While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3-kinase C2␤ (PI3K-C2␤) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2␤-AKT survival pathway. ITSN associated with PI3K-C2␤ on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2␤ activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2␤ and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.Intersectin (ITSN) is a modular scaffold with multiple protein interaction domains that is conserved among metazoa. At the amino terminus are two Eps15 homology (EH) domains that bind NPF motifs on proteins such as epsin (36). The EH domains are followed by a coiled-coil domain that enables ITSN to homo-and heterodimerize with proteins such as Eps15 (24). The carboxy terminus consists of five Src homology 3 (SH3) domains that interact with Pro-rich motifs on a variety of proteins, several of which are involved in regulating endocytosis. Indeed, a subset of ITSNЈs SH3 domains are potent inhibitors of clathrin-coated pit formation (26). Recent studies on the Drosophila melanogaster ortholog of ITSN, Dap160, indicate that this scaffold functions as a stabilizing or recruitment factor for components of the clathrin-coated pit (14, 17). The loss of Dap160 function results in fewer coated vesicles, as well as enlarged vesicles, indicating that ITSN functions in both the formation and maturation of endocytic vesicles. Consistent with this role in Drosophila, silencing ITSN expression in mammalian cells results in defects in epidermal growth factor receptor (EGFR) internalization (18).Although ITSN is clearly linked with endocytosis, increasing evidence suggests that ITSNЈs role within the cell is not limited to this process (1,10,18,19,(27)(28)(29)32). The cloning of mammalian ITSN revealed a longer, spliced product containing a guanine nucleotide exchange factor domain specific for Cdc42 (10, 27). Through the EH domains, ITSN activates a Jun Nterminal protein kinase-dependent pathway that cooper...

The ubiquitin-interacting motif (UIM) is a short peptide motif with the dual function of binding ubiquitin and promoting ubiquitylation. This motif is conserved throughout eukaryotes and is present in numerous proteins involved in a wide variety of cellular processes including endocytosis, protein trafficking, and signal transduction. We previously reported that the UIMs of epsin were both necessary and sufficient for its ubiquitylation. In this study, we found that many, but not all, UIM-containing proteins were ubiquitylated. When expressed as chimeric fusion proteins, most UIMs promoted ubiquitylation of the chimera. In contrast to previous studies, we found that UIMs do not exclusively promote monoubiquitylation but rather a mixture of mono-, multi-, and polyubiquitylation. However, UIMdependent polyubiquitylation does not lead to degradation of the modified protein. UIMs also bind polyubiquitin chains of varying lengths and to different degrees, and this activity is required for UIM-dependent ubiquitylation. Mutational analysis of the UIM revealed specific amino acids that are important for both polyubiquitin binding and ubiquitin conjugation. Finally we provide evidence that UIM-dependent ubiquitylation inhibits the interaction of UIM-containing proteins with other ubiquitylated cellular proteins. Our results suggest a new model for the ubiquitylation of UIM-containing proteins.The UIM 1 was first described as a peptide sequence consisting of a highly conserved ⌽-X-X-A-X-X-X-S-X-X-Ac core where ⌽ represents a hydrophobic residue and Ac is an acidic residue (1). It was identified based on the ubiquitin binding region of the RPN10 subunit of the 26 S proteasome (2, 3). The presence of UIMs in numerous proteins ranging from the MachadoJoseph disease protein (MJD1/ataxin3) to USP25, a member of the deubiquitylating enzyme family, suggests that this region is involved in regulating protein function. Indeed our previous studies and those of others have demonstrated an important role for UIMs in both ubiquitylation and in ubiquitin binding (2-11).Ubiquitylation is a post-translational modification resulting in the covalent attachment of ubiquitin through its COOHterminal Gly to the ⑀-NH 2 group of a Lys residue in a target protein. This process involves a multienzyme cascade that begins with the activation of ubiquitin in the presence of ATP and an E1 ubiquitin-activating enzyme. Subsequently the ubiquitin is transferred through a thiol-ester bond to a ubiquitin-conjugating enzyme (E2) and through the action of an E3 ubiquitin ligase is attached to the substrate by an isopeptide bond. Polyubiquitylation, the attachment of multimeric chains of ubiquitin, leads to the proteolytic destruction of proteins when Lys 48 of ubiquitin is the site of chain formation. However, ubiquitin chains formed through Lys 63 are not involved in protein degradation but rather a variety of processes including DNA repair, translation, IB kinase activation, endocytosis, and protein transport (for a review, see Ref. 12). In contrast to po...

The covalent attachment of ubiquitin to proteins is an evolutionarily conserved signal for rapid protein degradation. However, additional cellular functions for ubiquitination are now emerging, including regulation of protein trafficking and endocytosis. For example, recent genetic studies suggested a role for ubiquitination in regulating epsin, a modular endocytic adaptor protein that functions in the assembly of clathrin-coated vesicles; however, biochemical evidence for this notion has been lacking. Epsin consists of an epsin NH(2)-terminal homology (ENTH) domain that promotes the interaction with phospholipids, several AP2 binding sites, two clathrin binding sequences, and several Eps15 homology (EH) domain binding motifs. Interestingly, epsin also possesses several recently described ubiquitin-interacting motifs (UIMs) that have been postulated to bind ubiquitin. Here, we demonstrate that epsin is predominantly monoubiquitinated and resistant to proteasomal degradation. The UIMs are necessary for epsin ubiquitination but are not the site of ubiquitination. Finally, we demonstrate that the isolated UIMs from both epsin and an unrelated monoubiquitinated protein, Eps15, are sufficient to promote ubiquitination of a chimeric glutathione-S-transferase (GST)-UIM fusion protein. Thus, our data suggest that UIMs may serve as a general signal for ubiquitination.

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Osteoconductive materials play a critical role in promoting integration with surrounding bone tissue and resultant bone repair in vivo. However, the impact of 3D osteoconductive substrates coupled with soluble signals on progenitor cell differentiation is not clear. In this study, we investigated the influence of bone morphogenetic protein-2 (BMP-2) concentration on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) when seeded in carbonated apatite-coated polymer scaffolds. Mineralized scaffolds were more hydrophilic and adsorbed more BMP-2 compared to nonmineralized scaffolds. Changes in alkaline phosphatase (ALP) activity within stimulated hMSCs were dependent on the dose of BMP-2 and the scaffold composition. We detected more cell-secreted calcium on mineralized scaffolds at all time points, and higher BMP-2 concentrations resulted in increased ALP and calcium levels. RUNX2 and IBSP gene expression within hMSCs was affected by both substrate and soluble signals, SP7 by soluble factors, and SPARC by substrate-mediated cues. The present data indicate that a combination of apatite and BMP-2 do not simply enhance the osteogenic response of hMSCs, but act through multiple pathways that may be both substrate- and growth factor-mediated. Thus, multiple signaling strategies will likely be necessary to achieve optimal bone regeneration.

The ubiquitin proteasome system (UPS) actively controls protein dynamics and local abundance via regulated protein degradation. This study investigates UPS roles in the regulation of postsynaptic function and molecular composition in the Drosophila neuromuscular junction (NMJ) genetic system. To specifically impair UPS function postsynaptically, the UAS/GAL4 transgenic method was employed to drive postsynaptic expression of proteasome β2 and β6 subunit mutant proteins, which operate through a dominant negative mechanism to block proteasome function. When proteasome mutant subunits were constitutively expressed, excitatory junctional current (EJC) amplitudes were increased, demonstrating that postsynaptic proteasome function limits neurotransmission strength. Interestingly, the alteration in synaptic strength was calcium-dependent and miniature EJCs had significantly smaller mean amplitudes and more rapid mean decay rates. Postsynaptic levels of the Drosophila PSD-95/SAP97 homologue, discs large (DLG), and the GluRIIB-containing glutamate receptor were increased, but GluRIIA-containing receptors were unaltered. With acute postsynaptic proteasome inhibition using an inducible transgenic system, neurotransmission was similarly elevated with the same specific increase in postsynaptic GluRIIB abundance. These findings demonstrate postsynaptic proteasome regulation of glutamatergic synaptic function that is mediated through specific regulation of GluRIIB-containing glutamate receptors.

Introduction. The impact of interval (INT) vs. continuous (CONT) exercise training on endothelial function in relation to glucose metabolism prior to clinically meaningful weight loss is unknown in adults with prediabetes. Methods. Twenty-six subjects with prediabetes (60±1 y; 33±1 kg/m2; 2-hr-PG OGTT: 145±7 mg/dl) were randomized to 60 min of CONT (n=12; 70% of HRpeak) or work-matched INT exercise training (n=14; alternating 3 min at 90 and 50% HRpeak) for 2 weeks. Aerobic fitness (VO2peak) and body composition (bioelectrical impedance) were assessed before and after training. Flow-mediated dilation (FMD) was measured during a 2 h 75 g OGTT (0, 60, and 120 min) to assess endothelial function. Postprandial FMD was calculated as incremental area under the curve (iAUC). Glucose tolerance and insulin were also calculated by iAUC. Fasting plasma VCAM, ICAM, and hs-CRP were also assessed as indicators of vascular/systemic inflammation. Results. Both interventions increased VO2peak (P=0.002) but had no effect on body fat (P=0.20). Although both treatments improved glucose tolerance (P=0.06) and insulin iAUC (P=0.02), VCAM increased (P=0.01). There was no effect of either treatment on ICAM, hs-CRP, or fasting as well as postprandial FMD. However, 57% of people improved fasting and iAUC FMD following CONT compared with only 42% after INT exercise (each: P=0.04). Elevated VCAM was linked to blunted fasting FMD after training (r=−0.38, P=0.05). But, there was no correlation between fasting FMD or postprandial FMD with glucose tolerance (r=0.17, P=0.39 and r=0.02, P=0.90, respectively) or insulin iAUC following training (r=0.34, P=0.08 and r=0.04, P=0.83, respectively). Conclusion. Endothelial function is not improved consistently after short-term training, despite improvements in glucose and insulin responses to the OGTT in obese adults with prediabetes.

Adults with metabolic syndrome (MetS) have increased fasting arterial stiffness and altered central hemodynamics that contribute, partly, to increased cardiovascular disease (CVD) risk. Although insulin affects aortic wave reflections in healthy adults, the effects in individuals with MetS are unclear. We hypothesized that insulin stimulation would reduce measures of pressure waveforms and hemodynamics in people with MetS. Thirty-five adults with obesity (27F; 54.2 ± 6.0 yr; 37.1 ± 4.8 kg/m2) were selected for MetS (ATP III criteria) following an overnight fast. Pulse wave analysis was assessed using applanation tonometry before and after a 2hr euglycemic-hyperinsulinemic clamp (90 mg/dl, 40 mU/m2/min). Deconvolution analysis was used to decompose the aortic waveform (augmentation index corrected to heart rate of 75 bpm (AIx@75); augmentation pressure (AP)) into backward and forward pressure components. Aerobic fitness (VO2max), body composition (DXA), and blood biochemistries were also assessed. Insulin significantly reduced augmentation index (AIx@75, 28.0 ± 9.6 vs. 23.0 ± 9.9 %, P<0.01), augmentation pressure (14.8 ± 6.4 vs. 12.0 ± 5.7 mmHg, P<0.01), pulse pressure amplification (1.26 ± 0.01 vs. 0.03 ± 0.01, P=0.01), and inflammation (hsCRP: P=0.02; MMP-7: P=0.03) compared to fasting. In subgroup analyses to understand HTN influence, there were no insulin stimulation differences on any outcome. VO2max, visceral fat, and blood potassium correlated with fasting AIx@75 (r=-0.39, P=0.02; r=0.41, P=0.03; r=-0.53, P=0.002). Potassium levels were also associated with insulin-mediated reductions in AP (r=0.52, P=0.002). Our results suggest insulin stimulation improves indices of aortic reflection in adults with MetS.