Abstract
Tubby-like proteins (TULPs) are characterized by a conserved C-terminal domain that binds phosphoinositides. Collectively, mammalian TULP1-4 proteins play essential roles in intracellular transport, cell differentiation, signaling, and motility. Yet, little is known about how the function of these proteins is regulated in cells. Here, we present the protein–protein interaction network of TULP3, a protein that is responsible for the trafficking of G-protein-coupled receptors to cilia and whose aberrant expression is associated with severe developmental disorders and polycystic kidney disease. We identify several protein interaction nodes linked to TULP3 that include enzymes involved in acetylation and ubiquitination. We show that acetylation of two key lysine residues on TULP3 by p300 increases TULP3 protein abundance and that deacetylation of these sites by HDAC1 decreases protein levels. Furthermore, we show that one of these sites is ubiquitinated in the absence of acetylation and that acetylation inversely correlates with ubiquitination of TULP3. This mechanism is evidently conserved across species and is active in zebrafish during development. Finally, we identify this same regulatory module in TULP1, TULP2, and TULP4 and demonstrate that the stability of these proteins is similarly modulated by an acetylation switch. This study unveils a signaling pathway that links nuclear enzymes to ciliary membrane receptors via TULP3, describes a dynamic mechanism for the regulation of all tubby-like proteins, and explores how to exploit it pharmacologically using drugs.
Highlights
The tubby phenotype, characterized by mature-onset obesity, insulin resistance, sterility, and hearing and vision impairment, was first observed in an inbred strain of C57BL/6J mice [1,2,3,4]
We show that TULP1, TULP2, and TULP4 protein levels are regulated by a parallel lysine acetylation switch
We observed an approximate twofold increase in the levels of all three proteins in response to the drug, an effect that was slightly more pronounced for TULP1 and TULP4 than TULP2 (Fig. 7G). These results demonstrate that in addition to TULP3, p300 plays a role in modulating protein levels of TULP1, TULP2, and TULP4, while HDAC1 displays differential effects on different tubby-like proteins (TULPs) paralogs
Summary
To better understand the biological role of TULP3 in the nucleus and to identify potential regulatory mechanisms for this protein, we performed an IP-MS experiment to profile its protein interactome. We used the top 35 of these 221 hits, ordered by Protein Score, to generate a STRING [31] network diagram (Fig. 1, B–C) This analysis revealed several nodes containing proteins involved in the IFT-A complex, nucleic acid repair, transcription, and splicing, mediators of cell signaling, and components of the Cullin-3 RING ubiquitin ligase complex (CRL-3) [32] (Fig. 1C). To explore potential acetylation of TULP3, we performed an immunoprecipitation of HA-tagged TULP3 from 293T cells in the absence or presence of tagged histone acetyltransferase (HAT) enzymes including p300, PCAF, and GCN5 and probed the resulting western blot with a pan-acetyl antibody. We observed a strong acetylation signal resulting from p300 stimulation (Fig. 2A), but not the other HATs, which overlapped with the predicted molecular weight of TULP3 This effect was associated with an apparent increase in the total protein levels of TULP3 in the input of the experiment (Fig. 2A). BCR WDR35 TTC21B ANKRD54 PPM1G SLAIN2 RAD18 CSNK2A2 AKT1S1 COPS2 GTPBP1 TCF25 JMJD6
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