Characterization of the G2L1 Interactome Leads to the Discovery That CLIP2, G2L1, and EB1 Undergo Insulin-Stimulated Phosphorylation

G2L1 is a member of the plus-end tracking (+TIP) microtubule (MT)-associated protein family. G2L1 coordinates cooperativity between MTs and actin and was recently detected in an adipocyte CLASP2 interactome. Since CLASP2 has been linked to insulin action, and both actin and MTs play an unknown role in insulin action, we chose to study G2L1 within 3T3-L1 adipocytes by performing the first-ever G2L1 interactome. Using a commercially available antibody for G2L1 and two antibodies for epitope-tagged, overexpressed G2L1, we performed multiple affinity purification coupled with mass spectrometry experiments in combination with label-free quantitative proteomics and analysed the data with the bioinformatics tool Significance Analysis of Interactome. In concordance with findings in other cell types, we discovered that G2L1 co-IPs the master integrators of +TIP assembly, the end-binding (EB) proteins, with a strong preference for EB1 over EB3 in adipocytes. Subsequent EB1 interactome analysis confirmed reciprocal co-IP of G2L1. We discovered that G2L1 co-IPs the interaction partners CLASP2 and CLIP2, both of which are also +TIP family members, data which was confirmed by IP and western blot. Real-time total internal reflection fluorescence microscopy revealed colocalization of G2L1 and CLASP2 in adipocytes at growing MT ends. Since CLASP2 undergoes insulin-regulated phosphorylation in adipocytes, we hypothesized that the G2L1/CLIP2/EB1 network of +TIPs are also candidates for involvement in insulin-regulated control of MT dynamics. Using targeted quantitative phosphoproteomics, we discovered that insulin stimulates the phosphorylation of G2L1 at Ser597/599, CLIP2 at Ser552, and EB1 at Ser155. This now expands the novel network of insulin-affected +TIP family members to CLASP2, G2L1, CLIP2, and EB1 and introduces the new hypothesis that a coordinated network of MT-associated proteins cooperate to mediate insulin-regulated MT dynamics.