Extracellular signals induce dynamic ER remodeling through αTAT1-dependent microtubule acetylation.

Dynamic changes in the endoplasmic reticulum (ER) morphology are central to maintaining cellular homeostasis. Microtubules (MT) facilitate the continuous remodeling of the ER network into sheets and tubules by coordinating with many ER-shaping protein complexes, although how this process is controlled by extracellular signals remains unknown. Here we report that TAK1, a kinase responsive to various growth factors and cytokines including TGF-ß and TNF-α, triggers ER tubulation by activating αTAT1, an MT-acetylating enzyme that enhances ER-sliding. We show that this TAK1/αTAT1-dependent ER remodeling promotes cell survival by actively downregulating BOK, an ER membrane-associated proapoptotic effector. While BOK is normally protected from degradation when complexed with IP3R, it is rapidly degraded upon their dissociation during the ER sheets-to-tubules conversion. These findings demonstrate a distinct mechanism of ligand-induced ER remodeling and suggest that the TAK1/αTAT1 pathway may be a key target in ER stress and dysfunction.

Phosphoproteomic profiling identifies DNMT1 as a key substrate of beta IV spectrin-dependent ERK/MAPK signaling in suppressing angiogenesis.

ßIV-spectrin is a membrane-associated cytoskeletal protein that maintains the structural stability of cell membranes and integral proteins such as ion channels and transporters. Its biological functions are best characterized in the brain and heart, although recently we discovered a fundamental new role in the vascular system. Using cellular and genetic mouse models, we reported that ßIV-spectrin acts as a critical regulator of developmental and tumor-associated angiogenesis. ßIV-spectrin was shown to selectively express in proliferating endothelial cells (EC) and suppress VEGF/VEGFR2 signaling by enhancing receptor internalization and degradation. Here we examined how these events impact the downstream kinase signaling cascades and target substrates. Based on quantitative phosphoproteomics, we found that ßIV-spectrin significantly affects the phosphorylation of epigenetic regulatory enzymes in the nucleus, among which DNA methyltransferase 1 (DNMT1) was determined as a top substrate. Biochemical and immunofluorescence results showed that ßIV-spectrin inhibits DNMT1 function by activating ERK/MAPK, which in turn phosphorylates DNMT1 at S717 to impede its nuclear localization. Given that DNMT1 controls the DNA methylation patterns genome-wide, and is crucial for vascular development, our findings suggest that epigenetic regulation is a key mechanism by which ßIV-spectrin suppresses angiogenesis.

Glycosaminoglycan modifications of betaglycan regulate ectodomain shedding to fine-tune TGF-ß signaling responses in ovarian cancer.

In pathologies including cancer, aberrant Transforming Growth Factor-ß (TGF-ß) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-ß responses. Betaglycan/type III TGF-ß receptor (TßRIII), is an established co-receptor for the TGF-ß superfamily known to bind directly to TGF-ßs 1-3 and inhibin A/B. Betaglycan can be membrane-bound and also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. Its extracellular domain undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. We report the unexpected discovery that the heparan sulfate glycosaminoglycan chains on betaglycan are critical for the ectodomain shedding. In the absence of such glycosaminoglycan chains betaglycan is not shed, a feature indispensable for the ability of betaglycan to suppress TGF-ß signaling and the cells' responses to exogenous TGF-ß ligands. Using unbiased transcriptomics, we identified TIMP3 as a key inhibitor of betaglycan shedding thereby influencing TGF-ß signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-ß signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan chains of betaglycan for shedding and influence on TGF-ß signaling responses. Dysregulated shedding of TGF-ß receptors plays a vital role in determining the response and availability of TGF-ßs', which is crucial for prognostic predictions and understanding of TGF-ß signaling dynamics.

Development of adaptive anoikis resistance promotes metastasis that can be overcome by CDK8/19 Mediator kinase inhibition.

Anoikis resistance or evasion of cell death triggered by cell detachment into suspension is a hallmark of cancer that is concurrent with cell survival and metastasis. The effects of frequent matrix detachment encounters on the development of anoikis resistance in cancer remains poorly defined. Here we show using a panel of ovarian cancer models, that repeated exposure to suspension stress in vitro followed by attached recovery growth leads to the development of anoikis resistance paralleling in vivo development of anoikis resistance in ovarian cancer ascites. This resistance is concurrent with enhanced invasion, chemoresistance and the ability of anoikis adapted cells to metastasize to distant sites. Adapted anoikis resistant cells show a heightened dependency on oxidative phosphorylation and can also evade immune surveillance. We find that such acquired anoikis resistance is not genetic, as acquired resistance persists for a finite duration in the absence of suspension stress. Transcriptional reprogramming is however essential to this process, as acquisition of adaptive anoikis resistance in vitro and in vivo is exquisitely sensitive to inhibition of CDK8/19 Mediator kinase, a pleiotropic regulator of transcriptional reprogramming. Our data demonstrate that growth after recovery from repeated exposure to suspension stress is a direct contributor to metastasis and that inhibition of CDK8/19 Mediator kinase during such adaptation provides a therapeutic opportunity to prevent both local and distant metastasis in cancer.

Heparan sulfate modifications of betaglycan promote TIMP3-dependent ectodomain shedding to fine-tune TGF-ß signaling.

In pathologies such as cancer, aberrant Transforming Growth Factor-ß (TGF-ß) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pivotal pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-ß responses. Betaglycan/type III TGF-ß receptor (TßRIII), is an established co-receptor for the TGF-ß superfamily known to bind directly to TGF-ßs 1-3 and inhibin A/B. While betaglycan can be membrane-bound, it can also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. The extracellular domain of betaglycan undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. Here we report the unexpected discovery that the heparan sulfate modifications are critical for the ectodomain shedding of betaglycan. In the absence of such modifications, betaglycan is not shed. Such shedding is indispensable for the ability of betaglycan to suppress TGF-ß signaling and the cells' responses to exogenous TGF-ß ligands. Using unbiased transcriptomics, we identified TIMP3 as a key regulator of betaglycan shedding and thereby TGF-ß signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-ß signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan modifications of betaglycan for shedding and influence on TGF-ß signaling responses. Dysregulated shedding of TGF-ß receptors plays a vital role in determining the response and availability of TGF-ßs', which is crucial for prognostic predictions and understanding of TGF-ß signaling dynamics.

Beta IV spectrin inhibits the metastatic growth of melanoma by suppressing VEGFR2-driven tumor angiogenesis.

BACKGROUND: Tumor-associated angiogenesis mediates the growth and metastasis of most solid cancers. Targeted therapies of the VEGF pathways can effectively block these processes but often fail to provide lasting benefits due to acquired resistance and complications. RESULTS: Recently, we discovered ßIV -spectrin as a powerful regulator of angiogenesis and potential new target. We previously reported that ßIV -spectrin is dynamically expressed in endothelial cells (EC) to induce VEGFR2 protein turnover during development. Here, we explored how ßIV -spectrin influences the tumor vasculature using the murine B16 melanoma model and determined that loss of EC-specific ßIV -spectrin dramatically promotes tumor growth and metastasis. Intraperitoneally injected B16 cells formed larger tumors with increased tumor vessel density and greater propensity for metastatic spread particularly to the chest cavity and lung compared to control mice. These results support ßIV -spectrin as a key regulator of tumor angiogenesis and a viable vascular target in cancer.

Structural remodeling of the endoplasmic reticulum in response to extracellular signals requires αTAT1-induced microtubule acetylation.

Dynamic changes in the endoplasmic reticulum (ER) morphology are central to maintaining cellular homeostasis. Microtubules (MT) facilitate the continuous remodeling of the ER network into sheets and tubules by coordinating with many ER-shaping protein complexes, although how this process is controlled by extracellular signals remains unknown. Here we report that TAK1, a kinase responsive to numerous growth factors and cytokines including TGF-ß and TNF-α, triggers ER tubulation by activating αTAT1, an MT-acetylating enzyme that enhances ER-sliding. We show that this TAK1/αTAT-dependent ER remodeling promotes cell survival by actively downregulating BOK, an ER membrane-associated proapoptotic effector. While BOK is normally protected from degradation when complexed with IP3R, it is rapidly degraded upon their dissociation during the ER sheets-to-tubules conversion. These findings demonstrate a distinct mechanism of ligand-induced ER remodeling and suggest that the TAK1/αTAT pathway may be a key target in ER stress and dysfunction.

Endothelial tip/stalk cell selection requires BMP9-induced ßIV-spectrin expression during sprouting angiogenesis.

ßIV-Spectrin is a membrane cytoskeletal protein with specialized roles in the nervous system and heart. Recent evidence also indicates a fundamental role for ßIV-spectrin in angiogenesis as its endothelial-specific gene deletion in mice enhances embryonic lethality due to hypervascularization and hemorrhagic defects. During early vascular sprouting, ßIV-spectrin is believed to inhibit tip cell sprouting in favor of the stalk cell phenotype by mediating VEGFR2 internalization and degradation. Despite these essential roles, mechanisms governing ßIV-spectrin expression remain unknown. Here we identify bone morphogenetic protein 9 (BMP9) as a major inducer of ßIV-spectrin gene expression in the vascular system. We show that BMP9 signals through the ALK1/Smad1 pathway to induce ßIV-spectrin expression, which then recruits CaMKII to the cell membrane to induce phosphorylation-dependent VEGFR2 turnover. Although BMP9 signaling promotes stalk cell behavior through activation of hallmark stalk cell genes ID-1/3 and Hes-1 and Notch signaling cross-talk, we find that ßIV-spectrin acts upstream of these pathways as loss of ßIV-spectrin in neonate mice leads to retinal hypervascularization due to excessive VEGFR2 levels, increased tip cell populations, and strong Notch inhibition irrespective of BMP9 treatment. These findings demonstrate ßIV-spectrin as a BMP9 gene target critical for tip/stalk cell selection during nascent vessel sprouting.

Reciprocal SOX2 regulation by SMAD1-SMAD3 is critical for anoikis resistance and metastasis in cancer.

Growth factors in tumor environments are regulators of cell survival and metastasis. Here, we reveal the dichotomy between TGF-ß superfamily growth factors BMP and TGF-ß/activin and their downstream SMAD effectors. Gene expression profiling uncovers SOX2 as a key contextual signaling node regulated in an opposing manner by BMP2, -4, and -9 and TGF-ß and activin A to impact anchorage-independent cell survival. We find that SOX2 is repressed by BMPs, leading to a reduction in intraperitoneal tumor burden and improved survival of tumor-bearing mice. Repression of SOX2 is driven by SMAD1-dependent histone H3K27me3 recruitment and DNA methylation at SOX2's promoter. Conversely, TGF-ß, which is elevated in patient ascites, and activin A can promote SOX2 expression and anchorage-independent survival by SMAD3-dependent histone H3K4me3 recruitment. Our findings identify SOX2 as a contextual and contrastingly regulated node downstream of TGF-ß members controlling anchorage-independent survival and metastasis in ovarian cancers.

EPDR1 is a noncanonical effector of insulin-mediated angiogenesis regulated by an endothelial-specific TGF-ß receptor complex.

Insulin signaling in blood vessels primarily functions to stimulate angiogenesis and maintain vascular homeostasis through the canonical PI3K and MAPK signaling pathways. However, angiogenesis is a complex process coordinated by multiple other signaling events. Here, we report a distinct crosstalk between the insulin receptor and endoglin/activin receptor-like kinase 1 (ALK1), an endothelial cell-specific TGF-ß receptor complex essential for angiogenesis. While the endoglin-ALK1 complex normally binds to TGF-ß or bone morphogenetic protein 9 (BMP9) to promote gene regulation via transcription factors Smad1/5, we show that insulin drives insulin receptor oligomerization with endoglin-ALK1 at the cell surface to trigger rapid Smad1/5 activation. Through quantitative proteomic analysis, we identify ependymin-related protein 1 (EPDR1) as a major Smad1/5 gene target induced by insulin but not by TGF-ß or BMP9. We found endothelial EPDR1 expression is minimal at the basal state but is markedly enhanced upon prolonged insulin treatment to promote cell migration and formation of capillary tubules. Conversely, we demonstrate EPDR1 depletion strongly abrogates these angiogenic effects, indicating that EPDR1 is a crucial mediator of insulin-induced angiogenesis. Taken together, these results suggest important therapeutic implications for EPDR1 and the TGF-ß pathways in pathologic angiogenesis during hyperinsulinemia and insulin resistance.

Hypoxia-induced inhibin promotes tumor growth and vascular permeability in ovarian cancers.

Hypoxia, a driver of tumor growth and metastasis, regulates angiogenic pathways that are targets for vessel normalization and ovarian cancer management. However, toxicities and resistance to anti-angiogenics can limit their use making identification of new targets vital. Inhibin, a heteromeric TGFß ligand, is a contextual regulator of tumor progression acting as an early tumor suppressor, yet also an established biomarker for ovarian cancers. Here, we find that hypoxia increases inhibin levels in ovarian cancer cell lines, xenograft tumors, and patients. Inhibin is regulated primarily through HIF-1, shifting the balance under hypoxia from activins to inhibins. Hypoxia regulated inhibin promotes tumor growth, endothelial cell invasion and permeability. Targeting inhibin in vivo through knockdown and anti-inhibin strategies robustly reduces permeability in vivo and alters the balance of pro and anti-angiogenic mechanisms resulting in vascular normalization. Mechanistically, inhibin regulates permeability by increasing VE-cadherin internalization via ACVRL1 and CD105, a receptor complex that we find to be stabilized directly by inhibin. Our findings demonstrate direct roles for inhibins in vascular normalization via TGF-ß receptors providing new insights into the therapeutic significance of inhibins as a strategy to normalize the tumor vasculature in ovarian cancer.

ßIV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling.

Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report ßIV-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific ßIV-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, ßIV-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, ßIV-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.

Regulation of mitochondrial fission by GIPC-mediated Drp1 retrograde transport.

Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission, a large cytoplasmic GTPase recruited to the mitochondrial surface via transmembrane adaptors to initiate scission. While Brownian motion likely accounts for the local interactions between Drp1 and the mitochondrial adaptors, how this essential enzyme is targeted from more distal regions like the cell periphery remains unknown. Based on proteomic interactome screening and cell-based studies, we report that GAIP/RGS19-interacting protein (GIPC) mediates the actin-based retrograde transport of Drp1 toward the perinuclear mitochondria to enhance fission. Drp1 interacts with GIPC through its atypical C-terminal PDZ-binding motif. Loss of this interaction abrogates Drp1 retrograde transport resulting in cytoplasmic mislocalization and reduced fission despite retaining normal intrinsic GTPase activity. Functionally, we demonstrate that GIPC potentiates the Drp1-driven proliferative and migratory capacity in cancer cells. Together, these findings establish a direct molecular link between altered GIPC expression and Drp1 function in cancer progression and metabolic disorders.

A bioinformatic analysis of the inhibin-betaglycan-endoglin/CD105 network reveals prognostic value in multiple solid tumors.

Inhibins and activins are dimeric ligands belonging to the TGFß superfamily with emergent roles in cancer. Inhibins contain an α-subunit (INHA) and a ß-subunit (either INHBA or INHBB), while activins are mainly homodimers of either ßA (INHBA) or ßB (INHBB) subunits. Inhibins are biomarkers in a subset of cancers and utilize the coreceptors betaglycan (TGFBR3) and endoglin (ENG) for physiological or pathological outcomes. Given the array of prior reports on inhibin, activin and the coreceptors in cancer, this study aims to provide a comprehensive analysis, assessing their functional prognostic potential in cancer using a bioinformatics approach. We identify cancer cell lines and cancer types most dependent and impacted, which included p53 mutated breast and ovarian cancers and lung adenocarcinomas. Moreover, INHA itself was dependent on TGFBR3 and ENG/CD105 in multiple cancer types. INHA, INHBA, TGFBR3, and ENG also predicted patients' response to anthracycline and taxane therapy in luminal A breast cancers. We also obtained a gene signature model that could accurately classify 96.7% of the cases based on outcomes. Lastly, we cross-compared gene correlations revealing INHA dependency to TGFBR3 or ENG influencing different pathways themselves. These results suggest that inhibins are particularly important in a subset of cancers depending on the coreceptor TGFBR3 and ENG and are of substantial prognostic value, thereby warranting further investigation.

TGFß signaling networks in ovarian cancer progression and plasticity.

Epithelial ovarian cancer (EOC) is a leading cause of cancer-related death in women. Late-stage diagnosis with significant tumor burden, accompanied by recurrence and chemotherapy resistance, contributes to this poor prognosis. These morbidities are known to be tied to events associated with epithelial-mesenchymal transition (EMT) in cancer. During EMT, localized tumor cells alter their polarity, cell-cell junctions, cell-matrix interactions, acquire motility and invasiveness and an exaggerated potential for metastatic spread. Key triggers for EMT include the Transforming Growth Factor-ß (TGFß) family of growth factors which are actively produced by a wide array of cell types within a specific tumor and metastatic environment. Although TGFß can act as either a tumor suppressor or promoter in cancer, TGFß exhibits its pro-tumorigenic functions at least in part via EMT. TGFß regulates EMT both at the transcriptional and post-transcriptional levels as outlined here. Despite recent advances in TGFß based therapeutics, limited progress has been seen for ovarian cancers that are in much need of new therapeutic strategies. Here, we summarize and discuss several recent insights into the underlying signaling mechanisms of the TGFß isoforms in EMT in the unique metastatic environment of EOCs and the current therapeutic interventions that may be relevant.

Strength and duration of GIPC-dependent signaling networks as determinants in cancer.

GIPC is a PDZ-domain containing adaptor protein that regulates the cell surface expression and endocytic trafficking of numerous transmembrane receptors and signaling complexes. Interactions with over 50 proteins have been reported to date including VEGFR, insulin-like growth factor-1 receptor (IGF-1R), GPCRs, and APPL, many of which have essential roles in neuronal and cardiovascular development. In cancer, a major subset of GIPC-binding receptors and cytoplasmic effectors have been shown to promote tumorigenesis or metastatic progression, while other subsets have demonstrated strong tumor-suppressive effects. Given that these diverse pathways are widespread in normal tissues and human malignancies, precisely how these opposing signals are integrated and regulated within the same tumor setting likely depend on the strength and duration of their interactions with GIPC. This review highlights the major pathways and divergent mechanisms of GIPC signaling in various cancers and provide a rationale for emerging GIPC-targeted cancer therapies.

Context-dependent activation of SIRT3 is necessary for anchorage-independent survival and metastasis of ovarian cancer cells.

Tumor cells must alter their antioxidant capacity for maximal metastatic potential. Yet the antioxidant adaptations required for ovarian cancer transcoelomic metastasis, which is the passive dissemination of cells in the peritoneal cavity, remain largely unexplored. Somewhat contradicting the need for oxidant scavenging are previous observations that expression of SIRT3, a nutrient stress sensor and regulator of mitochondrial antioxidant defenses, is often suppressed in many primary tumors. We have discovered that this mitochondrial deacetylase is specifically upregulated in a context-dependent manner in cancer cells. SIRT3 activity and expression transiently increased following ovarian cancer cell detachment and in tumor cells derived from malignant ascites of high-grade serous adenocarcinoma patients. Mechanistically, SIRT3 prevents mitochondrial superoxide surges in detached cells by regulating the manganese superoxide dismutase (SOD2). This mitochondrial stress response is under dual regulation by SIRT3. SIRT3 rapidly increases SOD2 activity as an early adaptation to cellular detachment, which is followed by SIRT3-dependent increases in SOD2 mRNA during sustained anchorage-independence. In addition, SIRT3 inhibits glycolytic capacity in anchorage-independent cells thereby contributing to metabolic changes in response to detachment. While manipulation of SIRT3 expression has few deleterious effects on cancer cells in attached conditions, SIRT3 upregulation and SIRT3-mediated oxidant scavenging are required for anoikis resistance in vitro following matrix detachment, and both SIRT3 and SOD2 are necessary for colonization of the peritoneal cavity in vivo. Our results highlight the novel context-specific, pro-metastatic role of SIRT3 in ovarian cancer.

Chronic heart failure increases negative chronotropic effects of adenosine in canine sinoatrial cells via A1R stimulation and GIRK-mediated IKado.

AIMS: Bradycardia contributes to tachy-brady arrhythmias or sinus arrest during heart failure (HF). Sinoatrial node (SAN) adenosine A1 receptors (ADO A1Rs) are upregulated in HF, and adenosine is known to exert negative chronotropic effects on the SAN. Here, we investigated the role of A1R signaling at physiologically relevant ADO concentrations on HF SAN pacemaker cells. MAIN METHODS: Dogs with tachypacing-induced chronic HF and normal controls (CTL) were studied. SAN tissue was collected for A1R and GIRK mRNA quantification. SAN cells were isolated for perforated patch clamp recordings and firing rate (bpm), slope of slow diastolic depolarization (SDD), and maximum diastolic potential (MDP) were measured. Action potentials (APs) and currents were recorded before and after addition of 1 and 10 µM ADO. To assess contributions of A1R and G protein-coupled Inward Rectifier Potassium Current (GIRK) to ADO effects, APs were measured after the addition of DPCPX (selective A1R antagonist) or TPQ (selective GIRK blocker). KEY FINDINGS: A1R and GIRK mRNA expression were significantly increased in HF. In addition, ADO induced greater rate slowing and membrane hyperpolarization in HF vs CTL (p < 0.05). DPCPX prevented ADO-induced rate slowing in CTL and HF cells. The ADO-induced inward rectifying current, IKado, was observed significantly more frequently in HF than in CTL. TPQ prevented ADO-induced rate slowing in HF. SIGNIFICANCE: An increase in A1R and GIRK expression enhances IKAdo, causing hyperpolarization, and subsequent negative chronotropic effects in canine chronic HF at relevant [ADO]. GIRK blockade may be a useful strategy to mitigate bradycardia in HF.

Multifaceted roles of TAK1 signaling in cancer.

Context-specific signaling is a prevalent theme in cancer biology wherein individual molecules and pathways can have multiple or even opposite effects depending on the tumor type. TAK1 represents a particularly notable example of such signaling diversity in cancer progression. Originally discovered as a TGF-ß-activated kinase, over the years it has been shown to respond to numerous other stimuli to phosphorylate a wide range of downstream targets and elicit distinct cellular responses across cell and tissue types. Here we present a comprehensive review of TAK1 signaling and provide important therapeutic perspectives related to its function in different cancers.

Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes.

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, and led to microtubule stabilization. Our studies introduce insulin-stimulated microtubule stabilization and plus-end trailing of +TIPs as new modes of insulin action and reveal the likelihood that a network of microtubule-associated proteins synergize to coordinate insulin-regulated microtubule dynamics.