Probing protein phosphatase substrate binding: affinity pull-down of ILKAP phosphatase 2C with phosphopeptides

2012 ◽  
Vol 8 (5) ◽  
pp. 1452 ◽  
Author(s):  
Kim B. Højlys-Larsen ◽  
Kasper K. Sørensen ◽  
Knud J. Jensen ◽  
Steen Gammeltoft
Keyword(s):  
Binding Affinity ◽  
Protein Phosphatase ◽  
Substrate Binding ◽  
Phosphatase Substrate

scholarly journals Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase

2021 ◽  
Author(s):  
Chih-Wei Huang ◽  
Chi-Ching Hwang ◽  
Yung-Lung Chang ◽  
Jen-Tzu Liu ◽  
Sheng-Peng Wu ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Functional Role ◽  
Substrate Binding ◽  
Critical Role ◽  
Intermediate Formation ◽  
Hydroxyl Group ◽  
Double Mutation ◽  
Closed Conformation ◽  
Bond Network

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E’s Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

Characterization and improvement of substrate-binding affinity of d-aspartate oxidase of the thermophilic fungus Thermomyces dupontii

2019 ◽  
Vol 103 (10) ◽  
pp. 4053-4064 ◽  
Author(s):  
Shouji Takahashi ◽  
Kohei Osugi ◽  
Yuya Shimekake ◽  
Akira Shinbo ◽  
Katsumasa Abe ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Substrate Binding ◽  
Thermophilic Fungus ◽  
Thermomyces Dupontii

scholarly journals Hyaluronan–CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9

2002 ◽  
Vol 158 (6) ◽  
pp. 1133-1144 ◽  
Author(s):  
Paola Spessotto ◽  
Francesca Maria Rossi ◽  
Massimo Degan ◽  
Raffaele Di Francia ◽  
Roberto Perris ◽  
...  
Keyword(s):  
Cell Migration ◽  
Bone Resorption ◽  
Cell Motility ◽  
Binding Affinity ◽  
Antisense Oligonucleotides ◽  
Substrate Binding ◽  
Down Regulation ◽  
Multinucleated Cells ◽  
Cell Substrate

Osteoclast (OC) precursors migrate to putative sites of bone resorption to form functionally active, multinucleated cells. The preOC FLG 29.1 cells, known to be capable of irreversibly differentiating into multinucleated OC-like cells, displayed several features of primary OCs, including expression of specific integrins and the hyaluronan (HA) receptor CD44. OC-like FLG 29.1 cells adhered to and extensively migrated through membranes coated with fibronectin, vitronectin, and laminins, but, although strongly binding to HA, totally failed to move on this substrate. Moreover, soluble HA strongly inhibited OC-like FLG 29.1 cell migration on the permissive matrix substrates, and this behavior was dependent on its engagement with CD44, as it was fully restored by function-blocking anti-CD44 antibodies. HA did not modulate the cell–substrate binding affinity/avidity nor the expression levels of the corresponding integrins. MMP-9 was the major secreted metalloproteinase used by OC-like FLG 29.1 cells for migration, because this process was strongly inhibited by both TIMP-1 and GM6001, as well as by MMP-9–specific antisense oligonucleotides. After HA binding to CD44, a strong down-regulation of MMP-9 mRNA and protein was detected. These findings highlight a novel role of the HA–CD44 interaction in the context of OC-like cell motility, suggesting that it may act as a stop signal for bone-resorbing cells.

scholarly journals CK2 Is the Regulator of SIRT1 Substrate-Binding Affinity, Deacetylase Activity and Cellular Response to DNA-Damage

PLoS ONE ◽  
2009 ◽  
Vol 4 (8) ◽  
pp. e6611 ◽  
Author(s):  
Hyeog Kang ◽  
Jae-Won Jung ◽  
Myung K. Kim ◽  
Jay H. Chung
Keyword(s):  
Dna Damage ◽  
Binding Affinity ◽  
Cellular Response ◽  
Substrate Binding

Single-molecule photon-fueled DNA nanoscissors for DNA cleavage based on the regulation of substrate binding affinity by azobenzene

2013 ◽  
Vol 49 (77) ◽  
pp. 8716 ◽  
Author(s):  
Yuan Zou ◽  
Jie Chen ◽  
Zhi Zhu ◽  
Lianyu Lu ◽  
Yishun Huang ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Substrate Binding

scholarly journals SUMOylation of RepoMan during late telophase regulates dephosphorylation of lamin A

2021 ◽  
Author(s):  
Takanobu Moriuchi ◽  
Fumiko Hirose
Keyword(s):  
Binding Affinity ◽  
Protein Phosphatase ◽  
Regulatory Mechanism ◽  
Nuclear Lamina ◽  
Regulatory Subunit ◽  
Lamin A ◽  
Temporal Regulation ◽  
Late Telophase ◽  
Spatio Temporal

Dephosphorylation of lamin A, which triggers nuclear lamina reconstitution, is crucial for the completion of mitosis. However, the specific phosphatase and regulatory mechanism that allow timely lamin A dephosphorylation remain unclear. Here, we report that RepoMan, a regulatory subunit of protein phosphatase 1γ (PP1γ) is transiently modified with SUMO-2 at K762 during late telophase. SUMOylation of RepoMan markedly enhanced its binding affinity with lamin A. Moreover, the SUMOylated RepoMan/PP1γ contributes to lamin A recruitment to telophase chromosomes and dephosphorylation of the mitotic lamin A phosphorylation. Expression of a SUMO-2 mutant defective in interaction with SUMO interacting motif (SIM) resulted in failure of the lamin A and RepoMan association, along with abrogation of lamin A dephosphorylation and subsequent nuclear lamina formation. These findings strongly suggested that RepoMan/PP1γ recruits lamin A through SUMO-SIM interaction. Thus, transient SUMOylation of RepoMan plays an important role in the spatio-temporal regulation of lamin A dephosphorylation and the subsequent nuclear lamina formation at the end of mitosis.

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