Endolysosomal targeting of Protein Kinase A by ARHGAP36

• Applicant: Dr. Oliver Rocks
• Subject Area: Biochemistry ; Cell Biology
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 420526360

Protein kinase A (PKA) is a key mediator of cAMP signalling downstream of G protein coupled receptors, controlling development, metabolism and other essential processes. PKA activity must be precisely regulated to elicit its cellular responses. Failure to keep it under control contributes to serious disorders, including cancer. We have previously demonstrated that the Rho GTPase activating protein ARHGAP36 is a novel negative regulator of PKA. It binds the PKA catalytic subunit (PKAC) via a pseudosubstrate motif to block its kinase activity, and further, mediates its rapid ubiquitin-dependent degradation. Ubiquitylation of PKAC has not been described before. This bimodal antagonism of PKAC leads to inhibition of PKA signalling responses downstream. It may also contribute to medulloblastoma formation upon ARHGAP36 upregulation. Surprisingly, PKAC is not degraded via the proteasome but the endolysosomal system, a pathway that is typically reserved for transmembrane proteins. The underlying molecular and cellular mechanisms are unclear. The goal of this proposal is to resolve the mechanism of ARHGAP36-mediated PKAC ubiquitylation and to elucidate how the kinase is delivered from the cytosol to lysosomes. We have already demonstrated the involvement of the endosomal sorting complexes required for transport (ESCRT) and of K63 ubiquitylation in the transport of PKAC. We now aim to 1) identify the E3 ligase responsible for PKAC ubiquitylation and to study its regulation, 2) understand how ARHGAP36 mediates the encounter of the kinase with the ligase and its entry to the endosomal system and 3) to explore how ARHGAP36/PKAC is sequestered to multivesicular bodies and how ARHGAP36 ultimately segregates from PKAC to escape from degradation. The results of this work will not only reveal a new layer of regulation of a fundamental ubiquitous signalling pathway but also further unravel an unexpected cellular transport and degradation route that could potentially be targeted to interfere with aberrant PKA function and other signalling pathways.

• DFG Programme: Research Grants