Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function

Travers, Timothy; Shao, Hanshuang; Joughin, Brian A.; Lauffenburger, Douglas A.; Wells, Alan; Camacho, Carlos J.

doi:10.1126/scisignal.aaa1977

Cited by 28 publications

(29 citation statements)

References 62 publications

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“…Phosphoproteomic analysis revealed that phosphorylation of one site can enhance the phosphorylation of neighboring sites within ∼10 amino acids ( 49 ). Moreover, recent studies have found evidence for such phosphorylation priming in an actin cross-linking protein ( 50 ) and within the two tyrosines of the ITAM ( 51 ).…”

Section: Discussionmentioning

confidence: 99%

Multisite Phosphorylation Modulates the T Cell Receptor ζ-Chain Potency but not the Switchlike Response

Mukhopadhyay

Wet

Clemens

et al. 2016

Biophysical Journal

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Multisite phosphorylation is ubiquitous in cellular signaling and is thought to provide signaling proteins with additional regulatory mechanisms. Indeed, mathematical models have revealed a large number of mechanisms by which multisite phosphorylation can produce switchlike responses. The T cell antigen receptor (TCR) is a multisubunit receptor on the surface of T cells that is a prototypical multisite substrate as it contains 20 sites that are distributed on 10 conserved immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR ζ-chain is a homodimer subunit that contains six ITAMs (12 sites) and exhibits a number of properties that are predicted to be sufficient for a switchlike response. We have used cellular reconstitution to systematically study multisite phosphorylation of the TCR ζ-chain. We find that multisite phosphorylation proceeds by a nonsequential random mechanism, and find no evidence that multiple ITAMs modulate a switchlike response but do find that they alter receptor potency and maximum phosphorylation. Modulation of receptor potency can be explained by a reduction in molecular entropy of the disordered ζ-chain upon phosphorylation. We further find that the tyrosine kinase ZAP-70 increases receptor potency but does not modulate the switchlike response. In contrast to other multisite proteins, where phosphorylations act in strong concert to modulate protein function, we suggest that the multiple ITAMs on the TCR function mainly to amplify subsequent signaling.

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Section: Discussionmentioning

confidence: 99%

Multisite Phosphorylation Modulates the T Cell Receptor ζ-Chain Potency but not the Switchlike Response

Mukhopadhyay

Wet

Clemens

et al. 2016

Biophysical Journal

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“…For example, the CTs of α‐ and β‐tubulin are also IDRs that regulate microtubules via intermolecular interactions . Local charge, for example the phosphorylation state of a residue within the IDR or the IDP, is frequently associated with the control of conformation and serves as a regulatory switch for protein–protein interactions and functionality . In the case of the γ‐CT polypeptide, a Y > D substitution at a known in vivo phosphorylation site causes it to undergo global cooperative transitions between different conformational states.…”

Section: Discussionmentioning

confidence: 99%

Concerted millisecond timescale dynamics in the intrinsically disordered carboxyl terminus of γ‐tubulin induced by mutation of a conserved tyrosine residue

et al. 2017

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Tubulins are an ancient family of eukaryotic proteins characterized by an amino-terminal globular domain and disordered carboxyl terminus. These carboxyl termini play important roles in modulating the behavior of microtubules in living cells. However, the atomic-level basis of their function is not well understood. These regions contain multiple acidic residues and their overall charges are modulated in vivo by post-translational modifications, for example, phosphorylation. In this study, we describe an application of NMR and computer Monte Carlo simulations to investigate how the modification of local charge alters the conformational sampling of the γ-tubulin carboxyl terminus. We compared the dynamics of two 39-residue polypeptides corresponding to the carboxyl-terminus of yeast γ-tubulin. One polypeptide comprised the wild-type amino acid sequence while the second contained a Y > D mutation at Y11 in the polypeptide (Y445 in the full protein). This mutation introduces additional negative charge at a site that is phosphorylated in vivo and produces a phenotype with perturbed microtubule function. NMR relaxation measurements show that the Y11D mutation produces dramatic changes in the millisecond-timescale motions of the entire polypeptide. This observation is supported by Monte Carlo simulations that-similar to NMR-predict the WT γ-CT is largely unstructured and that the substitution of Tyr 11 with Asp causes the sampling of extended conformations that are unique to the Y11D polypeptide.

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“…One of the effects of phosphorylation on IDR could be transition of higher-order structures (10, 47, 48). Phosphorylation in IDR has been known to cause disorder-to-order or order-to-disorder transition.…”

Section: Discussionmentioning

confidence: 99%

Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates

Yamazaki

Kosako

Yoshimura

2019

Preprint

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16Protein phosphorylation plays inevitable roles in the regulation and the progression of mitosis. More than ten 17 thousands of phosphorylated residues and their responsible kinases have so far been identified by a number of 18 proteomic analyses. While some of those phosphosites have been demonstrated to affect either protein-protein 19 interaction or catalytic activity of the substrate protein, the effect of most mitotic phosphosites remain 20 unclarified. In this study, we tried to extract structural properties of mitotic phosphosites and neighboring 21 residues, and find a role of heavy phosphorylation at non-structured domain. A quantitative mass spectrometry 22 of mitosis-arrested and non-arrested HeLa cells identified more than 4,100 and 1,900 residues which are 23 significantly phosphorylated, and dephosphorylated at mitotic entry, respectively. Calculating the disorder score 24 of the neighboring amino acid sequence of individual phosphosites revealed that more than 70% of the 25 phosphosites which are dephosphorylated existed in disordered regions, whereas 50% of phosphorylated sites 26 existed in non-structured domain. There was a clear inversed co-relation between probability in non-structured 27 domain and increment of phosphorylation in mitosis. These results indicate that at the entry of mitosis, a 28 significant amount of phosphate group is removed from non-structured domains and attached to more structured 29 domains. GO term analysis revealed that mitosis-related proteins are heavily phosphorylated, whereas RNA-30 related proteins are both dephosphorylated and phosphorylated, implying that heavy 31 phosphorylation/dephosphorylation in non-structured domain of RNA-binding proteins may play a role in 32 dynamic rearrangement of RNA-containing organelle, as well as other intracellular environments. 33 34 65been identified (reviewed in (17-21)). Dephosphorylation also plays essential roles both at the entry and the 66 exit of mitosis (22, 23) (24, 25). More than 1,000 kinds of phosphosites are dephosphorylated during anaphase 67 (26), and more than five hundreds are dephosphorylated at the entry of mitosis (2). For example, CDK1 is 68 activated by dephosphorylation by Cdc25B/C (27, 28), and Cdc25C is partially activated by protein phosphatase 69 1 (27). These results indicate that a vast number of phosphate groups are transferred to and from the substrate 70 proteins during mitotic entry. 71One of the unclarified problems on mitotic phosphorylation could be why and how a vast number of 72 phosphate groups are transferred from a set of proteins to a different set of proteins, and whether these different 73 sets of mitotic phosphosites are structurally different or not. Especially, the distribution of phosphosites in 74 structured or non-structured regions of the substrate proteins is an important cue to understand the structural 75 effect of phosphorylation/dephosphorylation. In this study, we performed phosphoproteomic analysis of cellular 76 proteins to quantify and compare individual phosphosi...

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Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function

Cited by 28 publications

References 62 publications

Multisite Phosphorylation Modulates the T Cell Receptor ζ-Chain Potency but not the Switchlike Response

Multisite Phosphorylation Modulates the T Cell Receptor ζ-Chain Potency but not the Switchlike Response

Concerted millisecond timescale dynamics in the intrinsically disordered carboxyl terminus of γ‐tubulin induced by mutation of a conserved tyrosine residue

Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates

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