Insulin increases glucose uptake into muscle by enhancing the surface recycling of GLUT4 transporters. In myoblasts, insulin signals bifurcate downstream of phosphatidylinositol 3-kinase into separate Akt and Rac/actin arms. Akt-mediated Rab-GAP AS160 phosphorylation and Rac/actin are required for net insulin gain of GLUT4, but the specific steps (vesicle recruitment, docking or fusion) regulated by Rac, actin dynamics, and AS160 target Rab8A are unknown. In L6 myoblasts expressing GLUT4myc, blocking vesicle fusion by tetanus toxin cleavage of VAMP2 impeded GLUT4myc membrane insertion without diminishing its build-up at the cell periphery. Conversely, actin disruption by dominant negative Rac or Latrunculin B abolished insulin-induced surface and submembrane GLUT4myc accumulation. Expression of non-phosphorylatable AS160 (AS160-4P) abrogated membrane insertion of GLUT4myc and partially reduced its cortical build-up, an effect magnified by selective Rab8A knockdown. We propose that insulin-induced actin dynamics participates in GLUT4myc vesicle retention beneath the membrane, whereas AS160 phosphorylation is essential for GLUT4myc vesicle-membrane docking/fusion and also contributes to GLUT4myc cortical availability through Rab8A.

Interactions between α11 integrins and the Smad-dependent TGF-β2 signalling may contribute to the formation of pro-fibrotic myofibroblasts and the development of a fibrotic interstitium in diabetic cardiomyopathy.

Insulin induces GLUT4 translocation to the muscle cell surface. Using differential amino acid labeling and mass spectrometry, we observed insulin-dependent co-precipitation of actinin-4 (ACTN4) with GLUT4 (Foster, L. J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L. M., Bilan, P. J., Mann, M., and Klip, A. (2006) J. Proteome Res. 5, 64 -75). ACTN4 links F-actin to membrane proteins, and actin dynamics are essential for GLUT4 translocation. We hypothesized that ACTN4 may contribute to insulin-regulated GLUT4 traffic. In L6 muscle cells insulin, but not platelet-derived growth factor, increased co-precipitation of ACTN4 with GLUT4. Small interfering RNA-mediated ACTN4 knockdown abolished the gain in surface-exposed GLUT4 elicited by insulin but not by platelet-derived growth factor, membrane depolarization, or mitochondrial uncoupling. In contrast, knockdown of ␣-actinin-1 (ACTN1) did not prevent GLUT4 translocation by insulin. GLUT4 colocalized with ACTN4 along the insulin-induced cortical actin mesh and ACTN4 knockdown prevented GLUT4-actin colocalization without impeding actin remodeling or Akt phosphorylation, maintaining GLUT4 in a tight perinuclear location. We propose that ACTN4 contributes to GLUT4 traffic, likely by tethering GLUT4 vesicles to the cortical actin cytoskeleton. Insulin-regulated glucose transporter 4 (GLUT4)4 is a member of the SLC2A facilitative glucose transporter family (2) and is responsible for glucose entry into muscle and fat tissues (3-5). GLUT4 continuously cycles to/from the cell membrane through a series of endosomal compartments. In response to insulin there is a rapid increase in the steady-state level of GLUT4 at the cell surface, at the expense of intracellular pools (6 -9). This process is defective in insulin resistance and type 2 diabetes (10 -12). Stimuli other than insulin such as muscle contraction, depolarization, or hypoxia also increase surface GLUT4 (13-16). Whereas insulin largely increases the exocytic arm of GLUT4 cycling (17), hypoxia or membrane depolarization preferentially reduce GLUT4 endocytosis in muscle cells (16,18,19). Moreover, although insulin-dependent GLUT4 translocation requires dynamic remodeling of filamentous actin (20 -23), the gain in surface GLUT4 elicited by plateletderived growth factor (PDGF), depolarization, or mitochondrial uncouplers is independent of actin dynamics (24 -26).Intensive research has recently focused on identifying the individual mechanisms participating in GLUT4 traffic and the specific events regulated by insulin (27)(28)(29)(30)(31). Hypothesizing that GLUT4 traffic may be regulated by interaction with partner proteins, it is of fundamental and clinical interest to identify such proteins. Accordingly, we recently applied the novel SILAC (stable isotope labeling by amino acids in cell culture) approach (32) to search for proteins that associate with GLUT4 in an insulin-regulated manner (1). The study took advantage of the stable expression in L6 muscle cells of GLUT4 encoding an myc tag that faces the extrace...

Interleukin-1 (IL-1) induces extracellular matrix degradation as a result of increased expression of matrix metalloproteinases (MMPs).Reversible phosphorylation of proteins on tyrosine residues is a pivotal, post-translational modification in many signal transduction pathways. The extent of these modifications is determined by the balance between the activities of proteintyrosine kinases and phosphatases (1, 2). Whereas proteintyrosine kinases are thought to regulate the amplitude of responses to extracellular signals, protein-tyrosine phosphatases (PTPs) 2 may determine the rate and duration of these responses (3). For IL-1 signaling in adherent cells, tyrosine phosphorylation of focal adhesion proteins such as the focal adhesion kinase is a critical, rate-limiting process (4). Tyrosine phosphorylated proteins, such as focal adhesion kinase, paxillin, and Src family kinases, which are enriched in focal adhesions (5), influence the assembly, maturation, and disassembly of these adhesive structures (6 -9) and also impact signaling through focal adhesions (10). The dynamic and reversible nature of tyrosine phosphorylation of focal adhesion proteins suggests an important role for protein-tyrosine kinases and PTPs in focal adhesion-dependent signaling (11). There are numerous PTPs in focal adhesions, and, in particular, SHP-2 is recruited to focal adhesions upon integrin engagement (12-14). In the absence of SHP-2, the number of focal adhesions and actin stress fibers increases, which is associated with diminished spreading and motility (15, 16). Expression of a dominant-negative SHP-2 enhances the formation of focal adhesions and stress fibers (17). The dynamics of focal adhesion assembly and IL-1-induced signaling pathways, including Ca 2ϩ release and ERK phosphorylation, are dependent on phosphorylation of Tyr 542 of 18,19), which can in turn affect the phosphatase activity of SHP-2 (20).We recently reported that another PTP, namely PTP␣, plays a prominent role in the regulation of focal adhesions during cell adhesion, spreading, and motility (21). PTP␣ is a receptor-like PTP that can activate Src family kinases (e.g. Src and Fyn) via dephosphorylation of an inhibitory C-terminal tyrosine residue (22)(23)(24)(25)(26)(27). We have also shown that through its interactions with Src, PTP␣ controls IL-1 induced phosphorylation of the inositol 1,4,5-phosphate receptor and consequently, Ca 2ϩ release (28). Further, PTP␣ regulates formation of focal adhesions in response to mechanical force, strengthens connections between integrins and the cytoskeleton, and modulates cytoskeletal reorganization in response to integrin ligation (26,27). In the absence of PTP␣, fibroblasts demonstrated reduced spreading, increased numbers of abnormal focal adhesions, decreased tyrosine phosphorylation of focal * This work was supported by grants from the Canadian Institutes of Health Research (MOP 84254; to C. A. M. and G. P. D.). This work was also supported in part by National Institutes of Health Grant HL090669 (to G. P. D.) and funds ...

The adhesion of cardiac fibroblasts to the glycated collagen interstitium in diabetics is associated with de novo expression of the α11 integrin, myofibroblast formation and cardiac fibrosis. We examined how methylglyoxal-glycated collagen regulates α11 integrin expression. In cardiac fibroblasts plated on glycated collagen but not glycated fibronectin, there was markedly increased α11 integrin and α-smooth muscle actin expression. Compared with native collagen, binding of purified α11β1 integrin to glycated collagen was reduced by >fourfold, which was consistent with reduced fibroblast attachment to glycated collagen. Glycated collagen strongly enhanced the expression of TGF-β2 but not TGF-β1 or TGF-β3. The increased expression of TGF-β2 was inhibited by triple helical collagen peptides that mimic the α11β1 integrin binding site on type I collagen. In cardiac fibroblasts transfected with α11 integrin luciferase promoter constructs, glycated collagen activated the α11 integrin promoter. Analysis of α11 integrin promoter truncation mutants showed a novel Smad2/3 binding site located between -809 and -1300 nt that was required for promoter activation. We conclude that glycated collagen in the cardiac interstitium triggers an autocrine TGF-β2 signaling pathway that stimulates α11 integrin expression through Smad2/3 binding elements in the α11 integrin promoter, which is important for myofibroblast formation and fibrosis.