TY - JOUR
T1 - Characterization of the CLASP2 protein interaction network identifies SOGA1 as a microtubule-associated protein
AU - Kruse, Rikke
AU - Krantz, James
AU - Barker, Natalie
AU - Coletta, Richard L.
AU - Rafikov, Ruslan
AU - Luo, Moulun
AU - Højlund, Kurt
AU - Mandarino, Lawrence J.
AU - Langlais, Paul R.
N1 - Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2017/10
Y1 - 2017/10
N2 - 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 GTPaseactivating 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 cytoskeletonassociated protein biology.
AB - 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 GTPaseactivating 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 cytoskeletonassociated protein biology.
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U2 - 10.1074/mcp.RA117.000011
DO - 10.1074/mcp.RA117.000011
M3 - Article
C2 - 28550165
AN - SCOPUS:85030312079
SN - 1535-9476
VL - 16
SP - 1718
EP - 1735
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
IS - 10
ER -