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We explore mechanisms that enable cancer cells to tolerate PI3K or Akt inhibitors. Prolonged treatment of breast cancer cells with PI3K or Akt inhibitors leads to increased expression and activation of a kinase termed SGK3 that is related to Akt. Under these conditions, SGK3 is controlled by hVps34 that generates PtdIns(3)P, which binds to the PX domain of SGK3 promoting phosphorylation and activation by its upstream PDK1 activator. Furthermore, under conditions of prolonged PI3K/Akt pathway inhibition, SGK3 substitutes for Akt by phosphorylating TSC2 to activate mTORC1. We characterise 14h, a compound that inhibits both SGK3 activity and activation in vivo, and show that a combination of Akt and SGK inhibitors induced marked regression of BT‐474 breast cancer cell‐derived tumours in a xenograft model. Finally, we present the kinome‐wide analysis of mRNA expression dynamics induced by PI3K/Akt inhibition. Our findings highlight the importance of the hVps34‐SGK3 pathway and suggest it represents a mechanism to counteract inhibition of PI3K/Akt signalling. The data support the potential of targeting both Akt and SGK as a cancer therapeutic.

Sorting nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting nexin 9 (SNX9) is involved in the late stages of clathrin-mediated endocytosis in non-neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane-remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro. Elucidation of the protein superdomain structure, together with mutational analysis and biochemical and cell biological experiments, demonstrated how the SNX9 PX and BAR domains work in concert in targeting and tubulation of phosphoinositide-containing membranes. The study provides insights into the SNX9-induced membrane modulation mechanism.

KIF16B is a newly identified kinesin that regulates the intracellular motility of early endosomes. KIF16B is unique among kinesins in that its cargo binding is mediated primarily by the strong interaction of its PX domain with endosomal lipids. To elucidate the structural basis of this unique endosomal anchoring activity of KIF16B-PX, we determined the crystal structure of the PX domain and performed in vitro and cellular membrane binding measurements for KIF16B-PX and mutants. The most salient structural feature of KIF16B-PX is that two neighboring residues, L1248 and F1249, on the membrane-binding surface form a protruding hydrophobic stalk with a large solvent-accessible surface area. This unique structure, arising from the complementary stacking of the two side chains and the local conformation, allows strong hydrophobic membrane interactions and endosome tethering. The presence of similar hydrophobic pairs in the amino-acid sequences of other membrane-binding domains and proteins suggests that the same structural motif may be shared by other membrane-binding proteins, whose physiological functions depend on strong hydrophobic membrane interactions.

Insulin stimulates glucose uptake in fat and muscle cells via the translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the cell surface. The signaling pathways linking the insulin receptor to GLUT4 translocation in adipocytes involve activation of the Rho family GTPases TC10alpha and beta. We report here the identification of TCGAP, a potential effector for Rho family GTPases. TCGAP consists of N-terminal PX and SH3 domains, a central Rho GAP domain and multiple proline-rich regions in the C-terminus. TCGAP specifically interacts with cdc42 and TC10beta through its GAP domain. Although it has GAP activity in vitro, TCGAP is not active as a GAP in intact cells. TCGAP translocates to the plasma membrane in response to insulin in adipocytes. The N-terminal PX domain interacts specifically with phos phatidylinositol-(4,5)-bisphosphate. Overexpression of the full-length and C-terminal fragments of TCGAP inhibits insulin-stimulated glucose uptake and GLUT4 translocation. Thus, TCGAP may act as a downstream effector of TC10 in the regulation of insulin-stimulated glucose transport.

p47(phox) is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X-ray structure of the p47(phox) PX domain revealed two distinct basic pockets on the membrane-binding surface, each occupied by a sulfate. These two pockets have different specificities: one preferentially binds phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and is analogous to the phophatidylinositol 3-phosphate (PtdIns3P)-binding pocket of p40(phox), while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH. Simultaneous occupancy of the two phospholipid-binding pockets radically increases membrane affinity. Strikingly, measurements for full-length p47(phox) show that membrane interaction by the PX domain is masked by an intramolecular association with the C-terminal SH3 domain (C-SH3). Either a site-specific mutation in C-SH3 (W263R) or a mimic of the phosphorylated form of p47(phox) [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater affinity than the isolated PX domain.

Sorting nexins (SNXs) comprise a family of proteins characterized by the presence of a phox-homology domain, which mediates the association of these proteins with phosphoinositides and recruits them to specific membranes or vesicular structures within cells. Although only limited information about SNXs and their functions is available, they seem to be involved in membrane trafficking and sorting processes by directly binding to target proteins such as certain growth factor receptors. We show that SNX17 binds to the intracellular domain of some members of the low-density lipoprotein receptor (LDLR) family such as LDLR, VLDLR, ApoER2 and LDLR-related protein. SNX17 resides on distinct vesicular structures partially overlapping with endosomal compartments characterized by the presence of EEA1 and rab4. Using rhodamine-labeled LDL, it was possible to demonstrate that during endocytosis, LDL passes through SNX17-positive compartments. Functional studies on the LDLR pathway showed that SNX17 enhances the endocytosis rate of this receptor. Our results identify SNX17 as a novel adaptor protein for LDLR family members and define a novel mechanism for modulation of their endocytic activity.

We describe a method for identifying tyrosine kinase substrates using anti-phosphotyrosine antibodies to screen tyrosine-phosphorylated cDNA expression libraries. Several potential Src substrates were identified including Fish, which has five SH3 domains and a recently discovered phox homology (PX) domain. Fish is tyrosine-phosphorylated in Src-transformed fibroblasts (suggesting that it is a target of Src in vivo) and in normal cells following treatment with several growth factors. Treatment of cells with cytochalasin D also resulted in rapid tyrosine phosphorylation of Fish, concomitant with activation of Src. These data suggest that Fish is involved in signalling by tyrosine kinases, and imply a specialized role in the actin cytoskeleton.

The superoxide‐producing phagocyte NADPH oxidase is activated during phagocytosis to destroy ingested microbes. The adaptor protein p40 phox associates via the PB1 domain with the essential oxidase activator p67 phox , and is considered to function by recruiting p67 phox to phagosomes; in this process, the PX domain of p40 phox binds to phosphatidylinositol 3‐phosphate [PtdIns(3)P], a lipid abundant in the phagosomal membrane. Here we show that the PtdIns(3)P‐binding activity of p40 phox is normally inhibited by the PB1 domain both in vivo and in vitro . The crystal structure of the full‐length p40 phox reveals that the inhibition is mediated via intramolecular interaction between the PB1 and PX domains. The interface of the p40 phox PB1 domain for the PX domain localizes on the opposite side of that for the p67 phox PB1 domain, and thus the PB1‐mediated PX regulation occurs without preventing the PB1–PB1 association with p67 phox .