The Rab family of small GTP binding proteins represents key components of vesicular trafficking, mediating a range of diverse functions such as vesicle formation, vesicle transport, organelle motility, vesicle docking and fusion, and exocytosis and endocytosis (43
). Different Rab proteins can participate in these processes depending on the specific functions of the protein involved and its unique subcellular localization. For example, Rab7 and Rab9 are located in late endosomes, participating in late endosome Golgi transport, while Rab11 is observed in recycling endosomes and participates in this process (26
). Thus, Rab family proteins can be key determinants of directional movement, regulation, and the identity of intracellular vesicles.
GLUT4 proteins reside in specific vesicular compartments in fat and skeletal muscle tissues, and GLUT4 vesicles contain a number of associated proteins, including Rab4, Rab5, Rab7, and Rab11, and it has been reported that Rab4 and Rab5 can be translocated to the plasma membrane in response to GTPγS treatment in 3T3-L1 adipocytes (27
). Several previous studies (6
), including some from our own laboratory (42
), have already demonstrated that Rab4 plays an important role in insulin-mediated GLUT4 exocytosis, whereas Rab5 is important for the endocytotic process of GLUT4 internalization (16
). Consistent with these previous studies, the present results confirm the necessity of Rab4 function for insulin-stimulated GLUT4 translocation by showing that overexpression of dominant-negative Rab4 inhibits the stimulatory effects of insulin on this process.
We also show that insulin stimulates Rab4 activation as well as the association of Rab4 with PKC-λ. Our data also support a role for PKC-λ in GLUT4 translocation and glucose transport in 3T3-L1 adipocytes. Thus, microinjection of anti-PKC-λ antibodies inhibited GLUT4 translocation (20
), whereas adenovirus-mediated expression of dominant-negative PKC-λ inhibited, and constitutively active PKC-λ stimulated, glucose transport activity. These results are consistent with several other studies in the literature showing that PKC-λ plays an important role in the insulin-induced GLUT4 translocation process (2
) and that insulin can stimulate the translocation of PKC-λ to GLUT4 vesicles (38
). On the other hand, Tsuru et al. presented evidence showing that interference with PKC-λ activity does not influence GLUT4 translocation (40
), and it seems that further studies will be necessary to reconcile these different results.
Microtubules provide an intracellular structure along which trafficking vesicles can move under the influence of a number of motor proteins (12
). Intracellular vesicle trafficking is dependent on a specific motor protein function which coordinates movement of cargo along the cellular cytoskeletal structure, and different classes of motor proteins demonstrate specific functions with regard to movement, velocity, and direction along the microtubules (13
). For example, it has been shown that kinesin II moves toward the plus end of microtubules, whereas dynein traffics toward the minus end (15
). A role for motor proteins in facilitating the exocytosis and endocytosis of GLUT4 has been described in previous reports (8
). Along these lines, we found that KIF3 (kinesin II in mice) is important for the insulin-stimulated exocytosis of GLUT4 vesicles to the cell surface. It has been shown that the KIF3 motor protein functions only in anterograde transport (14
), and this is fully consistent with our findings that microinjection of the anti-KAP3A or -KIF3B antibody inhibits exocytosis but does not affect the endocytosis of GLUT4 proteins.
Insulin stimulation of KIF3's microtubule binding activity proceeds through a process involving PI3-kinase-dependent activation of PKC-λ. Although the precise molecular mechanisms of these events are still unclear, it is interesting that protein phosphatase 2A can affect the dynein motor, suggesting that motor protein activity can be regulated by alterations in serine/threonine phosphorylation (20a
). It is possible that PKC-λ-mediated phosphorylation of one or more of the KIF3 subunits is important in these stimulatory events, and this formulation is consistent with the data showing that expression of DN-PKC-λ blocks insulin-stimulated activation of KIF3 as well as insulin-induced GLUT4 translocation.
A number of previous papers have evaluated the role of microtubules in GLUT4 translocation, and, while several of these have provided evidence demonstrating the necessity for microtubule function in GLUT4 exocytosis (10
), other studies have come to different conclusions (28
). For example, several studies have used nocodazole to depolymerize microtubules, and others have used different methods to disrupt microtubule function, all showing that an intact microtubule cytoskeletal system is necessary for GLUT4 translocation (10
). On the other hand, Molero et al. (28
) have presented evidence indicating that nocodazole inhibition of GLUT4 translocation is independent of microtubule-depolymerizing effects, whereas Shigematsu et al. (37
) found little effect of nocodazole on GLUT4 translocation. Although the present studies do not directly address this issue and, therefore, cannot shed light on these divergent results, given the apparent role of motor proteins in the GLUT4 exocytotic and endocytotic itinerary, it would be logical to propose some role for microtubules in these processes.
Various kinds of linker proteins (binding partners) which couple the motor proteins to their cargo have been described (1
), and previous studies of other systems have indicated that various Rab proteins can serve as motor protein binding partners to facilitate motor protein-cargo interactions (7
). Furthermore, Rab proteins, particularly Rab4 and Rab5, have been identified in GLUT4 vesicles (27
). Previous work from our laboratory has demonstrated the interaction of Rab5 with the motor protein dynein to mediate endocytosis of GLUT4 proteins (16
), and the present report presents data indicating that Rab4 interacts with KIF3 to effect exocytosis of GLUT4 vesicles. Since there are many studies showing various roles of Rab4 and kinesin, it is likely that, in addition to the GLUT4 translocation system, both Rab4 and KIF3 interact with other compartments in other systems or cell types to function in membrane cycling events. Based on these data, one can propose a model in which insulin leads to GTP loading and activation of Rab4 within GLUT4 vesicles, thereby enabling recognition of the GLUT4 vesicle by the motor protein kinesin. PI3-kinase-dependent activation of PKC-λ facilitates this process and may promote the activation of kinesin with respect to its microtubule binding and motility functions.
In earlier studies, we showed that Rab5 can interact with the motor protein dynein to facilitate retrograde movement, or endocytosis, of GLUT4 proteins and that insulin can attenuate this process (16
). The present study, showing that insulin stimulates the Rab4-KIF3 interaction, which facilitates the exocytotic movement of GLUT4 proteins, provides a balanced picture of exocytosis and endocytosis in which Rab proteins participate in the coupling of motor proteins to their cargo to facilitate GLUT4 translocation to and from the cell surface under the influence of insulin.