The CLASP2 Protein Interaction Network in Adipocytes Links CLIP2 to AGAP3, CLASP2 to G2L1, MARK2, and SOGA1, and Identifies SOGA1 as a Microtubule-Associated Protein

‡ The Section of Molecular Diabetes & Metabolism, Department of Clinical Research and Institute of Molecular Medicine, University of Southern Denmark, DK-5000 Odense, Denmark ¶ Department of Endocrinology, Odense University Hospital, DK-5000 Odense, Denmark ǂ School of Life Sciences, Arizona State University, Tempe, Arizona 85787 ǁ Department of Medicine, Division of Translational and Regenerative Medicine, University of Arizona College of Medicine, Tucson, Arizona 85721 § Department of Medicine, Division of Endocrinology, University of Arizona College of Medicine, Tucson, Arizona 85721


MTOC microtubule organization center
NIgG non-immune serum

Introduction
Microtubules are versatile cytoskeletal structures known to serve the needs of the particular subcellular context they are situated in. Whether they act as a cellular highway tasked with the trafficking of molecular cargo to specific destinations or function to support the structure of the cell, proper microtubule dynamics are essential for normal cell function. The insulinstimulated glucose uptake system has distinct effects on the cytoskeleton. Insulin mediates acute glucose uptake in part by mobilizing insulin-stimulated glucose transporter 4 (GLUT4) 1 storage vesicles (GSVs) from intracellular pools to the plasma membrane. Insulin stimulates actin to reassemble into filamentous cortical projections, resulting in the physical effect of ruffling the plasma membrane. Insulin signaling proteins such as WASP, Arp3, PI 3-K, PIP 3 , Akt, GLUT4 and additional proteins all colocalize with reorganized actin at the membrane ruffle (1)(2)(3). Inhibition of actin reorganization with the drugs latrunculin B and jasplakinolide in 3T3-L1 adipocytes (4,5), L6 myotubes (1,6), rat adipocytes (7), and rat skeletal muscle (8) supports the dependence of insulin-stimulated GLUT4 translocation and glucose uptake on actin reorganization. Conversely, microtubules undergo rapid depolymerization and polymerization cycles, resulting in the shortening and lengthening of the microtubule, which is assisted by microtubule associating proteins (9). Total internal reflection fluorescence microscopy (TIRFM) revealed that microtubules respond to insulin stimulation with a substantial increase in microtubule density and curvature directly underneath the plasma membrane (10). Long range movement of GSVs along microtubules has been established (11,12) and real-time TIRFM in living 3T3-L1 adipocytes proved insulin-stimulated fusion of GSVs occurs proximal to microtubules at the plasma membrane. Non-specific inhibition of the kinesin motor protein family (13), and later the conventional kinesin KIF5B (14) and KIF3 (15), which transport vesicles outwards towards the growing plus end of the microtubule, decreased detection of GLUT4 at the cell surface in response to insulin. However, nocodazole-induced disassembly of microtubules did not significantly decrease the number of GSV fusion events stimulated by insulin, leading to the hypothesis that microtubules are not vital for the insertion of GSVs into the plasma membrane, but rather play a more important role in site selection for delivery of GLUT4 prior to fusion (10). Even with these substantial discoveries, the function of the insulin-stimulated effects on the cytoskeleton in GLUT4 trafficking and glucose uptake are completely unknown.
The adipocyte has the distinct property of storing large amounts of lipid, secretion of a number of hormones, and in addition, serves as a secondary site for insulin-stimulated glucose uptake and storage (57). Knockdown of CLASP2 protein expression in 3T3-L1 adipocytes inhibited insulin-stimulated glucose transport (16). Here we report label-free quantification of interactome experiments that have been processed with the Significance Analysis of Interactome (SAINT) scoring system (58-61). By performing a series of strategic, successive and confirmatory reciprocal interactome experiments on key novel proteins, we have constructed a 3T3-L1 adipocyte CLASP2 protein network that has led to the identification of suppressor of glucose by autophagy 1 (SOGA1) as a microtubule-associated protein.

Experimental Procedures
Cell culture, immunoprecipitation, and Western blot analysis. 3T3-L1 fibroblasts were cultured in growth media of DMEM (Thermo Fisher Scientific  min at 37 °C. Cells were lysed with 500 L of lysis buffer containing 40 mM HEPES (pH 7.6), 120 mM NaCl, 0.3% CHAPS, 10 mM NaF, 10 mM β-glycerol phosphate, 1 mM EDTA (pH 8.0), 2 mM sodium orthovanadate, 17 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM PMSF. Cell lysates were rotated at 4 °C for 20 min followed by centrifugation (14,000 RPM, 4 °C, 20 min), and the clarified supernatants were used for immunoprecipitation (IP). For each IP, cell lysate from 1x150mm tissue culture dish (approximately 3.5-5mg) was incubated with 5 g of specific antibodies conjugated to 25 L protein A or protein G-agarose beads for 3 h at 4 °C with gentle rotation. IPs were washed three times with 1 mL of ice-cold PBS and the proteins bound to beads were eluted by heating at 95°C for 4 min in 8 L SDS sample loading buffer (4%SDS, 0.0625M Tris-HCl, 10% glycerol, 0.02% bromophenol blue, 8M Urea). The eluate was extracted and the antibody/beads were subjected to a second elution at 95°C for 4 min in 8 L SDS sample loading buffer. The second eluate was extracted and the two eluates were combined and separated by 10% SDS-PAGE and the gels were either stained with Bio-Safe Coomassie G-250 Stain (Bio-Rad, Hercules, CA) or transferred to a nitrocellulose membrane for subsequent Western blotting. For Western blots, proteins were transferred to a nitrocellulose membrane for 1 h at 105 V and blocked with 5% nonfat dry milk in Tris-buffered saline with 0.2% Tween-20 for 1 h at room temperature.
Membranes were probed with primary antibodies for 1h in blocking buffer at room temperature.
Blots were washed in Tris-buffered saline with 0.2% Tween-20 (three times for 10 min), and probed with either goat anti-mouse (Santa Cruz Technologies, Santa Cruz, CA) or donkey antirabbit (GE Healthcare, Waukesha, WI) secondary antibodies (both at dilutions of 1:1500) conjugated to horseradish peroxidase and detected with Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate kit (Perkin Elmer, Waltham, Massachusetts). Primary antibodies used: anti-CLASP2 "Antibody #1" (the immunogen recognized by this antibody maps to a region between residue 275 and 325 of human CLASP2 using the numbering given in entry BAG11221.1 (GeneID 23122), cat. # 21395, Novus Biologicals, Littleton, CO), anti-CLASP2 "Antibody #2"  After removal of the ACN by aspiration, the gel pieces were dried in a vacuum centrifuge at 60 °C for 30 min. Trypsin (250 ng; Sigma-Aldrich) in 20 l of 40 mm NH 4 HCO 3 was added, and the samples were maintained at 4 °C for 15 min prior to the addition of 50-100 l of 40 mm NH 4 HCO 3 .
The digestion was allowed to proceed at 37 °C overnight and was terminated by addition of 10 l of 5% formic acid (FA). After further incubation at 37 °C for 30 min and centrifugation for 1 min, each supernatant was transferred to a clean LoBind polypropylene tube. The extraction procedure was repeated using 40 l of 0.5% FA, and the two extracts were combined and dried down to approximately 5-10 L followed by the addition of 10 L 0.05% heptafluorobutyric acid:5% FA  To reconstruct regions of SOGA1 non-homologous to 4RH7, BLAST was used to retrieve homologous sequences, create a multiple sequence alignment, and enter the sequences into a "discrimination of secondary structure class" prediction algorithm. The side chain loops were then optimized and added. The resulting structure was subjected to a combined steepest descent algorithm and refined with simulated annealing minimizations within surrounding water.
Experimental design and statistical rationale. Using the mean and standard deviations for the spectrum counts of the 39 proteins identified by SAINT analysis as significantly enriched between the CLASP2 Antibody #1 IPs (Group 1, 343 ± 280) and the NIgG IPs (Group 2, 26 ± 21), the effect size is equal to 1.60. With this robust effect size, a sample size of four biological replicates per group provided more than sufficient power to detect differences between groups. Based on the high degree of reproducibility, we also used a sample size of two and three biological replicates.
Each IP had a respective NIgG or appropriate tag antibody negative control IP, two to four biological replicates each, and no process and technical replicates due to the strong reproducibility of the data. The varying statistical analyzes for the different experiments are listed in the Figure Legends and were justifiable based upon the data and basic experiment performed.
For spectral counting measurements, modified peptides, semi-tryptic peptides, and shared peptides were included.

Results
The CLASP2 Interactomes. We devised a streamlined, label-free proteomic technique to identify and quantify protein interaction partners and applied this system to discover proteins that may be linked to regulation of the microtubule-associated protein CLASP2. This method combined cell lysis with a CHAPS detergent-containing buffer together with IP, SDS-PAGE-based fractionation, subsequent in-gel tryptic digestion and bottom-up mass spectrometry analysis ( Figure 1A). This approach possesses the caveat that proteins non-specifically bound to the Sepharose resin, Protein A/G, and non-targeting regions of the antibody are eluted together with the prey proteins, rendering the final interactome sample full of contaminating proteins which need systematic elimination (65,66). To avoid artefacts resulting from overexpression of epitope-tagged proteins, we used two different commercially available CLASP2 antibodies to independently IP endogenous CLASP2. On the other hand, since antibodies targeted to endogenous protein can disrupt protein-protein interactions and possess non-specific cross-reactivity (66, 67), we also adenovirally-overexpressed HA-epitope and GFP-tagged CLASP2 to create an anti-HA antibody GFP-CLASP2-HA interactome. These three independent CLASP2 interactomes act as cross references, to narrow down the list of potential CLASP2 interaction partners for follow-up confirmation experiments. Western blot of these CLASP2 protein IPs are shown in Figures 1B, C, and D. For a detailed breakdown of the proteomic numbers associated with the experimental approach adopted for these interactome studies, using the CLASP2 Antibody #1 experiment numbers as an example, please refer to Supplemental Figure 1 and the accompanying text. For all interactome experiments presented in this study, each gel slice's protein yield, total number of peptides identified, total number of spectra, and % identification rate are included in Supplemental   Tables (CLASP2 Antibody #1 data are in Supplemental Tables 1, 2, 3, and 4, GFP-CLASP2-HA   data are in Supplemental Tables 6, 7, 8, and 9, CLASP2 Antibody #2 data are in Supplemental Tables 11,12,13,and 14). These numbers are provided throughout the remainder of this report for all interactomes performed $$ . CLIP1 and CLIP2 are the original CLASP associated proteins (17), so we chose to use CLIP2 as a high-confidence CLASP2 interacting partner to test the validity of the current experimental conditions and approach, using the CLASP2 Antibody #1 interactome data as an example. Western blot analysis of CLIP2 in the CLASP2 Antibody #1 basal and insulin IPs showed strong enrichment of CLIP2 in the CLASP2 Antibody #1 IPs, with no effect of insulin on the interaction (Supplemental Figure 2A, and densitometry results from co-IP/Western blot experiments are shown in Supplemental Figure 2B). The spectral count data (Supplemental Figure 2C) is very similar to the results from the co-IP/Western blot experiments, in that CLIP2 shows a strong enrichment in the CLASP2 Antibody #1 basal and insulin IPs over the NIgG negative controls and no effect of insulin on the interaction. CLIP2 averaged 134 spectral counts versus only 13 for CLIP1 (CLIP1 lacked an effect of insulin as well). The strong co-IP of CLIP2 with CLASP2 supports the validity of the experimental conditions used.
We used the established bioinformatic tool Significance Analysis of Interactome (SAINT) (58-61) to analyze our interactome data for protein enrichment. SAINT assigns each protein a SAINT Probability Score ("P-Score"), on a scale of 0 to 1, with 1 being the top score. Based upon review of the literature, we chose 0.65 as a SAINT cut-off score for enrichment (68)(69)(70)(71)(72)(73). With the approach used here, all proteins have two P-Scores, one each for basal or insulin enrichment over the respective negative controls. A detailed breakdown of SAINT results typical for our interactome approach are outlined in Supplemental Figure 3, again using CLASP2 Antibody #1 interactome data as an example. Protein SAINT scores and spectrum counts for all interactomes performed in this study are included in Supplemental Tables (CLASP2 Antibody #1 in  Supplemental Table 5, GFP-CLASP2-HA data in Supplemental Table 10, CLASP2 Antibody #2 data in Supplemental Table 15).
We sought to analyze the raw spectral count data of all proteins with SAINT scores greater than a 0.65 in both the basal and insulin samples (which we refer to as "SAINT-qualified") in a hierarchical manner, with spectral count results from all experiments individually plotted. We therefore devised the "Spectrum Count Profile " (SCP), an easily readable format for the visualization of whole sets of raw spectral count data for interactome experiments, and applied it to the CLASP2 Antibody #1 interactome data (Figure 2A). Using the same interactome processing approach, we generated SCPs for the two remaining CLASP2 interactomes we had prepared as cross references, the adenovirally overexpressed HA-epitope and GFP-tagged CLASP2 anti-HA IP ( Figure 2B) and the second, alternative CLASP2 Antibody #2 IP ( Figure 2C). We first searched the three SAINT-qualified CLASP2 interactomes for proteins previously linked to CLASP2 (for reviews on CLASP2 and members of the plus-end tracking microtubule-associated protein family, please refer to (49, 51-54, 74)) and found MARE1/EB1, SLAIN2, MARK2, MARK3, CLIP1, CKAP5/ch-TOG, PHLDB1/LL5, MACF1, GCC2, CLASP1, CLIP1, and CLIP2. The CLASP2 Antibody #1 interactome had more SAINT-qualified proteins, although reciprocal co-IP follow-up interactome experiments were unsuccessful for two of these, namely TUG/ASPC1, which we chose due to a reported role in insulin action (75,76), and FAM13A since this protein exhibited a significant increase in abundance in the CLASP2 IPs upon insulin stimulation (data not shown).
TUG and FAM13A were exclusive to the CLASP2 Antibody #1 interactome, so we took advantage of Cytoscape (77) for the integration and visualization of all three CLASP2 interactomes and followed up on SAINT-qualified proteins that were present in more than one of the individual CLASP2 interactomes (Figure 3). This approach helps overcome false positives resulting from cross-reactivity or non-specificity from a single antibody. Proteins with established links to CLASP2 that were present in multiple CLASP2 interactomes include the binding partners SLAIN2 and CKAP5/ch-TOG (78), CLASP1, MARK2 (21), GCC2 (also known as GCC185) (32), and of course CLIP1 and CLIP2 (17). In addition, we present the novel discoveries that CLASP2 co-IPs Arf-GAP with GTPase, ANK repeat and PH domain-containing proteins 1 and 3 (AGAP1 and AGAP3), the microtubule/actin-regulating protein GAS2-like protein 1 (GA2L1/G2L1), and the protein "suppressor of glucose by autophagy" (SOGA1), all of which together represent a new subset of CLASP2-associated proteins that do not have any previously established connection to CLASP2.
The AGAP3 and CLIP2 Interactomes. AGAP1, AGAP2, and AGAP3 are GAP proteins with links to membrane traffic and actin (79). Since both AGAP1 and AGAP3 were present in all three CLASP2 interactomes, and GAP proteins are important regulators of cytoskeletal dynamics and vesicle trafficking, we chose to analyze the AGAP3 interactome with a commercially available antibody, using the same SAINT-based interactome approach as described above for CLASP2 ( Figure 4A and accompanying Supplemental Tables 16,17,18,19,and 20). SCP analysis of the SAINT-qualified AGAP3 interactome confirmed successful immunoprecipitation of AGAP3 and while CLASP1 but not CLASP2 was detected, CLIP2 was highly enriched, as was AGAP1, albeit to a lesser extent. This finding introduced the possibility that AGAP3 was enriched in the CLASP2 interactomes due to binding CLIP2 rather than direct interaction with CLASP2. To test this, we analyzed the CLIP2 interactome with a commercially available antibody, again, using the same interactome approach (Figure 4B and accompanying Supplemental Tables 21, 22, 23, 24, and 25). We report the novel finding that AGAP3, and to a lesser extent, AGAP1, can strongly co-IP with CLIP2. Since much less CLASP2 was present in the CLIP2 interactome as compared to the CLASP2 interactomes, and the amount of AGAP3 in the CLIP2 interactome was at least triple the detected levels of AGAP3 in the CLASP2 IPs, these findings suggest a preference of AGAP3 for CLIP2 over CLASP2. In order to visualize interaction partners shared among CLASP2, CLIP2, and AGAP3, we integrated the corresponding SAINT-qualified interactomes with Cytoscape ( Figure 4C). This led to the discovery that G2L1, the novel protein identified in each of the three CLASP2 interactomes, as well as both CKAP5 and SLAIN2, were present in both the CLIP2 and CLASP2 interactomes. This leads to the novel hypothesis that CLIP2-CLASP2-SLAIN2-CKAP5-G2L1 are a protein network, although more evidence is needed supporting a direct link between G2L1, CKAP5 and SLAIN2. Cytoscape integration also revealed that both CLIP2 and AGAP3 share association with CLIP1, CLASP1, and AGAP1, as well as with members of the protein representing a very strong enrichment of GYS1 in the mCherry-MARK2 interactome. We modified the SCP of mCherry-MARK2 to include these two proteins for Cytoscape-based integration of the two MARK2 interactomes ( Figure 5C). We confirmed the previous report that MARK2 associates with MTCL1 (81). In addition, suppressor of glucose by autophagy 1 (SOGA1), a protein enriched in two out of the three CLASP2 interactomes, was also identified as a SAINT-qualified protein within both MARK2 interactomes tested. SOGA1 has been linked to adiponectin-mediated inhibition of glucose production by enhancing suppression of autophagy in an insulin-dependent manner in hepatocytes (82), although an association between SOGA1 and microtubules or microtubule-associated proteins has yet to be shown. To test for reciprocal co-IP of CLASP2 and MARK2 with SOGA1, we analyzed the SOGA1 interactome with a commercially available antibody (Figure 6A and accompanying Supplemental Tables 36, 37 To better understand the SOGA1 structure-function relationship, we developed a homology model of full-length SOGA1 protein structure. Due to the absence of an x-ray crystallography structure for SOGA1, we utilized the YASARA Structure software package (83) to perform three-dimensional homology modeling of SOGA1 (Uniprot Accession E1U8D0) in silico.
Screening of SOGA1 against the Protein Data Bank resulted in a best alignment/coverage score for the Cytoplasmic Dynein 2 Motor Domain (PDB ID 4RH7) (64). Using the dynein 2 domain as a template, the complete structure of SOGA1 was built in silico. Our analysis of the resulting structure indicates that SOGA1 shares a close similarity to the main features of dynein such as microtubule binding, stalk and strut domains (Figure 9). SOGA1 sequence length is less than dynein 2, due to SOGA1 lacking the massive linker, neck, and tail portions of the dynein 2 Nterminus. The SOGA1 structure predicts long N-terminal alpha-helixes of structural similarity to Tropomyosin chains, which could hypothetically interact with actin fibers after release from the observed "clamp" structure ( Figure 9, top right inset). Further structure-function experiments for SOGA1 will be the focus of the follow up studies. The molecular modeling data indicating SOGA1 is structurally similar to a known microtubule binding protein like dynein 2 is in agreement with the findings that SOGA1 colocalizes with CLASP2 and tubulin and supports the identification of SOGA1 as a new microtubule associated protein.

Discussion
We have developed and successfully executed a streamlined interactome approach to characterize the CLASP2 interactome in a 3T3-L1 adipocyte system. We compared NIgG antibody negative control IPs against two different CLASP2 antibody IPs and combined these interactomes with one null anti-HA antibody IP versus a GFP-CLASP2-HA anti-HA antibody IP and analyzed the raw interactome data with SAINT to identify proteins enriched in the CLASP2 IPs. To aid in the visualization of the raw spectral count data from the interactome experiments, we developed the Spectrum Count Profile, which allowed for facilitated interpretation of the raw spectral count results. By performing these in-depth, multiple antibody interactome experiments, we discovered that different antibodies for the same protein, in this case CLASP2 and MARK2, can present individually diverse interactomes, while also containing cross-correlating proteins as well. Taking this into account for the CLASP2 data, we narrowed down our focus to AGAP3 as one of the proteins of interest in the CLASP2 interactome. Subsequent analysis of the AGAP3 interactome revealed a strong enrichment of CLIP2 but not CLASP2, opening up the likelihood that AGAP3 appeared in the CLASP2 interactome as a result of associating with CLIP2 (which was highly present in all three CLASP2 interactomes tested). In support of this, successive experiments performed to characterize the CLIP2 interactome revealed robust enrichment of AGAP3. AGAP3 co-immunoprecipitated AGAP1 very strongly, which may support the proposed functional cooperativity between AGAP family members (84), as AGAP1 and AGAP3 were also detected in tandem in the CLIP2 interactome and all three CLASP2 interactomes. The AGAPs, also referred to as the centaurins (85) and the GGAPs (86), have been shown to act as GAPs for Arf family members (79,84) and have intrinsic GTPase property as well (85,86), although GTPase activity has been questioned (79,84). The fact that AGAP1 and AGAP3 consistently coimmunoprecipitated with the microtubule-associated proteins CLIP2 and CLASP2 now places these two proteins in position to be tested for roles in regulating microtubule dynamics.
TBC1D4/AS160 is a GAP whose inhibitory control of Rab function in GLUT4 translocation is deactivated by insulin-stimulated AKT phosphorylation (87). Based on our interactome findings, we hypothesize that both AGAP1 and AGAP3 are linked to CLASP2 through CLIP2, and since CLASP2 undergoes strong insulin-stimulated phosphorylation (16), it will be of interest to explore whether the proposed GTPase activity of the AGAP proteins is under the control of insulinstimulated AGAP phosphorylation.
Another novel finding was that G2L1 (Uniprot gene name GAS2L1; protein name also known as GAR22) exhibited enrichment in all three CLASP2 interactomes as well as in the CLIP2 interactome. G2L1, like CLASP2, contains microtubule-tip localization sequences, which supports likely co-localization and possible cooperativity between these two proteins, and perhaps CLIP2 as well. G2L1 has been shown to bind with both filamentous actin (F-actin) and microtubules (88)(89)(90)(91) to support microtubule and actin coalignment (88,92). G2L1 mediates actin and microtubule crosstalk by promoting microtubule guidance along actin, which is of interest to insulin action since it has been proposed that the GSV could at some point transfer from microtubules to actin during the vesicle translocation process (93). This hypothesis is supported by the findings that knockdown of the actin-based motor protiens Myo1c and Myo5, which are responsible for cargo transport along actin, reduced cell surface levels of GLUT4 (94)(95)(96). It is also worth noting that both CLASP1 and CLASP2 have been found to interact with actin filaments (97). Future studies will investigate the functional consequences for the proposed CLASP2-CLIP2-G2L1 protein network, and the importance this complex has in regulating the function of these individual proteins.
With regards to the known associating partners for CLASP2, CLIP1 and CLIP2, both had excellent enrichment in the CLASP2 IPs, although there was no observable insulin effect on the co-IP of the two CLIPs with CLASP2 as tested, nor did insulin affect CLASP2 (or CLASP1) co-IP in the CLIP2 interactome. There has been a reported association between insulin action and the CLIP proteins, in that CLIP1 restores insulin-stimulated GLUT4 translocation in TSC2 -/-cells, a mechanism postulated to involve mTOR-regulated microtubule organization (98) since mTOR can phosphorylate CLIP1 (99). CLIP2 and CLASP2 both shared association with CLIP1 and CLASP1, as well as with the binding partners SLAIN2 and CKAP5/ch-TOG, which is agreement with previous findings implicating that the microtubule plus end-tracking proteins SLAIN2, CKAP5, CLIP2, and CLASP2 can complex together (78). Another novel finding resulting from this study was the shared association of CLIP2 and AGAP3 with ANR28, PP6R3 and PPP6, all of which are subunits of the serine/threonine-protein phosphatase 6 holoenzyme complex. Seeing as phosphorylation is a known modulator of GAP activity, the protein phosphatase 6 holoenzyme complex may contribute to the dynamic regulation of AGAP function.
We present the discovery that SOGA1, a protein with no previous connection to cytoskeletal elements, is enriched in the CLASP2 interactome. This finding was strengthened by successive analysis and confirmation of the presence of CLASP2 in the reciprocal SOGA1 interactome, SOGA1 is a relatively uncharacterized protein that has been linked to the regulation of autophagy in hepatocytes (82). In response to nutritional deprivation, autophagy can initiate the release of glucose from the liver by promoting the hydrolysis of proteins, glycogen, and triglycerides (100)(101)(102). SOGA1 expression was shown to be increased upon adiponectinstimulated activation of the insulin signaling pathway in hepatocytes, and this increase in SOGA1 protein levels contributes to the reduction of glucose production by inhibiting autophagy through an as-of-yet unknown mechanism (82). These previous findings highlight the importance of our discovery that glycogen synthase (GYS1), the key biosynthetic enzyme for the synthesis of glycogen (103), is enriched in the SOGA1 interactome. While the role of glycogen autophagy in adipocytes was only recently examined (104), it is possible to hypothesize that SOGA1 may regulate glucose and glycogen metabolism by directly cooperating with glycogen synthase and the glycogen synthase-associated protein glycogenin, which was also detected in the SOGA1 interactome. Since increased expression of SOGA1 was found to enhance inhibition of glycogen autophagy (82), SOGA1 binding to glycogen synthase and glycogenin could hypothetically participate in the protection of glycogen from autophagy, although these new proposed interactions from our proteomics studies will require further validation. In addition, this mechanism likely involves a microtubule element since SOGA1 subcellular localization studies presented here revealed SOGA1 as a new microtubule-associated protein and molecular modeling studies predict SOGA1 to have a structure similar to the known microtubule binding protein dynein. This hypothesis is further strengthened by the additional discovery of MARK2 and MARK3 in the SOGA1 interactome, both of which are kinases known to regulate microtubule dynamics.
Whereas CLASP2 promotes microtubule stability, MARK family members can disrupt microtubule growth (105), a phenomenon we showed that also results in the subcellular displacement of both SOGA1 and CLASP2 in adipocytes. The MARK family of protein kinases do have an established connection to metabolism, as MARK2, MARK3, and MARK4 knockout mice each exhibit enhanced peripheral insulin sensitivity and resistance to high-fat diet induced obesity (106)(107)(108).
In addition, the MARK family of kinases are closely related to the energy sensor and metabolic regulator AMPK, as these kinases share a common consensus phosphorylation motif (109).
These observations, together with the discovery presented here that MARK2, like SOGA1, can co-IP glycogen synthase and glycogenin, introduce the proposed SOGA1-MARK2-GYS1-GLYG network and microtubules as a target for studies aimed at determining the metabolic function of this potentially novel protein complex.
By taking advantage of proteomics and the advancements that are being made in interactome studies and bioinformatic resources, this study has made progress in identifying proteins that potentially associate with CLASP2, which has allowed us to propose a series of novel protein networks ( Figure 10). The discoveries presented here will lead to future studies aimed at further confirming these findings and understanding the purpose of the functional relationship these new protein networks have with microtubule dynamics, actin reorganization, glucose and glycogen metabolism, autophagy, and insulin action.   Basal NIgG IPs (green), insulin NIgG IPs (magenta), basal CLASP2 Antibody #1 IPs (red), and insulin CLASP2 Antibody #1 IPs (turquoise). B, Anti-HA antibody IPs for GFP-CLASP2-HA were performed as described in Figure 1. Tandem mass spectrometry on the IPs was performed as described in Experimental Procedures (n=3) and SCP analysis of the 18 SAINT-qualified proteins is shown. C, CLASP2 Antibody #2 IPs were performed as described in Figure 1. Tandem mass spectrometry on the IPs was performed as described in Experimental Procedures (n=4) and SCP analysis of the 17 SAINT-qualified proteins is shown.       to build a homology model of SOGA1 structure. Homology modeling of the SOGA1 sequence results in a structure similar to dynein 2 (PDB ID 4RH7). The SOGA1 model has high similarity with structural characteristics of dynein 2 including a microtubule binding region (yellow) and both stalk (orange) and strut (green) domains. SOGA1 is predicted to contain only one ATP binding site (red), whereas dynein 2 posesses six distinctive ATP binding sites. SOGA1 contains both Serine-and Glutamine-rich sequences (purple) and a long N-terminal alpha-helix structure that has structural and sequence similarity to the actin binding protein tropomyosin. SOGA1 is predicted to contain a "clamp" region around the long N-terminal tropomyosin-like alpha-helix structure (the clamp feature is colored in blue within the top right inset). and both CLASP1 and CLIP1 exist together to coordinate cytoskeletal events between the plus end of microtubules and perhaps insulin-stimulated cortically-reorganized actin. We also hypothesize that CLASP2 can associate with both MARK2 and SOGA1 in a separate complex, to coordinate dynamic stability of the microtubule. An alternative complex of MARK2/SOGA1, localized to microtubules, may functionally link glycogen synthase, glycogenin, and microtubules to glycogen management. The final complex revolves around the observed relationship between AGAP3 and CLIP2, which includes AGAP1, CLASP1, CLASP2, CLIP1 and members of the protein phosphatase 6 holoenzyme, specifically PPP6/PP6C, PP6R3, and ANR28. The hypothetical function of this complex may be to integrate the regulation of the dynamic instability of the plus end of the microtubule together with plus end-associated proteins and the GTPase activity of the AGAP1 and AGAP3 proteins.