The role of lysine palmitoylation/myristoylation in the function of the TEAD transcription factors

Abstract

The TEAD transcription factors are the most downstream elements of the Hippo pathway. Their transcriptional activity is modulated by different regulator proteins and by the palmitoylation/myristoylation of a specific cysteine residue. In this report, we show that a conserved lysine present in these transcription factors can also be acylated, probably following the intramolecular transfer of the acyl moiety from the cysteine. Using Scalloped (Sd), the Drosophila homolog of human TEAD, as a model, we designed a mutant protein (Glu352GlnSd) that is predominantly acylated on the lysine (Lys350Sd). This protein binds in vitro to the three Sd regulators—Yki, Vg and Tgi—with a similar affinity as the wild type Sd, but it has a significantly higher thermal stability than Sd acylated on the cysteine. This mutant was also introduced in the endogenous locus of the sd gene in Drosophila using CRISPR/Cas9. Homozygous mutants reach adulthood, do not present obvious morphological defects and the mutant protein has both the same level of expression and localization as wild type Sd. This reveals that this mutant protein is both functional and able to control cell growth in a similar fashion as wild type Sd. Therefore, enhancing the lysine acylation of Sd has no detrimental effect on the Hippo pathway. However, we did observe a slight but significant increase of wing size in flies homozygous for the mutant protein suggesting that a higher acylation of the lysine affects the activity of the Hippo pathway. Altogether, our findings indicate that TEAD/Sd can be acylated either on a cysteine or on a lysine, and suggest that these two different forms may have similar properties in cells.

N‐terminal β‐strand in YAP is critical for stronger binding to scalloped relative to TEAD transcription factor

“…TEAD and Sd are known to be acylated (myristoylated or palmitoylated) on a conserved cysteine (Cys367 TEAD4 or Cys373 Sd ) and this modification is important for the function of these transcription factors (Chan et al, 2016; Noland et al, 2016). Recently, it has been shown that Lys350 Sd can be acylated: the effect of this modification on Sd function has been studied (Mesrouze et al, 2022). Only 50% of the Sd protein used in our assays was acylated on Lys350Ala Sd (the rest is acylated on Cys373 Sd ) (Mesrouze et al, 2022), but this acylation may nevertheless enhance the binding of YAP to Sd.…”

“…Recently, it has been shown that Lys350 Sd can be acylated: the effect of this modification on Sd function has been studied (Mesrouze et al, 2022). Only 50% of the Sd protein used in our assays was acylated on Lys350Ala Sd (the rest is acylated on Cys373 Sd ) (Mesrouze et al, 2022), but this acylation may nevertheless enhance the binding of YAP to Sd. To check this possibility, we measured the affinity of YAP 50‐171 for the Lys350Ala Sd mutant, which is only acylated on Cys367 Sd (Mesrouze et al, 2022).…”

“…Only 50% of the Sd protein used in our assays was acylated on Lys350Ala Sd (the rest is acylated on Cys373 Sd ) (Mesrouze et al, 2022), but this acylation may nevertheless enhance the binding of YAP to Sd. To check this possibility, we measured the affinity of YAP 50‐171 for the Lys350Ala Sd mutant, which is only acylated on Cys367 Sd (Mesrouze et al, 2022). The affinity of Lys350Ala Sd for YAP 50‐171 , K d = 3.6 ± 0.2 nM (Table 1), is similar to the one measured for wt Sd (Table 1), suggesting that the presence of acylated Lys350 Sd is not required for YAP to bind more tightly to Sd.…”

In vivoseamless genetic engineering via CRISPR-triggered single-strand annealing

“…This approached has been proven efficient in cell culture {Li, 2018 #527}, but its application in vivo remains unexplored. As a proof of principle, we have recently generated a point mutant in the scalloped ( sd ) locus using SEED/harvest (Mesrouze et al, 2022) (Supplementary figure 3A). In this case, the region to be mutated was duplicated at both sides of the SEED cassette (Supplementary Figure 3B).…”

Allosteric Modulation of YAP/TAZ-TEAD Interaction by Palmitoylation and Small Molecule Inhibitors

“…16 Additionally, recent research suggested that acylation could occur instead at a nearby conserved lysine, resulting in more stabilized TEAD compared to the Cys-palmitoylated protein. 18,19 One study posits Cys-palmitoylation rendered TEAD in a “transient active form”, while Lys-palmitoylation resulted in a “stable active form”. 19 Liberelle et al recently analyzed available TEAD crystal structures and revealed that the fatty acid could reside freely in the PBP, form a covalent bond with the conserved cysteine, or covalently bind to the conserved lysine.…”