoveries that came from our work over the
past three years. First is the discovery that Cβ subunits, a family of previously unexplored splice variants that
account for ~50% of PKA signaling in neurons, are linked to a neurodegenerative phenotype that abolishes
Sonic hedgehog (Shh) signaling. With imaging in human retina we then validated that Cβ is highly expressed
in neurons, that it localizes differently than C, and that Cβ4/Cβ4ab are enriched at mitochondria. We are now
characterizing the neuron-specific Cβ4 isoforms and the specific mutants that correlate with Shh signaling.
Another new and potentially related discovery is that there is a functional PKI-like sequence embedded in the
C-terminal tail of Smoothened, the GPCR that is associated with Shh signaling. A final discovery that the RI
subunit undergoes liquid:liquid phase separation that contributes to cAMP buffering in cells opens another new
frontier for non-canonical PKA signaling in cells. We find that RIβ also forms biomolecular condensates that
are distinct from RIα, and we are now characterizing an RIβ mutant, RIβ(R335W), that is associated with
dementia and autism. An essential part of our strategy is to use a multi-scale approach that includes not only
biochemical characterizations and structure solutions but also high-resolution imaging in human tissues where
we can hopefully correlate changes in localization and expression with pathogenic mutations in Cβ and RIβ. In
parallel, we will build on our cryo-EM structure ...

## Key facts

- **NIH application ID:** 10878751
- **Project number:** 5R35GM130389-07
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** SUSAN S. TAYLOR
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $632,000
- **Award type:** 5
- **Project period:** 2019-01-01 → 2027-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10878751

## Citation

> US National Institutes of Health, RePORTER application 10878751, Lessons Learned from PKA: Assembly of Dynamic Macromolecular Switches (5R35GM130389-07). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10878751. Licensed CC0.

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