Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiledcoil regions near the enzymatically active heads at the N terminus and the regulatory tails at the C terminus bring these globular elements in proximity and stabilize the folded conformation. However, it has remained a mystery how kinesin-1's microtubule-stimulated ATPase activity is regulated in this folded conformation. Here, we present evidence for a direct interaction between the kinesin-1 head and tail. We photochemically cross-linked heads and tails and produced an 8-Å cryoEM reconstruction of the cross-linked head-tail complex on microtubules. These data demonstrate that a conserved essential regulatory element in the kinesin-1 tail interacts directly and specifically with the enzymatically critical Switch I region of the head. This interaction suggests a mechanism for tail-mediated regulation of the ATPase activity of kinesin-1. In our structure, the tail makes simultaneous contacts with the kinesin-1 head and the microtubule, suggesting the tail may both regulate kinesin-1 in solution and hold it in a paused state with high ADP affinity on microtubules. The interaction of the Switch I region of the kinesin-1 head with the tail is strikingly similar to the interactions of small GTPases with their regulators, indicating that other kinesin motors may share similar regulatory mechanisms.cross-linking ͉ electron microscopy ͉ regulation ͉ switch

The Akt substrate AS160 (TCB1D4) regulates Glut4 exocytosis; shRNA knockdown of AS160 increases surface Glut4 in basal adipocytes. AS160 knockdown is only partially insulinmimetic; insulin further stimulates Glut4 translocation in these cells. Insulin regulates translocation as follows: 1) by releasing Glut4 from retention in a slowly cycling/noncycling storage pool, increasing the actively cycling Glut4 pool, and 2) by increasing the intrinsic rate constant for exocytosis of the actively cycling pool (k ex ). Kinetic studies were performed in 3T3-L1 adipocytes to measure the effects of AS160 knockdown on the rate constants of exocytosis (k ex ), endocytosis (k en ), and release from retention into the cycling pool. AS160 knockdown released Glut4 into the actively cycling pool without affecting k ex or k en . Insulin increased k ex in the knockdown cells, further increasing cell surface Glut4. Inhibition of phosphatidylinositol 3-kinase or Akt affected both k ex and release from retention in control cells but only k ex in AS160 knockdown cells. Glut4 vesicles accumulate in a primed pre-fusion pool in basal AS160 knockdown cells. Akt regulates the rate of exocytosis of the primed vesicles through an AS160-independent mechanism. Therefore, there is an additional Akt substrate that regulates the fusion of Glut4 vesicles that remain to be identified. Mathematical modeling was used to test the hypothesis that this substrate regulates vesicle priming (release from retention), whereas AS160 regulates the reverse step by stimulating GTP turnover of a Rab protein required for vesicle tethering/docking/fusion. Our analysis indicates that fusion of the primed vesicles with the plasma membrane is an additional non-Akt-dependent insulinregulated step.Glucose uptake in muscle and adipose tissue is rate-limited by the number of facilitative glucose transport proteins present in the plasma membrane (1). In adipocytes, the majority of glucose uptake occurs via the insulin-responsive glucose transporter 4 (Glut4). Insulin regulates glucose uptake by changing the steady state distribution of Glut4 from predominantly an intracellular, perinuclear localization to the plasma membrane, a process known as Glut4 translocation. Under basal conditions, less than 5% of the total Glut4 is found at the PM, 2 whereas after insulin stimulation 30 -50% is localized to the PM. In basal cells, most of the Glut4 is sequestered into a noncycling/slowly cycling pool in specialized Glut4 storage vesicles (GSVs). A small fraction of the Glut4 is found in a more rapidly cycling pool that is distributed between the PM and endosomal compartments. Insulin increases cell surface Glut4 by two main mechanisms as follows: insulin releases Glut4 from retention in the sequestered GSVs into the actively cycling pool (2-5), and insulin also increases the rate constant of exocytosis, resulting in a further increase in surface Glut4 (2-6).Although the trafficking pathways followed by Glut4 are well characterized, the mechanism of regulation of Glut4 trafficking by ...

Insulin regulates glucose uptake through effects on the trafficking of the glucose transporter Glut4. To investigate the degree of overlap between Glut4 and the general endocytic pathways, the kinetics of trafficking of Glut4 and the receptors for transferrin (Tf) and ␣ 2 -macroglobulin (␣-2-M; LRP-1) were compared using quantitative flow cytometric assays. Insulin increased the exocytic rate constant (k ex ) for both Glut4 and Tf. However, the k ex of Glut4 was 5-15 times slower than Tf in both basal and insulin-stimulated cells. The endocytic rate constant (k en ) of Glut4 was also five times slower than Tf. Insulin did not affect the k en of either protein. In basal cells, the k en for ␣-2-M/ LRP-1 was similar to Glut4 but 5-fold slower than Tf. Insulin increased k en for ␣-2-M/LRP-1 by 30%. In contrast, the k ex for LRP-1 was five times faster than Glut4 in basal cells, and insulin did not increase this rate constant. Thus, although there is overlap in the protein machineries/compartments utilized, the differences in trafficking kinetics indicate that Glut4, the Tf receptor, and LRP-1 are differentially processed both within the cell and at the plasma membrane. It has been reported that insulin decreases the k en of Glut4 in adipocytes. However, the effect of exocytosis on the "internalization" assays was not considered. Because it is counterintuitive, the effect of exocytosis on these assays is often overlooked in endocytosis studies. Using mathematical modeling and simulation, we show that the reported decrease in Glut4 k en can be entirely accounted for by the well established increase in Glut4 k ex .

Background: Cell surface levels of glucose transporter Glut4 are tightly controlled in adipocytes. Results: The effects of insulin and differentiation on the trafficking kinetics of Glut4, the transferrin receptor, and LRP1 were measured to identify regulatory steps. Conclusion: Six independent steps determine cell surface Glut4; insulin stimulates three of these. Significance: These results provide a framework for functionally mapping treatments/proteins that affect Glut4 translocation.

A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by smooth muscle myosin light chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM (up-SMM) from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73 ± 9), the ratio was ~ 23–37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at ~1 nM free [Ca2+]. There were two MLCK pools that bound unphosphorylated SMM with Kd ~10 μM and 0.2 μM and phosphorylated SMM with a Kd ~ 20 μM and 0.2 μM. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK-CaM was activated by Ca2+ and phosphorylation of SMM occurred at a pCa50 of 6.1 and Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK-CaM-SMM. Using motility assays, co-sedimentation assays, and on-coverslip ELISA assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.