In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (∼60–100%) and humans (∼40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20–58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.

Glucose transporter 4 (GLUT4) is responsible for the uptake of glucose into muscle and adipose tissues. Under resting conditions, GLUT4 is dynamically retained through idle cycling among selective intracellular compartments, from whence it undergoes slow recycling to the plasma membrane (PM). This dynamic retention can be released by command from intracellular signals elicited by insulin and other stimuli, which result in 2-10-fold increases in the surface level of GLUT4. Insulin-derived signals promote translocation of GLUT4 to the PM from a specialized compartment termed GLUT4 storage vesicles (GSV). Much effort has been devoted to the characterization of the intracellular compartments and dynamics of GLUT4 cycling and to the signals by which GLUT4 is sorted into, and recruited from, GSV. This review summarizes our understanding of intracellular GLUT4 traffic during its internalization from the membrane, its slow, constitutive recycling, and its regulated exocytosis in response to insulin. In spite of specific differences in GLUT4 dynamic behavior in adipose and muscle cells, the generalities of its endocytic and exocytic itineraries are consistent and an array of regulatory proteins that regulate each vesicular traffic event emerges from these cell systems.

Background The epidermal growth factor (EGF) stimulates rapid tyrosine phosphorylation of EGF-receptor (EGFR). This event precedes signalling from both the plasma membrane and from endosomes, and it is essential for recruitment of an ubiquitin ligase, CBL, that sorts activated receptors to endosomes and degradation. Because hyper-phosphorylation of EGFR is involved in oncogenic pathways, we performed an unbiased screen of siRNA oilgonucleotides targeting all human tyrosine phosphatases. Results We report the identification of PTPRK and PTPRJ (DEP-1) as EGFR-targeting phosphatases. DEP-1 is a tumour suppressor that dephosphorylates, thereby stabilizes EGFR by hampering its ability to associate with the CBL-GRB2 ubiquitin ligase complex. DEP-1 silencing enhanced tyrosine phosphorylation of endosomal EGFRs and, accordingly, increased cell proliferation. In line with functional interactions, EGFR and DEP-1 form physical associations, and EGFR phosphorylates a substrtae trapping mutant of DEP-1. Interestingly, the interactions of DEP-1 and EGFR are followed by physical segregation: whereas EGFR undergoes endocytosis, DEP-1 remains confined to the cell surface. Conclusions EGFR and DEP-1 physically interact at the cell surface and maitain bidirectional enzyme-substrate interactions, which are relevant to their respective oncogenic and tumor suppressive functions. These observations highlight the emerging roles of vesicular trafficking in malignant processes.

The armadillo-family protein, p120 catenin (p120), binds to the juxtamembrane domain of classical cadherins and increases cellcell junction stability. Overexpression of p120 modulates the activity of Rho family GTPases and augments cell migratory ability. Here we show that down-regulation of p120 in epithelial MCF-7 cells by siRNA leads to a striking decrease in lamellipodial persistence and focal adhesion formation. Similar alterations in lamellipodial activity were observed in MCF-7 cells treated with siRNA to cortactin, an activator of Arp2/3-dependent actin polymerization. We found that, in many cell types, p120 is colocalized with cortactin-containing actin structures not only at cell-cell junctions, but also at extrajunctional sites including membrane ruffles and actin-rich halos around endocytotic vesicles. p120 depletion led to dramatic loss of cortactin and its partner, Arp3, from the cell leading edges. Cortactin and p120 are shown to directly interact with each other via the cortactin N-terminal region. We propose that the mechanism underlying p120 functions at the leading edge involves its cooperation with cortactin.

Insulin reduces the velocity of mobile Myo1c-positive GLUT4 vesicles beneath the muscle cell plasma membrane as visualized by total internal reflection fluorescence microscopy. Binding of vesicle-bound Myo1c to actin filaments underlies Myo1c's participation in GLUT4 vesicle tethering for subsequent productive docking and fusion of GLUT4 vesicles with the plasma membrane.