Time-resolved proteomic profiling of the ciliary Hedgehog response reveals that GPR161 and PKA undergo regulated co-exit from cilia

Abstract

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein-coupled receptor GPR161 in response to Hedgehog; and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the putative phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits

“…Regardless of the underlying mechanism, prior literature strongly supports a role for free PKA-C in cilia phosphorylating and inactivating GLI. Second, a recent study of the ciliary proteome [ 130 ] readily detected SMO, but failed to detect PKA-C. Given the evidence for free PKA-C in cilia (see above), a parsimonious interpretation is that (1) levels of free PKA-C in cilia, while sufficient to phosphorylate and inactivate GLI, must be extremely low in absolute terms; and (2) levels of SMO likely exceed those of PKA-C, particularly in the pathway “on” state in which steady-state SMO levels dramatically rise in cilia.…”

Targeting an anchored phosphatase-deacetylase unit restores renal ciliary homeostasis

“…Interestingly, AKAP79/150 has been implicated as a modulator of renal ciliary signaling ( Choi et al, 2011 ). However, proximity proteomic approaches identify AKAP220 in cilia, and knockout of the AKAP79/150 ortholog in mIMCD3 cells and mice does not impact cilia development ( May et al, 2020 and Figure 1—figure supplement 2 ). In contrast, our in vivo and in vitro data strongly implicate AKAP220 signaling in the modulation of aquaporin-2 trafficking and ciliogenesis.…”

“…A-kinase anchoring proteins (AKAPs) spatially constrain second messenger regulated kinases, protein phosphatases, and GTPase effector proteins within subcellular compartments ( Bucko and Scott, 2021 ; Langeberg and Scott, 2015 ; Omar and Scott, 2020 ; Smith et al, 2017 ; Smith et al, 2018 ; Taskén and Aandahl, 2004 ; Whiting et al, 2015 ). Several AKAPs participate in renal signaling, yet only a few anchoring proteins reside in cilia ( Choi et al, 2011 ; Gopalan et al, 2021 ; Jo et al, 2001 ; May et al, 2020 ; Stefan et al, 2007 ). Ciliary AKAPs are postulated to position protein kinase A (PKA) as a negative regulator of hedgehog signaling ( Breslow et al, 2018 ; Mukhopadhyay et al, 2013 ; Somatilaka et al, 2020 ).…”

Targeting an anchored phosphatase-deacetylase unit restores renal ciliary homeostasis

“…Interestingly, AKAP79/150 has been implicated as a modulator of renal ciliary signaling (Choi et al, 2011). However, proximity proteomic approaches identify AKAP220 in cilia, and knockout of the AKAP79/150 ortholog in mIMCD3 cells and mice does not impact this aspect of cilia development ((May et al, 2020) & Supp Fig 1). In contrast, our in vivo and in vitro data strongly implicate AKAP220 signaling in the modulation of aquaporin-2 trafficking and ciliogenesis.…”

“…A-kinase anchoring proteins (AKAPs) spatially constrain second messenger regulated kinases, protein phosphatases and GTPase effector proteins within subcellular compartments (Bucko and Scott, 2021; Langeberg and Scott, 2015; Omar and Scott, 2020; Smith et al, 2017, 2018; Taskén and Aandahl, 2004; Whiting et al, 2015). Several AKAPs participate in renal signaling, yet only a few anchoring proteins reside in cilia (Choi et al, 2011; Jo et al, 2001; May et al, 2020; Stefan et al, 2007). Ciliary AKAPs are postulated to position protein kinase A (PKA) as a negative regulator of hedgehog signaling (Breslow et al, 2018; Mukhopadhyay et al, 2013; Somatilaka et al, 2020).…”