Myosin 5 is a ubiquitously expressed non-muscle-type F-actin-interacting motor protein. There are three distinct subclasses (myosin 5a, -b, and -c) in vertebrates, which are differentially expressed with overlapping functions (1
). These isoforms are dimeric molecules, containing two conserved motor domains followed by six repeats of an IQ motif and two tail domains which participate in cargo binding. Myosin 5a plays an important role in anterograde membrane trafficking in specific transport pathways, such as melanosomes (9
), neurotransmitter vesicles (34
), and insulin-containing vesicles (43
). In the current study, we report that myosin 5a/5b play a role in anterograde GLUT4 vesicle trafficking in 3T3-L1 adipocytes. Our data further show that myosin 5a is a direct target for insulin-directed Akt2 activity and that insulin-induced myosin 5a phosphorylation stimulates its ability to interact with F-actin, consistent with its role as an actin cytoskeletal-based motor protein facilitating GLUT4 exocytosis.
Although the functional regulation of myosin 5 is incompletely understood, two major domains are known to participate in the motor activity of myosin 5a. A series of repeat IQ motifs are located near the motor protein head domain, and these motifs bind to calmodulins and specific myosin light chains (36
). The myosin 5a tail domain is important for cargo binding and cellular localization, and overexpression of this tail domain can inhibit myosin 5a activity (45
). Interestingly, by searching the myosin sequence databases, we observed that the myosin 5a tail domain contains two Akt substrate sequence repeats (RXRXXpS/T) at Ser 1650 and Ser 1812. Here we used three distinct approaches to test whether myosin 5a is a direct substrate of Akt. First, we showed a modest insulin-stimulated gel mobility shift of myosin 5a (Fig. ). Second, we utilized a phospho-Akt substrate-specific antibody and clearly demonstrated insulin-stimulated phosphorylation of Ser 1650 in a PI 3-kinase- and Akt2-dependent manner. Third, we purified the myosin 5a C-terminal domain (MGT) and used a direct in vitro phosphorylation assay to demonstrate that Akt2 phosphorylates myosin 5a at Ser 1650. Further, insulin-induced phosphorylation of myosin 5a stimulated the actin binding activity of this protein, and this insulin effect was inhibited by expression of dominant-negative Akt, or siRNA-mediated Akt2 knockdown, as well as expression of dominant-negative mutant myosin 5a. Insulin stimulation also caused association of myosin 5a with GLUT4 vesicles and translocation of myosin 5a to the plasma membrane along with GLUT4 and IRAP. Consistent with the motor function of myosin 5a, we found that siRNA-induced depletion of myosin 5a, or expression of dominant-negative myosin 5a, inhibited insulin-stimulated glucose transport and GLUT4 translocation without affecting the early steps of insulin signaling. From these results, we conclude that insulin induces myosin 5a activation via Akt2-mediated phosphorylation of Ser 1650 in the tail domain. In turn, activated myosin 5a then functions as a motor protein facilitating anterograde translocation of GLUT4 through the actin cytoskeleton network.
Activation of insulin receptor is rapidly followed by docking of insulin receptor substrates and stimulation of PI 3-kinase. Akt and PKCλ are serine/threonine kinases activated downstream of PI 3-kinase, and both are mediators of major metabolic actions of insulin, such as glucose transport and GLUT4 translocation (38
). However, the signaling events downstream of these two kinases remain to be fully elucidated. Recently, a new Akt substrate, Akt substrate of 160 kDa (AS160), has been identified (19
). Upon insulin stimulation, phosphorylation of AS160 through Akt leads to inactivation of AS160 GAP activity, which releases retention of GLUT4, promoting translocation (7
). Evidence shows that Rab8A and Rab14 are targets of AS160 and may be involved in GLUT4 translocation in the perinuclear region (16
). That report suggested that AS160 serves as a modulator of basal GLUT4 trafficking. More recently, Gonzalez et al. reported that inhibition of Akt impaired GLUT4 exocytosis in AS160 knockdown adipocytes, suggesting that additional Akt substrates, other than AS160, are involved in insulin regulation of GLUT4 exocytosis (8
). They also showed that Akt activity was specifically required for GLUT4 exocytosis within the region 250 nm from the plasma membrane, where F-actin is located in juxtaposition to the plasma membrane. These data are fully consistent with our results showing that myosin 5a is a new Akt2 substrate involved in GLUT4 translocation along F-actin.
It has been well demonstrated in several systems that vesicular trafficking is observed along microtubule and actin cytoskeletal structures (10
). Indeed, in adipocytes, disruption of microtubules (11
) or F-actin (20
) resulted in marked inhibition of insulin-stimulated GLUT4 translocation and glucose uptake. Since the plus ends of microtubules do not connect directly to the plasma membrane, vesicle cargo has to be transferred from microtubules to F-actin structures in order to reach the cell surface. Although the understanding of vesicle transfer mechanisms between these two systems is limited, it has been reported that melanosomes can be transported to melanocyte dendrites by the microtubule-based motor protein KIF3 and that the subsequent movement of these vesicles, and their tethering at the cell membrane, is dependent on myosin 5 and F-actin (46
). Further support for this microtubule/actin cytoskeletal “handoff” model has been provided by colocalization and binding studies that showed the direct interaction between the myosin 5a and KIF3 tail domains (13
). Close cooperation between the kinesin/microtubule and myosin 5/F-actin systems has also been observed in melanophore transport (32
). In our previous study, we found that GLUT4 vesicles are localized to the perinuclear Golgi region in nonstimulated cells and, after insulin stimulation, are transported in an anterograde fashion along microtubules, and this is dependent on the microtubule-based kinesin motor protein KIF3 (14
). In the overall process of GLUT4 exocytosis, this would account for GLUT4 movement along microtubules towards the cell periphery after insulin stimulation. The potential transfer mechanism of GLUT4 vesicles from microtubules to the F-actin system has not been addressed, and based on the current results, we suggest that PI 3-kinase-dependent KIF3/microtubule and myosin 5a/F-actin cooperation could be a model to translocate GLUT4 vesicles from their intracellular perinuclear loci to the plasma membrane via microtubular and then F-actin structures, under the influence of insulin.
Previous reports have shown that another microtubule motor protein, KIF5B, and the actin-based motor protein, myosin 1c, are involved in insulin-induced GLUT4 translocation (2
). In this regard, KIF5B was wortmannin insensitive, suggesting that a PI 3-kinase-independent pathway(s) mediates insulin's effect on this protein. Additionally, myosin 1c is now thought to facilitate the fusion of exocytic GLUT4-containing vesicles with the adipocyte plasma membrane (3
). Time-lapse total internal reflection microscopy studies (25
) in rat primary adipocytes demonstrated that GLUT4 vesicles rapidly move along microtubules, periodically tethering to the plasma membrane in the basal state, and that insulin halted this traffic by enhancing the tethering step. It is possible that myosin 5a participates in this tethering step.
Akt and PKCλ are both PI 3-kinase-dependent serine/threonine kinases, and numerous studies have demonstrated that both PKCλ and Akt are necessary for insulin-induced glucose transport (14
). These findings raise the question as to how these two similar serine/threonine kinases both play important roles in the process of insulin signaling to glucose transport. The current study, coupled with our previous reports of PKCλ signaling (14
), provide a working model to help understand these phenomena. In a previous study, we reported that insulin stimulation of PKCλ mediates the activation of a kinesin family motor protein, KIF3, to facilitate anterograde movement of GLUT4 along microtubule structures. In the current study, we find that insulin-induced Akt2 activation stimulates the ability of another motor protein, myosin 5a, to mediate anterograde movement of GLUT4 by the actin cytoskeleton. This suggests that sequential cooperation of PKCλ/KIF3 and Akt2/myosin 5a could participate in the movement of GLUT4 cargo through the microtubule system and then on through the actin cytoskeleton, thus providing an explanation for the role of both of these serine/threonine kinases in the overall process of GLUT4 translocation. Additional supporting evidence for this idea is the subcellular localization of these two kinases. Thus, PKCλ has been colocalized with microtubules (32
), whereas Akt is colocalized with F-actin (39
). Taken together, these differences in subcellular localizations of Akt and PKCλ, combined with the biochemical and biological data in the current and previous studies, are consistent with the working model proposed above.
The PKCλ/KIF3/microtubule and Akt2/myosin 5a/actin systems are biochemically and structurally distinct. Nevertheless, GLUT4 vesicles must be recognized by both systems under the influence of insulin to complete the full process of GLUT4 translocation from its initial perinuclear localization to the cell surface. Although the mechanisms for this remain incompletely understood, we have provided some evidence for a possible linker protein between GLUT4 vesicles and both KIF3 and myosin 5a motor proteins. Thus, we have previously shown that insulin stimulates Rab4 activation in 3T3-L1 adipocytes and that Rab4 may serve as an adaptor, between KIF3 and GLUT4 vesicles, in relationship to microtubule-based movement (14
). In the current study, we show that, under the influence of insulin, myosin 5a binds to GLUT4-containing vesicles and translocates to the plasma membrane. In the insulin-stimulated state, Rab4 can also coassociate with myosin 5a. This Rab4 association with myosin 5a was decreased in Akt2 knockdown but not in PKCλ knockdown cells, suggesting that the insulin-stimulated interaction of myosin 5a and Rab4 is downstream of Akt2. Thus, Rab4 may provide a common link between GLUT4 vesicles and the microtubular and actin-based motor proteins facilitating the transfer of GLUT4 vesicles from microtubules to the F-actin system in the process of GLUT4 translocation.
In summary, these results provide evidence that the actin-based motor protein myosin 5a is a direct substrate for insulin-stimulated Akt2. In turn, Akt2-dependent phosphorylation of myosin 5a enhances its ability to interact with the actin cytoskeleton and GLUT4 vesicles, and depletion of myosin 5a inhibits glucose transport stimulation. As such, these novel results are consistent with the view that myosin 5a is a new regulator of GLUT4 translocation, providing an important and direct functional link between the insulin-directed PI 3-kinase-Akt2 signaling pathway and GLUT4 translocation. In addition, taken together with our previous findings on the connection between activation of PKCλ and the microtubule-based motor protein KIF3, it is suggested that PKCλ and Akt2 sequentially cooperate to translocate GLUT4 vesicles from the perinuclear pool to the cell surface through a sequential KIF3/microtubule and myosin 5a/F-actin mechanism.