Dataset Information

Deciphering TRIM69 interactors and its phosphor-proteomic regulation in centrosome dynamics

ABSTRACT: Stringent control of centrosome duplication and separation is important for preventing chromosome instability. Structural and numerical alterations in centrosomes are hallmarks of neoplastic cells and contribute to tumorigenesis. We show that a Centrosome Amplification 20 (CA20) gene signature is associated with high expression of the Tripartite Motif (TRIM) family member E3 ubiquitin ligase, TRIM69. TRIM69-ablation in cancer cells leads to centrosome scattering and chromosome segregation defects. To identify potential effectors of TRIM69 in regulating centrosome dynamics, we performed mass-spectrometry analysis of immunopurified TRIM69 complexes and defined the TRIM69 protein-interaction network. We discovered Serine/threonine-protein kinase 3 (MST2) as a new direct binding partner of TRIM69 and TRIM69 redistributes MST2 to the perinuclear cytoskeleton. Additionally, we performed IP mass- spectrometry analysis to define the MST2 interactome in the presence and absence of co-expressed TRIM69A. We found that WT TRIM69A (but not the E3 ligase mutant) promoted complex formation between MST2 and PLK1 in the detergent-insoluble CSK compartment. PLK1-mediated phosphorylation of MST2 at S15, S18 and S316 is crucial for centrosome disjunction. Analysis of MST2 phospho-peptides from the LC-MS/MS experiment revealed increased phosphorylation of S15, S316 and several other residues in the detergent-insoluble fraction of TRIM69A-overexpressing cells. Our findings suggest that TRIM69 is a new proximal regulator of distinct signaling pathways that regulate centrosome dynamics and promote bipolar mitosis. Further studies are necessary to identify the putative TRIM69A substrate(s) whose ubiquitination is necessary for regulating and interacting MST2 and centrosome dynamics. To address this issue, we have performed an unbiased mass spectrometry screen for TRIM69A-induced ubiquitination events.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Christine Mills

LAB HEAD: Cyrus Vaziri

PROVIDER: PXD042098 | Pride | 2024-01-26

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20180306_PC567-Vaziri_Yang-Sample1.msf	Msf
	20180306_PC567-Vaziri_Yang-Sample1.pep.xml	Pepxml
	20180306_PC567-Vaziri_Yang-Sample1.raw	Raw
	20180306_PC567-Vaziri_Yang-Sample2.msf	Msf
	20180306_PC567-Vaziri_Yang-Sample2.pep.xml	Pepxml

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Publications

The TRIM69-MST2 signaling axis regulates centrosome dynamics and chromosome segregation.

Wang Yilin Y Risteski Patrik P Yang Yang Y Chen Huan H Droby Gaith G Walens Andrea A Jayaprakash Deepika D Troester Melissa M Herring Laura L Chernoff Jonathan J Tolić Iva M IM Bowser Jessica J Vaziri Cyrus C

Nucleic acids research 20231001 19

Stringent control of centrosome duplication and separation is important for preventing chromosome instability. Structural and numerical alterations in centrosomes are hallmarks of neoplastic cells and contribute to tumorigenesis. We show that a Centrosome Amplification 20 (CA20) gene signature is associated with high expression of the Tripartite Motif (TRIM) family member E3 ubiquitin ligase, TRIM69. TRIM69-ablation in cancer cells leads to centrosome scattering and chromosome segregation defect ...[more]

PMID: 37739411

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Project description:During development, neurons undergo expansive growth and shape change to achieve their mature morphology and physiological function. As cellular remodeling occurs, proteins are synthesized, transported, degraded, and recycled. As such, protein levels and localization are tightly regulated. E3 ubiquitin ligases play a key role in proteostasis by altering protein localization, intracellular trafficking, and protein lifetime via the addition of ubiquitin modifiers. TRIM9 and TRIM67 are brain-enriched E3 ubiquitin ligases implicated in numerous stages of neuronal morphogenesis. We previously demonstrated both proteins are required for axon pathfinding and growth cone shape changes in developing neurons. In particular, TRIM9 and TRIM67 regulate filopodia number and stability in early stages of neuronal development. Our published in vivo studies suggest TRIM9 and TRIM67 may function at the synapse as well. We observed distinct deficits in spatial learning and memory of Trim9-/- and Trim67-/- mice in the Morris Water Maze test, compared to their littermate controls. Furthermore, adult-born neurons in the dentate gyrus in Trim9-/- mice also displayed a decreased number of dendritic spines. Here we demonstrate TRIM9 and TRIM67 localize to the post-synaptic density (PSD), a structure attached to the post-synaptic membrane in dendritic spines. We identified 148 proteins that were significantly changed (p < 0.05) in the Trim67-/- mice compared to their Trim67+/+ littermates. Gene Ontology analysis of these proteins demonstrated enrichment of several cellular pathways, including peptidyl-amino acid modification, microtubule dynamics, and Ras signal transduction. Likewise, we identified 109 proteins that were significantly changed (p < 0.05) in the Trim9-/- mice compared to their Trim9+/+ littermates. Following Gene Ontology analysis, we observed the prominent enrichment of one pathway in our significantly different proteins: the actin cytoskeleton. Changes in these actin cytoskeleton proteins were bidirectional, suggesting the presence of altered cytoskeletal architecture and organization in the Trim9-/- PSD.