Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes[S]

• Published in: Molecular & cellular proteomics 2019-07, Vol.18 (7), p.1363-1381
• Main Authors: Parker, Sara S., Krantz, James, Kwak, Eun-A, Barker, Natalie K., Deer, Chris G., Lee, Nam Y., Mouneimne, Ghassan, Langlais, Paul R.
• Format: Article
• Language: English
• Subjects: 3T3-L1 Cells; Acetylation - drug effects; Adipocytes - drug effects; Adipocytes - metabolism; Affinity proteomics; Animals; CLASP2; G2L1; Insulin; Insulin - pharmacology; Interactome; Label-free quantification; Lysine - metabolism; Mice; Microtubule-Associated Proteins - metabolism; Microtubules; Microtubules - drug effects; Microtubules - metabolism; Phosphorylation; Phosphorylation - drug effects; Protein Binding - drug effects; Protein Interaction Mapping; Protein Transport - drug effects; Protein-Protein Interactions; Quantification; Tubulin - metabolism
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.RA119.001450

In adipocytes, our quantitative proteomics studies have revealed that insulin affects the phosphorylation of a new microtubule-associated protein network. Mapping microtubule plus-end interacting protein interactomes resulted in the live-cell based discovery that insulin has novel effects on microtubule plus-end protein and microtubule dynamics. In addition, we present microtubule stabilization as a new paradigm in insulin action. Taken together, our findings introduce new directions aimed at pursuing a better understanding of the relationship between insulin and the microtubule network. [Display omitted] Highlights •Insulin Affects the Phosphorylation of G2L1, MARK2, CLIP2, EB1, AGAP3, and CKAP5.•Insulin Increases CLASP2 +TIP Density and Decreases CLASP2 +TIP Velocity.•Insulin Stimulates CLASP2 and G2L1 Trailing Along Microtubules.•Insulin Stimulates α-Tubulin Acetylation at Lysine 40 and Microtubule Stabilization. Insulin-stimulated glucose uptake is known to involve microtubules, although the function of microtubules and the microtubule-regulating proteins involved in insulin action are poorly understood. CLASP2, a plus-end tracking microtubule-associated protein (+TIP) that controls microtubule dynamics, was recently implicated as the first +TIP associated with insulin-regulated glucose uptake. Here, using protein-specific targeted quantitative phosphoproteomics within 3T3-L1 adipocytes, we discovered that insulin regulates phosphorylation of the CLASP2 network members G2L1, MARK2, CLIP2, AGAP3, and CKAP5 as well as EB1, revealing the existence of a previously unknown microtubule-associated protein system that responds to insulin. To further investigate, G2L1 interactome studies within 3T3-L1 adipocytes revealed that G2L1 coimmunoprecipitates CLASP2 and CLIP2 as well as the master integrators of +TIP assembly, the end binding (EB) proteins. Live-cell total internal reflection fluorescence microscopy in adipocytes revealed G2L1 and CLASP2 colocalize on microtubule plus-ends. We found that although insulin increases the number of CLASP2-containing plus-ends, insulin treatment simultaneously decreases CLASP2-containing plus-end velocity. In addition, we discovered that insulin stimulates redistribution of CLASP2 and G2L1 from exclusive plus-end tracking to “trailing” behind the growing tip of the microtubule. Insulin treatment increases α-tubulin Lysine 40 acetylation, a mechanism that was observed to be regulated by a counterbalance between GSK3 and mTOR