Synthetic approaches to protein phosphorylation

Chen, Zan; Cole, Philip A.

doi:10.1016/j.cbpa.2015.07.001

Cited by 99 publications

(101 citation statements)

References 67 publications

(68 reference statements)

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“…To gain further insight into the role of pTyr369, we used protein semisynthesis (Muir et al, 1998) in the context of 2,3-linkerL-WW3-WW4-HECT WWP2 (aa 356-870) to site-specifically install a pTyr residue at this position. In this method, a synthetic peptide (aa 356-373) containing pTyr369 and a C-terminal thioester (Blanco-Canosa and Dawson, 2008) is chemoselectively ligated to an N-Cys containing WWP2 recombinant fragment (Figure 4B and 4C) (Chen and Cole, 2015). This ligation strategy introduces a Cys in place of Ser374, which was shown to be non-perturbing for WWP2 catalysis (Figure S5B).…”

Section: Resultsmentioning

confidence: 99%

“…Although Glu is typically of limited reliability as a mimic of pTyr (Chen and Cole, 2015), analysis of the autoinhibited WWP2 crystal structure suggested that Glu replacement of Tyr369 or Tyr392 could be similarly disruptive as tyrosine phosphorylation to the linker-HECT interactions. Replacement of either Tyr369 or Tyr392 with Glu as the single or double mutants in recombinant full-length WWP2 stimulated its autoubiquitination activity, although these Y/E mutants were still less active than full 2,3-linker deletion (Figure 4E).…”

Section: Resultsmentioning

confidence: 99%

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A Tunable Brake for HECT Ubiquitin Ligases

et al. 2017

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The HECT E3 ligases ubiquitinate numerous transcription factors and signaling molecules and their activity must be tightly controlled to prevent cancer, immune disorders, and other diseases. In this study we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities. Biochemical, structural, and cellular analysis has revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site. Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications, but complete removal of the brake can induce hyperactive autoubiquitination and E3 self-destruction. These results clarify the mechanisms of several HECT protein cancer associated mutations and provide a new framework for understanding how HECT ubiquitin ligases must be finely tuned to ensure normal cellular behavior.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A Tunable Brake for HECT Ubiquitin Ligases

et al. 2017

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“…This study also illustrates the power of expressed protein ligation in elucidating how a complex series of PTMs can alter the structure of a protein (24). Because mass spectrometry has identified hundreds of thousands of PTMs in proteins, it has become daunting to cope with illuminating the structural and functional effects of these PTMs at an individual protein level.…”

Section: Discussionmentioning

confidence: 99%

Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation

Chen

Dempsey

Thomas

et al. 2016

Journal of Biological Chemistry

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PTEN is a tumor suppressor that functions to negatively regulate the PI3K/AKT pathway as the lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate. Phosphorylation of a cluster of Ser/Thr residues (amino acids 380 -385) on the C-terminal tail serves to alter the conformational state of PTEN from an open active state to a closed inhibited state, resulting in a reduction of plasma membrane localization and inhibition of enzyme activity. The relative contribution of each phosphorylation site to PTEN autoinhibition and the structural basis for the conformational closure is still unclear. To further the structural understanding of PTEN regulation by C-terminal tail phosphorylation, we used protein semisynthesis to insert stoichiometric and site-specific phospho-Ser/Thr(s) in the C-terminal tail of PTEN. Additionally, we employed photo-cross-linking to map the intramolecular PTEN interactions of the phospho-tail. Systematic evaluation of the PTEN C-tail phospho-cluster showed autoinhibition, and conformational closure was influenced by the aggregate effect of multiple phospho-sites rather than dominated by a single phosphorylation site. Moreover, photo-crosslinking suggested a direct interaction between the PTEN C-tail and a segment in the N-terminal region of the catalytic domain. Mutagenesis experiments provided additional insights into how the PTEN phospho-tail interacts with both the C2 and catalytic domains. PTEN3 is a phosphatidylinositol 3,4,5-triphosphate (PIP3) lipid phosphatase that is frequently inactivated in cancer by mutation, epigenetic silencing, or post-translational modifications (PTMs) (1-8). Loss of PTEN function allows unabated production of PIP3 from PIP2 by PI3Ks and stimulates the AKT protein kinase signaling pathway and cancer cell proliferation (1, 9, 10). Understanding the mechanisms of PTEN regulation is therefore critical to the development of therapeutics that can treat a wide variety of cancers.PTEN is a ϳ45-kDa protein composed of an N-terminal PTP-type phosphatase domain, a midsection phospholipid membrane interacting C2 domain, and a 50-residue regulatory tail (1). A crystal structure of the core PTEN catalytic-C2 region has revealed intimate interactions between the catalytic and C2 domains but omitted the C-terminal tail, which is considered to be flexible and largely unstructured (11). One of the most intensively studied set of PTMs in PTEN is a cluster of four C-terminal phosphorylations at Ser 380 , Thr 382 , Thr 383 , and Ser 385 (12-15). These phosphorylation events have been suggested to be catalyzed by protein kinases casein kinase 2 and/or glycogen synthase kinase 3␤ and demonstrated to drive a conformational change that inhibits membrane interactions and reduces catalytic activity (12-15).Several reports that shed light on the molecular basis for this phospho-dependent regulatory event suggest that the PTEN tail phosphorylation induces conformational closure involving intramolecular interactions between the tail and the CBR3 loop of the C2 domain of PTEN (14 -16). H...

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“…[1] This also applies to mechanistic investigations of "phosphoregulation", in which access to site-specifically phosphorylated proteins is required. [2] Recombinant methods afford folded phosphoproteins. [3] However,t he limited availability of suitable protein kinases restricts the global analysis of alternative protein phosphoforms.F or example,t he SH3 domain of the Abl protein, an onreceptor tyrosine kinase critically involved in chronic myelogenous leukemia (CML), [4,5] is found phosphorylated at each of its three tyrosine residues (Y 7 ,Y 30 ,Y 52 ).…”

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confidence: 99%

Parallel Chemical Protein Synthesis on a Surface Enables the Rapid Analysis of the Phosphoregulation of SH3 Domains

Zitterbart

Seitz

2016

Angew Chem Int Ed

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Analysis of postranslationally modified protein domains is complicated by an availability problem, as recombinant methods rarely allow site-specificity at will. Although total synthesis enables full control over posttranslational and other modifications, chemical approaches are limited to shorter peptides. To solve this problem, we herein describe a method that combines a) immobilization of N-terminally thiolated peptide hydrazides by hydrazone ligation, b) on-surface native chemical ligation with self-purified peptide thioesters, c) radical-induced desulfurization, and d) a surface-based fluorescence binding assay for functional characterization. We used the method to rapidly investigate 20 SH3 domains, with a focus on their phosphoregulation. The analysis suggests that tyrosine phosphorylation of SH3 domains found in Abl kinases act as a switch that can induce both the loss and, unexpectedly, gain of affinity for proline-rich ligands.

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Synthetic approaches to protein phosphorylation

Cited by 99 publications

References 67 publications

A Tunable Brake for HECT Ubiquitin Ligases

A Tunable Brake for HECT Ubiquitin Ligases

Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation

Parallel Chemical Protein Synthesis on a Surface Enables the Rapid Analysis of the Phosphoregulation of SH3 Domains

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