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Mol Cell Proteomics. 2017 Oct;16(10):1718-1735. doi: 10.1074/mcp.RA117.000011. Epub 2017 May 26.

Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein.

Author information

1
From the ‡The Section of Molecular Diabetes & Metabolism, Department of Clinical Research and Institute of Molecular Medicine, University of Southern Denmark, DK-5000 Odense, Denmark.
2
§Department of Endocrinology, Odense University Hospital, DK-5000 Odense, Denmark.
3
¶Department of Medicine, Division of Endocrinology, University of Arizona College of Medicine, Tucson, Arizona 85721.
4
‖School of Life Sciences, Arizona State University, Tempe, Arizona 85787.
5
¶Department of Medicine, Division of Endocrinology, University of Arizona College of Medicine, Tucson, Arizona 85721; langlais@deptofmed.arizona.edu.

Abstract

CLASP2 is a microtubule-associated protein that undergoes insulin-stimulated phosphorylation and co-localization with reorganized actin and GLUT4 at the plasma membrane. To gain insight to the role of CLASP2 in this system, we developed and successfully executed a streamlined interactome approach and built a CLASP2 protein network in 3T3-L1 adipocytes. Using two different commercially available antibodies for CLASP2 and an antibody for epitope-tagged, overexpressed CLASP2, we performed multiple affinity purification coupled with mass spectrometry (AP-MS) experiments in combination with label-free quantitative proteomics and analyzed the data with the bioinformatics tool Significance Analysis of Interactome (SAINT). We discovered that CLASP2 coimmunoprecipitates (co-IPs) the novel protein SOGA1, the microtubule-associated protein kinase MARK2, and the microtubule/actin-regulating protein G2L1. The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and revealed MARK2 can co-IP SOGA1, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and with tubulin, which identifies SOGA1 as a new microtubule-associated protein. These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology.

PMID:
28550165
PMCID:
PMC5629260
DOI:
10.1074/mcp.RA117.000011
[Indexed for MEDLINE]
Free PMC Article

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