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Proteomics analysis of the ezrin interactome in B cells reveals a novel association with Myo18a?.

ABSTRACT: The molecular regulation of recruitment and assembly of signalosomes near the B cell receptor (BCR) is poorly understood. We have previously demonstrated a role for the ERM family protein ezrin in regulating antigen-dependent lipid raft coalescence in B cells. In this study, we addressed the possibility that ezrin may collaborate with other adaptor proteins to regulate signalosome dynamics at the membrane. Using mass spectrometry-based proteomics analysis, we identified Myo18a? as a novel binding partner of ezrin. Myo18a? is an attractive candidate as it has several protein-protein interaction domains and an intrinsic motor activity. The expression of Myo18a? varied during B cell development in the bone marrow and in mature B cell subsets suggesting functional differences. Interestingly, BCR stimulation increased the association between ezrin and Myo18a?, and induced co-segregation of Myo18a? with the BCR and phosphotyrosine-containing proteins. Our data raise an intriguing possibility that the Myo18a?/ezrin complex may facilitate BCR-mediated signaling by recruiting signaling proteins that are in close proximity of the antigen receptor. Our study is not only significant with respect to understanding the molecular regulation of BCR signaling but also provides a broader basis for understanding the mechanism of action of ezrin in other cellular systems.

SUBMITTER: Matsui K

PROVIDER: S-EPMC3181492 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Proteomics analysis of the ezrin interactome in B cells reveals a novel association with Myo18aα.

Matsui Ken K Parameswaran Neetha N Bagheri Nayer N Willard Belinda B Gupta Neetu N

Journal of proteome research 20110728 9

The molecular regulation of recruitment and assembly of signalosomes near the B cell receptor (BCR) is poorly understood. We have previously demonstrated a role for the ERM family protein ezrin in regulating antigen-dependent lipid raft coalescence in B cells. In this study, we addressed the possibility that ezrin may collaborate with other adaptor proteins to regulate signalosome dynamics at the membrane. Using mass spectrometry-based proteomics analysis, we identified Myo18aα as a novel bindin ...[more]

PMID: 21751808

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Project description:The dynamic modification of specific serine and threonine residues of intracellular proteins by O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) mitigates injury and promotes cytoprotection in a variety of stress models. The O-GlcNAc transferase (OGT) and the O-GlcNAcase are the sole enzymes that add and remove O-GlcNAc, respectively, from thousands of substrates. It remains unclear how just two enzymes can be specifically controlled to affect glycosylation of target proteins and signaling pathways both basally and in response to stress. Several lines of evidence suggest that protein interactors regulate these responses by affecting OGT and O-GlcNAcase activity, localization, and substrate specificity. To provide insight into the mechanisms by which OGT function is controlled, we have used quantitative proteomics to define OGT's basal and stress-induced interactomes. OGT and its interaction partners were immunoprecipitated from OGT WT, null, and hydrogen peroxide-treated cell lysates that had been isotopically labeled with light, medium, and heavy lysine and arginine (stable isotopic labeling of amino acids in cell culture). In total, more than 130 proteins were found to interact with OGT, many of which change their association upon hydrogen peroxide stress. These proteins include the major OGT cleavage and glycosylation substrate, host cell factor 1, which demonstrated a time-dependent dissociation after stress. To validate less well-characterized interactors, such as glyceraldehyde 3-phosphate dehydrogenase and histone deacetylase 1, we turned to parallel reaction monitoring, which recapitulated our discovery-based stable isotopic labeling of amino acids in cell culture approach. Although the majority of proteins identified are novel OGT interactors, 64% of them are previously characterized glycosylation targets that contain varied domain architecture and function. Together these data demonstrate that OGT interacts with unique and specific interactors in a stress-responsive manner.

Dataset Information

Proteomics analysis of the ezrin interactome in B cells reveals a novel association with Myo18a?.

Publications

Proteomics analysis of the ezrin interactome in B cells reveals a novel association with Myo18aα.

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