Regulation of trehalase activity by multi-site phosphorylation and 14-3-3 interaction

Abstract

Protein phosphorylation enables a rapid adjustment of cellular activities to diverse intracellular and environmental stimuli. Many phosphoproteins are targeted on more than one site, which allows the integration of multiple signals and the implementation of complex responses. However, the hierarchy and interplay between multiple phospho-sites are often unknown. Here, we study multi‐site phosphorylation using the yeast trehalase Nth1 and its activator, the 14-3-3 protein Bmh1, as a model. Nth1 is known to be phosphorylated by the metabolic kinase PKA on four serine residues and by the cell cycle kinase CDK on one residue. However, how these five phospho-sites adjust Nth1 activity remains unclear. Using a novel reporter construct, we investigated the contribution of the individual sites for the regulation of the trehalase and its 14-3-3 interactor. In contrast to the constitutively phosphorylated S20 and S83, the weaker sites S21 and S60 are only phosphorylated by increased PKA activity. For binding Bmh1, S83 functions as the high‐affinity “gatekeeper” site, but successful binding of the Bmh1 dimer and thus Nth1 activation requires S60 as a secondary site. Under nutrient-poor conditions with low PKA activity, S60 is not efficiently phosphorylated and the cell cycle dependent phosphorylation of S66 by Cdk1 contributes to Nth1 activity, likely by providing an alternative Bmh1 binding site. Additionally, the PKA sites S20 and S21 modulate the dephosphorylation of Nth1 on downstream Bmh1 sites. In summary, our results expand our molecular understanding of Nth1 regulation and provide a new aspect of the interaction of 14-3-3 proteins with their targets.

Discovering molecular features of intrinsically disordered regions by using evolution for contrastive learning

“…Qualitatively, we observed that the sequence logo of Max F87 resembles two overlapping PKA motifs. A recent study indicates that PKA is capable of phosphorylating two serines in a row (Dengler et al, 2021). The authors observe in these cases, one serine is constitutively phosphorylated, and the other serine requires increased PKA activity, and hypothesize this multisite phosphorylation might be a regulatory mechanism.…”

“…In visual exploration of the features learned by reverse homology models, we identified previously unknown features as well. We were interested Max Feature 231 (Figure 5B) that appears to recognize an RRRSS motif that we speculated might represent a basophilic phosphorylation site consensus, perhaps related to two overlapping PKA phosphorylation consensus motifs RRxS [70] [90]. To test this, we analyzed the frequency of two adjacent phosphorylated serines [89] in the 8-amino acid window around the maximally activated position of the top activating sequences and found 2.31 times more doubly phosphorylated regions (13 of 52 IDRs for RRRSSS vs. 15 of 139 IDRs for the feature matching the PKA consensus.…”

Discovering molecular features of intrinsically disordered regions by using evolution for contrastive learning

“…In visual exploration of the features learned by reverse homology models, we identified previously unknown features as well. We were interested Max Feature 231 ( Fig 5B ) that appears to recognize an RRRSS motif that we speculated might represent a basophilic phosphorylation site consensus, perhaps related to two overlapping PKA phosphorylation consensus motifs RRxS [ 70 ] [ 90 ]. To test this, we analyzed the frequency of two adjacent phosphorylated serines [ 89 ] in the 8-amino acid window around the maximally activated position of the top activating sequences and found 2.31 times more doubly phosphorylated regions (13 of 52 IDRs for RRRSSS vs. 15 of 139 IDRs for the feature matching the PKA consensus.…”

Characterization, heterologous expression and engineering of trehalase for biotechnological applications

“…Four residues phosphorylated by protein kinase A (PKA) within the disordered N-terminal fragment of Nth1 have been identified [70]. Site-directed mutagenesis indicated that S83 acts as a high-affinity gatekeeper site to bind Bamh1 (the yeast 14-3-3 isoforms), and S60 acts as a second site to bind to the Bmh1 dimer and activate Nth1 [71]. In addition, PKA sites S20 and S21 regulate the dephosphorylation of Nth1 at the downstream Bmh1 site.…”