The signalling factor PI3K is a specific regulator of the clathrin-independent dynamin-dependent endocytosis of IL-2 receptors

Abstract

SummaryReceptor-mediated endocytosis is an essential process used by eukaryotic cells to internalise many molecules. Several clathrinindependent endocytic routes exist, but the molecular mechanism of each pathway remains to be uncovered. The present study focuses on a clathrin-independent dynamin-dependent pathway used by interleukin 2 receptors (IL-2R), essential players of the immune response. Ras-related C3 botulinum toxin substrate (Rac1) and its targets, the p21-activated kinases (Pak), are specific regulators of this pathway, acting on cortactin and actin polymerization. The present study reveals a dual and specific role of phosphatidylinositol 3-kinase (PI3K) in IL-2R endocytosis. Inhibition of the catalytic activity of PI3K strongly affects IL-2R endocytosis, in contrast to transferrin (Tf) uptake, a marker of the clathrin-mediated pathway. Moreover, Vav2, a GTPase exchange factor (GEF) induced upon PI3K activation, is specifically involved in IL-2R entry. The second action of PI3K is through its regulatory subunit, p85a, which binds to and recruits Rac1 during IL-2R internalisation. Indeed, the overexpression of a p85a mutant missing the Rac1 binding motif leads to the specific inhibition of IL-2R endocytosis. The inhibitory effect of this p85a mutant could be rescued by the overexpression of either Rac1 or the active form of Pak, indicating that p85a acts upstream of the Rac1-Pak cascade. Finally, biochemical and fluorescent microscopy techniques reveal an interaction between p85a, Rac1 and IL-2R that is enhanced by IL-2. In summary, our results indicate a key role of class I PI3K in IL-2R endocytosis that creates a link with IL-2 signalling.

Essential role of endocytosis for Interleukin-4 receptor mediated JAK/STAT signalling

“…In agreement with this pathway also acting in our HEK293T model, most cortical endosomes overlapped with immunostaining against the activated phosphorylated form of the RacGEF Vav2 (Vav2-pY172, 95.7±3.9%, mean±s.d., n=5 cells) ( Fig. 6A; Table S3), which has previously been implicated in IL-2R endocytosis (Basquin et al, 2013).…”

“…Given that the IL-2Rγ subunit is known to interact with regulators of the endocytosis machinery (Basquin et al, 2013), it is likely that cortical endosomes are increased in size relative to the endogenous situation. In addition, local re-recruitment of the actin-branching machinery to the overexpressed IL-2Rγ chains, as reflected by the presence of phosphorylated Vav and Arp2/3, might cause extended retention of the cortical endosomes in the actin cortex, which could account for the presence of Rab11 in the periphery.…”

“…Receptor internalization also involves the regulatory phosphoinositide 3-kinase (PI3K) subunit p85 and the large modular guanine-nucleotide-exchange factor (GEF) Vav2 (Basquin et al, 2013), although a role for these proteins in pathway activation has not been addressed.…”

Clathrin-Independent Pathways of Endocytosis

“…It is likely that in this domain, an actin-based machinery, involving cortactin and neuronal Wiskott-Aldrich syndrome protein (N-WASP) is assembled to facilitate IL-2R entry. This is regulated by input from a IL-2R-activated PI3P signaling cascade that in turn activates RhoA, and consequently Rac1 (Basquin et al 2013). Subsequently p21-activated kinase 1 (Pak1), a downstream target of Rac1, phosphorylates cortactin, promoting its interaction with N-WASP (Grassart et al 2008(Grassart et al , 2010.…”

Novel Roles of Focal Adhesion Kinase in Cytoplasmic Entry and Replication of Influenza A Viruses

“…FAK activation mediates actin cytoskeletal rearrangement to facilitate receptor and vesicular internalization (39). During receptor internalization, localization of cortactin to the periphery of the cell, where it associates with cortical actin, increases (40) and can be used as a marker to show whether cortical actin was disrupted or altered by FAK inhibitor I treatment. Therefore, we assessed the colocalization of cortactin and F-actin at the periphery of the cell (Fig.…”