Phosphoinositides (PIPs) are key regulators of membrane traffic and signaling. The interconversion of PIPs by lipid kinases and phosphatases regulates their functionality. Phosphatidylinositol (PI) and PIPs have a unique enrichment of 1-stearoyl-2-arachidonyl acyl species; however, the regulation and function of this specific acyl profile remains poorly understood. We examined the role of the PI acyltransferase LYCAT in control of PIPs and PIPdependent membrane traffic. LYCAT silencing selectively perturbed the levels and localization of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ] and phosphatidylinositol-3-phosphate and the membrane traffic dependent on these specific PIPs but was without effect on phosphatidylinositol-4-phosphate or biosynthetic membrane traffic. The acyl profile of PI(4,5)P 2 was selectively altered in LYCAT-deficient cells, whereas LYCAT localized with phosphatidylinositol synthase. We propose that LYCAT remodels the acyl chains of PI, which is then channeled into PI(4,5)P 2 . Our observations suggest that the PIP acyl chain profile may exert broad control of cell physiology. INTRODUCTIONPhosphoinositides (PIPs) control many facets of cell physiology, such as nutrient uptake, receptor signaling, and cell adhesion by control of specific stages of membrane traffic (Di Paolo and De Camilli, 2006;Krauss and Haucke, 2007). Through the action of lipid kinases and phosphatases, PIPs can be interconverted into seven different species defined by phosphorylation of the inositol head group (Balla, 2013). Each of the seven PIPs exhibits unique enrichment within membrane compartments and helps to recruit a variety of cognate effector proteins. Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) and phosphatidylinositol-3-phosphate (PI(3)P) illustrate these concepts well.PI(4,5)P 2 predominates within the plasma membrane (PM) and regulates clathrin-mediated endocytosis (referred to here as endocytosis) to control the internalization of cell surface proteins such as transferrin (Tfn) receptor (TfR;Jost et al., 1998;Varnai et al., 2006;Zoncu et al., 2007;Posor et al., 2013). PI(4,5)P 2 binds to and recruits AP2 and other proteins, which, together with cargo molecules and clathrin, initiate the formation and assembly of clathrin-coated pits (CCPs; Gaidarov and Keen, 1999;Itoh et al., 2001;Jackson et al., 2010). CCPs couple cargo selection to membrane invagination and eventually undergo scission from the PM by the GTPase dynamin2 to yield endocytic vesicles (Conner and Schmid, 2003;McMahon and Boucrot, 2011). CCPs harbor lipid phosphatases such as synaptojanins and OCRL that mediate PI(4,5)P 2 turnover to control the efficiency of vesicle formation (Antonescu et al., 2011) and, afterMonitoring Editor

The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers.RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes.RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.

Clathrin-mediated endocytosis controls internalization of many receptors from the cell surface. It was found that clathrin-mediated endocytosis of epidermal growth factor receptor (EGFR) but not that of transferrin receptor requires EGFR-activated phospholipase Cγ1, Ca2+ signals, and protein kinase C. Thus EGFR elicits Ca2+ signals to distinctly regulate its own clathrin-mediated endocytosis.

The RET receptor tyrosine kinase is implicated in normal development and cancer. RET is expressed as two isoforms, RET9 and RET51, with unique C-terminal tail sequences that recruit distinct protein complexes to mediate signals. Upon activation, RET isoforms are internalized with distinct kinetics, suggesting differences in regulation. Here, we demonstrate that RET9 and RET51 differ in their abilities to recruit E3 ubiquitin ligases to their unique C-termini. RET51, but not RET9, interacts with, and is ubiquitylated by CBL, which is recruited through interactions with the GRB2 adaptor protein. RET51 internalization was not affected by CBL knockout but was delayed in GRB2-depleted cells. In contrast, RET9 ubiquitylation requires phosphorylation-dependent changes in accessibility of key RET9 C-terminal binding motifs that facilitate interactions with multiple adaptor proteins, including GRB10 and SHANK2, to recruit the NEDD4 ubiquitin ligase. We showed that NEDD4-mediated ubiquitylation is required for RET9 localization to clathrin-coated pits and subsequent internalization. Our data establish differences in the mechanisms of RET9 and RET51 ubiquitylation and internalization that may influence the strength and duration of RET isoform signals and cellular outputs.This article has an associated First Person interview with the first authors of the paper.

<p>Phosphoinositides (PIPs) are key regulators of membrane traffic and signaling. The interconversion of PIPs by lipid kinases and phosphatases regulates their functionality. Phosphatidylinositol (PI) and PIPs have a unique enrichment of 1-stearoyl-2-arachidonyl acyl species; however, the regulation and function of this specific acyl profile remains poorly understood. We examined the role of the PI acyltransferase LYCAT in control of PIPs and PIP-dependent membrane traffic. LYCAT silencing selectively perturbed the levels and localization of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-3-phosphate and the membrane traffic dependent on these specific PIPs but was without effect on phosphatidylinositol-4-phosphate or biosynthetic membrane traffic. The acyl profile of PI(4,5)P2 was selectively altered in LYCAT-deficient cells, whereas LYCAT localized with phosphatidylinositol synthase. We propose that LYCAT remodels the acyl chains of PI, which is then channeled into PI(4,5)P2 Our observations suggest that the PIP acyl chain profile may exert broad control of cell physiology.</p>