Fig. 5: Phosphorylation of TTP and its family members does not directly affect CCR4–NOT interaction or deadenylation activity.

a Phosphorylated TTP (pTTP) and its human family members (pZFP36L1, pZFP36L2) were expressed and isolated from eukaryotic systems (human HEK-293 and insect Sf21 cells). Phosphorylation status was confirmed by treating proteins with protein phosphatase (black circles) and comparing migration patterns to untreated samples via SDS-PAGE. TTP expressed in E. coli served as a non-phosphorylated control. b Diagram comparing previously reported phosphorylation sites in TTP based on the PhosphoSitePlus database⁷² to sites identified by mass spectrometry in TTP isolated from HEK-293 or Sf21 cells in this study. Overlapping sites (orange triangle), non-overlapping sites (red circles) and new sites (green rectangles) are indicated. c Deadenylation assays testing the activity of pTTP (expressed in HEK-293 or Sf21 cells) and phosphorylated pZFP36L1 and pZFP36L2 in targeted deadenylation. The activity of pTTP was also compared to non-phosphorylated TTP. All TTP proteins were used at 100 nM, in twofold excess over CCR4–NOT (50 nM) and substrate RNA (50 nM). Controls included reactions with TTP and RNA alone (C1–C5). d Pull-down assays demonstrating the interactions between phosphorylated TTP, pZFP36L1, and pZFP36L2, with CCR4–NOT modules (black circles). MBP was included as a negative control.