Abstract
The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms.
Highlights
Introduction c-Src is a non-receptor tyrosine kinase that has been implicated in pathways regulating angiogenesis, invasion and metastasis, cell migration, endocytosis, and many others [1,2,3,4,5,6]
We determined a mechanism whereby MTs and actin combine their efforts for efficient trafficking of c-Src-associated endosomes; we provide evidence of tight coordination between these mechanisms, whereby GEF-H1-dependent RhoB activation serves as a switch
As Scar1/Wave1 has been implicated in as important for mediating actin polymerization at c-Src vesicles, we examined its role in our system by utilizing an inhibitor of Arp2/3, CK-666
Summary
Src is a non-receptor tyrosine kinase that has been implicated in pathways regulating angiogenesis, invasion and metastasis, cell migration, endocytosis, and many others [1,2,3,4,5,6]. Activation of c-Src is associated with its translocation to the plasma membrane where c-Src interacts with a number of important effectors [7]. The subcellular localization of c-Src is critical to its function. Studies have indicated that inactive c-Src can be localized to the perinuclear region, colocalizing with endosomal and Golgi markers [8], and upon activation, it can be subsequently targeted to the cell periphery [9]. At present, the mechanism by which c-Src is localized.
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