Regulation of Src trafficking and activation by the endocytic regulatory proteins MICAL-L1 and EHD1

Abstract

Localization of the non-receptor tyrosine kinase Src to the cell periphery is required for its activation and to mediate focal adhesion turnover, cell spreading and migration. Inactive Src localizes to a perinuclear compartment and the movement of Src to the plasma membrane is mediated by endocytic transport. However, the precise pathways and regulatory proteins that are responsible for SRC transport are incompletely understood. Here, we demonstrate that Src partially colocalizes with the endocytic regulatory protein MICAL-L1 (molecule interacting with CasL-like protein 1) in mammalian cells. Furthermore, MICAL-L1 is required for growthfactor-and integrin-induced Src activation and transport to the cell periphery in HeLa cells and human fibroblasts. Accordingly, MICAL-L1 depletion impairs focal adhesion turnover, cell spreading and cell migration. Interestingly, we find that the MICAL-L1 interaction partner EHD1 (EH domain-containing protein 1) is also required for Src activation and transport. Moreover, the MICAL-L1-mediated recruitment of EHD1 to Src-containing recycling endosomes is required for the release of Src from the perinuclear endocytic recycling compartment in response to growth factor stimulation. Our study sheds new light on the mechanism by which Src is transported to the plasma membrane and activated, and provides a new function for MICAL-L1 and EHD1 in the regulation of intracellular non-receptor tyrosine kinases.

Dissecting the cell to nucleus, perinucleus and cytosol

“…It is well known that healthy people have cells with oncogenic K-Ras in different organs at rates far exceeding the rates of cancer development, and Magliano and Lonsdon (2013) report that the presence of oncogenic KRAS is not sufficient to transform cells, but the mechanism that removes barriers to tumorigenesis, is unknown48. Therefore, the perinuclear region as a periphery of the core nucleus may serve as a restriction site for potentially oncogenic proteins such as Ras or as was shown recently for Src8 or for the regulation of tumor suppressor proteins such as p53. In these experiments Src was localized exclusively in the perinuclear region.…”

Recycling endosomes

“…Tubulation of the Rab8a-dependent recycling system, which is responsible for recycling of non-clathrin-dependent endocytosed cargoes, such as MHC Class I, is regulated by MICAL-L1 [7,8]. MICAL-L1 recruits both Arf6 and EHD1 to recycling membranes and promotes tubulation [7,9,10 • ,11]. The association of MICAL-L1 with tubular endosomes is regulated by Rab35 [8,12].…”

Endocytosis and the Src family of non-receptor tyrosine kinases

“…Recently, we demonstrated that endogenous Src decorates MICAL-L1 tubules in HeLa cells, suggesting that Src is either a cargo or regulator of MICAL-L1-decorated TRE(54). Given that inactive Src overlaps in its localization with transferrin receptor, a bona-fide cargo protein of the ERC, this suggests that Src is likely a cargo.…”

“…This coincides with an increase in Src activation as measured by Src pY416, in agreement with previous findings(44). MICAL-L1- or EHD1-depletion caused Src to remain in the ERC following EGF treatment and also severely impaired Src activation(54). MICAL-L1-depletion in human fibroblasts decreased PDGF- and integrin-induced Src activation and also impaired Src-dependent processes such as PDGF-induced macropinocytosis, focal adhesion turnover, cell spreading and migration(54).…”

“…MICAL-L1- or EHD1-depletion caused Src to remain in the ERC following EGF treatment and also severely impaired Src activation(54). MICAL-L1-depletion in human fibroblasts decreased PDGF- and integrin-induced Src activation and also impaired Src-dependent processes such as PDGF-induced macropinocytosis, focal adhesion turnover, cell spreading and migration(54). Collectively, our data provide support for the idea that Src is an ERC cargo protein and that its localization, activation, and function are regulated by MICALL1 and EHD1.…”