TY - JOUR

T1 - Large-scale phosphomimetic screening identifies phospho-modulated motif-based protein interactions

AU - Kliche, Johanna

AU - Garvanska, Dimitriya Hristoforova

AU - Simonetti, Leandro

AU - Badgujar, Dilip

AU - Dobritzsch, Doreen

AU - Nilsson, Jakob

AU - Davey, Norman E.

AU - Ivarsson, Ylva

N1 - Publisher Copyright: © 2023 The Authors. Published under the terms of the CC BY 4.0 license.

PY - 2023

Y1 - 2023

N2 - Phosphorylation is a ubiquitous post-translation modification that regulates protein function by promoting, inhibiting or modulating protein–protein interactions. Hundreds of thousands of phosphosites have been identified but the vast majority have not been functionally characterised and it remains a challenge to decipher phosphorylation events modulating interactions. We generated a phosphomimetic proteomic peptide-phage display library to screen for phosphosites that modulate short linear motif-based interactions. The peptidome covers ~13,500 phospho-serine/threonine sites found in the intrinsically disordered regions of the human proteome. Each phosphosite is represented as wild-type and phosphomimetic variant. We screened 71 protein domains to identify 248 phosphosites that modulate motif-mediated interactions. Affinity measurements confirmed the phospho-modulation of 14 out of 18 tested interactions. We performed a detailed follow-up on a phospho-dependent interaction between clathrin and the mitotic spindle protein hepatoma-upregulated protein (HURP), demonstrating the essentiality of the phospho-dependency to the mitotic function of HURP. Structural characterisation of the clathrin-HURP complex elucidated the molecular basis for the phospho-dependency. Our work showcases the power of phosphomimetic ProP-PD to discover novel phospho-modulated interactions required for cellular function.

AB - Phosphorylation is a ubiquitous post-translation modification that regulates protein function by promoting, inhibiting or modulating protein–protein interactions. Hundreds of thousands of phosphosites have been identified but the vast majority have not been functionally characterised and it remains a challenge to decipher phosphorylation events modulating interactions. We generated a phosphomimetic proteomic peptide-phage display library to screen for phosphosites that modulate short linear motif-based interactions. The peptidome covers ~13,500 phospho-serine/threonine sites found in the intrinsically disordered regions of the human proteome. Each phosphosite is represented as wild-type and phosphomimetic variant. We screened 71 protein domains to identify 248 phosphosites that modulate motif-mediated interactions. Affinity measurements confirmed the phospho-modulation of 14 out of 18 tested interactions. We performed a detailed follow-up on a phospho-dependent interaction between clathrin and the mitotic spindle protein hepatoma-upregulated protein (HURP), demonstrating the essentiality of the phospho-dependency to the mitotic function of HURP. Structural characterisation of the clathrin-HURP complex elucidated the molecular basis for the phospho-dependency. Our work showcases the power of phosphomimetic ProP-PD to discover novel phospho-modulated interactions required for cellular function.

KW - clathrin

KW - phage display

KW - phosphomimetic mutation

KW - phosphorylation

KW - protein–protein interactions

U2 - 10.15252/msb.202211164

DO - 10.15252/msb.202211164

M3 - Journal article

C2 - 37219487

AN - SCOPUS:85159905516

VL - 19

JO - Molecular Systems Biology

JF - Molecular Systems Biology

SN - 1744-4292

IS - 7

M1 - e11164

ER -