Dataset Information

Diverse p120RasGAP interactions with doubly phosphorylated partners EphB4, p190RhoGAP, and Dok1.

ABSTRACT: RasGAP (p120RasGAP), the founding member of the GTPase-activating protein (GAP) family, is one of only nine human proteins to contain two SH2 domains and is essential for proper vascular development. Despite its importance, its interactions with key binding partners remains unclear. In this study we provide a detailed viewpoint of RasGAP recruitment to various binding partners and assess their impact on RasGAP activity. We reveal the RasGAP SH2 domains generate distinct binding interactions with three well-known doubly phosphorylated binding partners: p190RhoGAP, Dok1, and EphB4. Affinity measurements demonstrate a 100-fold weakened affinity for RasGAP-EphB4 binding compared to RasGAP-p190RhoGAP or RasGAP-Dok1 binding, possibly driven by single versus dual SH2 domain engagement with a dominant N-terminal SH2 interaction. Small-angle X-ray scattering reveals conformational differences between RasGAP-EphB4 binding and RasGAP-p190RhoGAP binding. Importantly, these interactions do not impact catalytic activity, implying RasGAP utilizes its SH2 domains to achieve diverse spatial-temporal regulation of Ras signaling in a previously unrecognized fashion.

SUBMITTER: Vish KJ

PROVIDER: S-EPMC10470053 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Diverse p120RasGAP interactions with doubly phosphorylated partners EphB4, p190RhoGAP, and Dok1.

Vish Kimberly J KJ Stiegler Amy L AL Boggon Titus J TJ

The Journal of biological chemistry 20230727 9

RasGAP (p120RasGAP), the founding member of the GTPase-activating protein (GAP) family, is one of only nine human proteins to contain two SH2 domains and is essential for proper vascular development. Despite its importance, its interactions with key binding partners remains unclear. In this study we provide a detailed viewpoint of RasGAP recruitment to various binding partners and assess their impact on RasGAP activity. We reveal the RasGAP SH2 domains generate distinct binding interactions with ...[more]

PMID: 37507023

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Project description:In the majority of bacterial species, the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target parS sequence(s), assists in the chromosome partitioning. ParB forms large nucleoprotein complexes at parS(s), located in the vicinity of origin of chromosomal replication (oriC), which after replication are subsequently positioned by ParA in cell poles. Remarkably, ParA and ParB participate not only in the chromosome segregation but through interactions with various cellular partners they are also involved in other cell cycle-related processes, in a species-specific manner. In this work, we characterized Pseudomonas aeruginosa ParB interactions with the cognate ParA, showing that the N-terminal motif of ParB is required for these interactions, and demonstrated that ParAB-parS-mediated rapid segregation of newly replicated ori domains prevented structural maintenance of chromosome (SMC)-mediated cohesion of sister chromosomes. Furthermore, using proteome-wide techniques, we have identified other ParB partners in P. aeruginosa, which encompass a number of proteins, including the nucleoid-associated proteins NdpA(PA3849) and NdpA2, MinE (PA3245) of Min system, and transcriptional regulators and various enzymes, e.g., CTP synthetase (PA3637). Among them are also NTPases PA4465, PA5028, PA3481, and FleN (PA1454), three of them displaying polar localization in bacterial cells. Overall, this work presents the spectrum of P. aeruginosa ParB partners and implicates the role of this protein in the cross-talk between chromosome segregation and other cellular processes. IMPORTANCE In Pseudomonas aeruginosa, a Gram-negative pathogen causing life-threatening infections in immunocompromised patients, the ParAB-parS system is involved in the precise separation of newly replicated bacterial chromosomes. In this work, we identified and characterized proteins interacting with partitioning protein ParB. We mapped the domain of interactions with its cognate ParA partner and showed that ParB-ParA interactions are crucial for the chromosome segregation and for proper SMC action on DNA. We also demonstrated ParB interactions with other DNA binding proteins, metabolic enzymes, and NTPases displaying polar localization in the cells. Overall, this study uncovers novel players cooperating with the chromosome partition system in P. aeruginosa, supporting its important regulatory role in the bacterial cell cycle.

Dataset Information

Diverse p120RasGAP interactions with doubly phosphorylated partners EphB4, p190RhoGAP, and Dok1.

Publications

Diverse p120RasGAP interactions with doubly phosphorylated partners EphB4, p190RhoGAP, and Dok1.

Similar Datasets

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