A conserved acetylation switch enables pharmacological control of tubby-like protein stability

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.

Progressive liver, kidney, and heart degeneration in children and adults affected by TULP3 mutations

“…The TULP3 interaction network suggests an association with several core DDR elements, including DDB1 and TP53 ( Figure S5 ). Among the identified protein interactions was the recently reported TULP3 interaction partner SIRT1, 34 a class III histone deacetylase that has broad reaching roles as a regulator of transcription and DDR by mediating deacetylation of TP53 and several histones. 35 , 36 , 37 We confirmed the interaction between TULP3 and SIRT1 through co-immunoprecipitation in HEK293T cells ( Figure 4 C).…”

HTR6 and SSTR3 ciliary targeting relies on both IC3 loops and C-terminal tails

“…Coexpression of wild-type TULP3-myc (or of myc-B9D1, a negative control) did not affect total levels of RABL2B-bound HTR6. However, reduced HTR6-Flag co-IP was seen with TULP3-KR-myc (K268A+R270A), a mutant abolishing TULP3’s phosphoinositide-binding ability, and perhaps also its acetylation ( Fig 12K ) ( 30 , 40 ). Moreover, for TULP3-WT-myc, and even more so for TULP3-KR-myc, we observed moderately reduced co-IP of HTR6-Flag’s lowest molecular weight band (roughly corresponding to its predicted unmodified size of 48.7 kD).…”

HTR6 and SSTR3 targeting to primary cilia

“…We also tested whether TULP3 affects HTR6-RABL2 binding. Overexpression of wild type TULP3 had no effect on it, but a reduction was seen with the TULP3-KR mutant (K268A + R270A), which cannot bind PIPs and, as recently shown, cannot be acetylated at K268 [59,66,70]. This suggests RABL2 and TULP3 may cooperate in ciliary HTR6 targeting.…”