IQGAP1 binds AMPK and is required for maximum AMPK activation.

AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.

Tyrosine phosphorylation of the scaffold protein IQGAP1 in the MET pathway alters function.

IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.

Ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the Rho GTPase CDC42.

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475, and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.

Ca2+-Dependent Switch of Calmodulin Interaction Mode with Tandem IQ Motifs in the Scaffolding Protein IQGAP1.

IQ domain GTPase-activating scaffolding protein 1 (IQGAP1) mediates cytoskeleton, cell migration, proliferation, and apoptosis events. Calmodulin (CaM) modulates IQGAP1 functions by binding to its four tandem IQ motifs. Exactly how CaM binds the IQ motifs and which functions of IQGAP1 CaM regulates and how are fundamental mechanistic questions. We combine experimental pull-down assays, mutational data, and molecular dynamics simulations to understand the IQ-CaM complexes with and without Ca2+ at the atomic level. Apo-CaM favors the IQ3 and IQ4 motifs but not the IQ1 and IQ2 motifs that lack two hydrophobic residues for interactions with apo-CaM's hydrophobic pocket. Ca2+-CaM binds all four IQ motifs, with both N- and C-lobes tightly wrapped around each motif. Ca2+ promotes IQ-CaM interactions and increases the amount of IQGAP1-loaded CaM for IQGAP1-mediated signaling. Collectively, we describe IQ-CaM binding in atomistic detail and feature the emergence of Ca2+ as a key modulator of the CaM-IQGAP1 interactions.

LAPTM4B is a PtdIns(4,5)P2 effector that regulates EGFR signaling, lysosomal sorting, and degradation.

Lysosomal degradation is essential for the termination of EGF-stimulated EGF receptor (EGFR) signaling. This requires EGFR sorting to the intraluminal vesicles (ILVs) of multi-vesicular endosomes (MVEs). Cytosolic proteins including the ESCRT machineries are key regulators of EGFR intraluminal sorting, but roles for endosomal transmembrane proteins in receptor sorting are poorly defined. Here, we show that LAPTM4B, an endosomal transmembrane oncoprotein, inhibits EGF-induced EGFR intraluminal sorting and lysosomal degradation, leading to enhanced and prolonged EGFR signaling. LAPTM4B blocks EGFR sorting by promoting ubiquitination of Hrs (an ESCRT-0 subunit), which inhibits the Hrs association with ubiquitinated EGFR. This is counteracted by the endosomal PIP kinase, PIPKIγi5, which directly binds LAPTM4B and neutralizes the inhibitory function of LAPTM4B in EGFR sorting by generating PtdIns(4,5)P2 and recruiting SNX5. PtdIns(4,5)P2 and SNX5 function together to protect Hrs from ubiquitination, thereby promoting EGFR intraluminal sorting. These results reveal an essential layer of EGFR trafficking regulated by LAPTM4B, PtdIns(4,5)P2 signaling, and the ESCRT complex and define a mechanism by which the oncoprotein LAPTM4B can transform cells and promote tumor progression.

IQGAP1 binds the Axl receptor kinase and inhibits its signaling.

Axl is a tyrosine kinase receptor that is important for hematopoiesis, the innate immune response, platelet aggregation, engulfment of apoptotic cells and cell survival. Binding of growth arrest-specific protein 6 (Gas6) activates Axl signaling, but the mechanism of inactivation of the Axl receptor is poorly understood. In the present study, we show that IQGAP1 modulates Axl signaling. IQGAP1 is a scaffold protein that integrates cell signaling pathways by binding several growth factor receptors and intracellular signaling molecules. Our in vitro analysis revealed a direct interaction between the IQ domain of IQGAP1 and Axl. Analysis by both immunoprecipitation and proximity ligation assays demonstrated an association between Axl and IQGAP1 in cells and this interaction was decreased by Gas6. Unexpectedly, reducing IQGAP1 levels in cells significantly enhanced the ability of Gas6 to stimulate both Axl phosphorylation and activation of Akt. Moreover, IQGAP1 regulates the interaction of Axl with the epidermal growth factor receptor. Our data identify IQGAP1 as a previously undescribed suppressor of Axl and provide insight into regulation of Axl function.

Calmodulin Lobes Facilitate Dimerization and Activation of Estrogen Receptor-α.

Estrogen receptor α (ER-α) is a nuclear hormone receptor that controls selected genes, thereby regulating proliferation and differentiation of target tissues, such as breast. Gene expression controlled by ER-α is modulated by Ca2+ via calmodulin (CaM). Here we present the NMR structure of Ca2+-CaM bound to two molecules of ER-α (residues 287-305). The two lobes of CaM bind to the same site on two separate ER-α molecules (residues 292, 296, 299, 302, and 303), which explains why CaM binds two molecules of ER-α in a 1:2 complex and stabilizes ER-α dimerization. Exposed glutamate residues in CaM (Glu-11, Glu-14, Glu-84, and Glu-87) form salt bridges with key lysine residues in ER-α (Lys-299, Lys-302, and Lys-303), which is likely to prevent ubiquitination at these sites and inhibit degradation of ER-α. Transfection of cells with full-length CaM slightly increased the ability of estrogen to enhance transcriptional activation by ER-α of endogenous estrogen-responsive genes. By contrast, expression of either the N- or C-lobe of CaM abrogated estrogen-stimulated transcription of the estrogen responsive genes pS2 and progesterone receptor. These data suggest that CaM-induced dimerization of ER-α is required for estrogen-stimulated transcriptional activation by the receptor. In light of the critical role of ER-α in breast carcinoma, our data suggest that small molecules that selectively disrupt the interaction of ER-α with CaM may be useful in the therapy of breast carcinoma.

Absence of IQGAP1 Protein Leads to Insulin Resistance.

Insulin binds to the insulin receptor (IR) and induces tyrosine phosphorylation of the receptor and insulin receptor substrate-1 (IRS-1), leading to activation of the PKB/Akt and MAPK/ERK pathways. IQGAP1 is a scaffold protein that interacts with multiple binding partners and integrates diverse signaling cascades. Here we show that IQGAP1 associates with both IR and IRS-1 and influences insulin action. In vitro analysis with pure proteins revealed that the IQ region of IQGAP1 binds directly to the intracellular domain of IR. Similarly, the phosphotyrosine-binding domain of IRS-1 mediates a direct interaction with the C-terminal tail of IQGAP1. Consistent with these observations, both IR and IRS-1 co-immunoprecipitated with IQGAP1 from cells. Investigation of the functional effects of the interactions revealed that in the absence of IQGAP1, insulin-stimulated phosphorylation of Akt and ERK, as well as the association of phosphatidylinositol 3-kinase with IRS-1, were significantly decreased. Importantly, loss of IQGAP1 results in impaired insulin signaling and glucose homeostasis in vivo Collectively, these data reveal that IQGAP1 is a scaffold for IR and IRS-1 and implicate IQGAP1 as a participant in insulin signaling.

IQGAP1 Binds to Yes-associated Protein (YAP) and Modulates Its Transcriptional Activity.

During development, the Hippo signaling pathway regulates key physiological processes, such as control of organ size, regeneration, and stem cell biology. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. The scaffold protein IQGAP1 interacts with more than 100 binding partners to integrate diverse signaling pathways. In this study, we report that IQGAP1 binds to YAP and modulates its activity. IQGAP1 and YAP co-immunoprecipitated from cells. In vitro analysis with pure proteins demonstrated a direct interaction between IQGAP1 and YAP. Analysis with multiple fragments of each protein showed that the interaction occurs via the IQ domain of IQGAP1 and the TEAD-binding domain of YAP. The interaction between IQGAP1 and YAP has functional effects. Knock-out of endogenous IQGAP1 significantly increased the formation of nuclear YAP-TEAD complexes. Transcription assays were performed with IQGAP1-null mouse embryonic fibroblasts and HEK293 cells with IQGAP1 knockdown by CRISPR/Cas9. Quantification demonstrated that YAP-TEAD-mediated transcription in cells lacking IQGAP1 was significantly greater than in control cells. These data reveal that IQGAP1 binds to YAP and modulates its co-transcriptional function, suggesting that IQGAP1 participates in Hippo signaling.

IQGAP1 is a novel phosphatidylinositol 4,5 bisphosphate effector in regulation of directional cell migration.

Phosphatidylinositol 4,5 bisphosphate (PIP2) is a key lipid messenger for regulation of cell migration. PIP2 modulates many effectors, but the specificity of PIP2 signalling can be defined by interactions of PIP2-generating enzymes with PIP2 effectors. Here, we show that type Iγ phosphatidylinositol 4-phosphate 5-kinase (PIPKIγ) interacts with the cytoskeleton regulator, IQGAP1, and modulates IQGAP1 function in migration. We reveal that PIPKIγ is required for IQGAP1 recruitment to the leading edge membrane in response to integrin or growth factor receptor activation. Moreover, IQGAP1 is a PIP2 effector that directly binds PIP2 through a polybasic motif and PIP2 binding activates IQGAP1, facilitating actin polymerization. IQGAP1 mutants that lack PIPKIγ or PIP2 binding lose the ability to control directional cell migration. Collectively, these data reveal a synergy between PIPKIγ and IQGAP1 in the control of cell migration.

The biology of IQGAP proteins: beyond the cytoskeleton.

IQGAP scaffold proteins are evolutionarily conserved in eukaryotes and facilitate the formation of complexes that regulate cytoskeletal dynamics, intracellular signaling, and intercellular interactions. Fungal and mammalian IQGAPs are implicated in cytokinesis. IQGAP1, IQGAP2, and IQGAP3 have diverse roles in vertebrate physiology, operating in the kidney, nervous system, cardio-vascular system, pancreas, and lung. The functions of IQGAPs can be corrupted during oncogenesis and are usurped by microbial pathogens. Therefore, IQGAPs represent intriguing candidates for novel therapeutic agents. While modulation of the cytoskeletal architecture was initially thought to be the primary function of IQGAPs, it is now clear that they have roles beyond the cytoskeleton. This review describes contributions of IQGAPs to physiology at the organism level.

PIP kinases define PI4,5P2signaling specificity by association with effectors.

Phosphatidylinositol 4,5-bisphosphate (PI4,5P2) is an essential lipid messenger with roles in all eukaryotes and most aspects of human physiology. By controlling the targeting and activity of its effectors, PI4,5P2modulates processes, such as cell migration, vesicular trafficking, cellular morphogenesis, signaling and gene expression. In cells, PI4,5P2has a much higher concentration than other phosphoinositide species and its total content is largely unchanged in response to extracellular stimuli. The discovery of a vast array of PI4,5P2 binding proteins is consistent with data showing that the majority of cellular PI4,5P2is sequestered. This supports a mechanism where PI4,5P2functions as a localized and highly specific messenger. Further support of this mechanism comes from the de novo synthesis of PI4,5P2which is often linked with PIP kinase interaction with PI4,5P2effectors and is a mechanism to define specificity of PI4,5P2signaling. The association of PI4,5P2-generating enzymes with PI4,5P2effectors regulate effector function both temporally and spatially in cells. In this review, the PI4,5P2effectors whose functions are tightly regulated by associations with PI4,5P2-generating enzymes will be discussed. This article is part of a Special Issue entitled Phosphoinositides.

Isoform 5 of PIPKIγ regulates the endosomal trafficking and degradation of E-cadherin.

Phosphatidylinositol phosphate kinases (PIPKs) have distinct cellular targeting, allowing for site-specific synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to activate specific signaling cascades required for cellular processes. Several C-terminal splice variants of PIPKIγ (also known as PIP5K1C) exist, and have been implicated in a multitude of cellular roles. PI(4,5)P2 serves as a fundamental regulator of E-cadherin transport, and PI(4,5)P2-generating enzymes are important signaling relays in these pathways. We present evidence that the isoform 5 splice variant of PIPKIγ (PIPKIγi5) associates with E-cadherin and promotes its lysosomal degradation. Additionally, we show that the endosomal trafficking proteins SNX5 and SNX6 associate with PIPKIγi5 and inhibit PIPKIγi5-mediated E-cadherin degradation. Following HGF stimulation, activated Src directly phosphorylates PIPKIγi5. Phosphorylation of the PIPKIγi5 C-terminus regulates its association with SNX5 and, consequently, E-cadherin degradation. Additionally, this PIPKIγi5-mediated pathway requires Rab7 to promote degradation of internalized E-cadherin. Taken together, the data indicate that PIPKIγi5 and SNX5 are crucial regulators of E-cadherin sorting and degradation. PIPKIγi5, SNX and phosphoinositide regulation of lysosomal sorting represent a novel area of PI(4,5)P2 signaling and research. PIPKIγi5 regulation of E-cadherin sorting for degradation might have broad implications in development and tissue maintenance, and enhanced PIPKIγi5 function might have pathogenic consequences due to downregulation of E-cadherin.

Phosphatidylinositol 4,5-bisphosphate: targeted production and signaling.

Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) is a key lipid signaling molecule that regulates a vast array of biological activities. PI4,5P(2) can act directly as a messenger or can be utilized as a precursor to generate other messengers: inositol trisphosphate, diacylglycerol, or phosphatidylinositol 3,4,5-trisphosphate. PI4,5P(2) interacts with hundreds of different effector proteins. The enormous diversity of PI4,5P(2) effector proteins and the spatio-temporal control of PI4,5P(2) generation allow PI4,5P(2) signaling to control a broad spectrum of cellular functions. PI4,5P(2) is synthesized by phosphatidylinositol phosphate kinases (PIPKs). The array of PIPKs in cells enables their targeting to specific subcellular compartments through interactions with targeting factors that are often PI4,5P(2) effectors. These interactions are a mechanism to define spatial and temporal PI4,5P(2) synthesis and the specificity of PI4,5P(2) signaling. In turn, the regulation of PI4,5P(2) effectors at specific cellular compartments has implications for understanding how PI4,5P(2) controls cellular processes and its role in diseases.

The IQGAP scaffolds: Critical nodes bridging receptor activation to cellular signaling.

The scaffold protein IQGAP1 assembles multiprotein signaling complexes to influence biological functions. Cell surface receptors, particularly receptor tyrosine kinases and G-protein coupled receptors, are common IQGAP1 binding partners. Interactions with IQGAP1 modulate receptor expression, activation, and/or trafficking. Moreover, IQGAP1 couples extracellular stimuli to intracellular outcomes via scaffolding of signaling proteins downstream of activated receptors, including mitogen-activated protein kinases, constituents of the phosphatidylinositol 3-kinase pathway, small GTPases, and ß-arrestins. Reciprocally, some receptors influence IQGAP1 expression, subcellular localization, binding properties, and post-translational modifications. Importantly, the receptor:IQGAP1 crosstalk has pathological implications ranging from diabetes and macular degeneration to carcinogenesis. Here, we describe the interactions of IQGAP1 with receptors, summarize how they modulate signaling, and discuss their contribution to pathology. We also address the emerging functions in receptor signaling of IQGAP2 and IQGAP3, the other human IQGAP proteins. Overall, this review emphasizes the fundamental roles of IQGAPs in coupling activated receptors to cellular homeostasis.

Insulin suppresses transcriptional activity of yes-associated protein in insulin target cells.

Yes-associated protein (YAP), the main transcriptional coactivator of the Hippo pathway, integrates multiple inputs from different signaling cascades. Evidence implicates YAP in the control of cellular nutrient and energy status, but the underlying mechanisms are not fully elucidated. Here we show that insulin modulates YAP transcriptional activity in classic insulin target cells, namely HepG2 and C2C12. Insulin increases YAP phosphorylation and significantly decreases YAP abundance in HepG2 cell nuclei. Proximity ligation assay analysis revealed a marked reduction in the interaction of YAP with TEA domain (TEAD) transcription factors in the nuclei of insulin-exposed cells. Consistent with these findings, insulin impaired both YAP/TEAD-mediated transcription and transcription of YAP target genes in HepG2 and C2C12 cells. Serum starvation abrogated the effect of insulin on YAP phosphorylation and YAP transcription. Both the expression of two gluconeogenesis genes, G6PC and PCK1, and the inhibitory effect of insulin on these genes were attenuated in YAP-deficient HepG2 cells. Our results identify insulin as a previously undescribed suppressor of YAP activity in insulin target cells and provide insight into cross-talk between the insulin and Hippo pathways.

Endogenous IQGAP1 and IQGAP3 do not functionally interact with Ras.

The Ras family of small GTPases modulates numerous essential processes. Activating Ras mutations result in hyper-activation of selected signaling cascades, which leads to human diseases. The high frequency of Ras mutations in human malignant neoplasms has led to Ras being a desirable chemotherapeutic target. The IQGAP family of scaffold proteins binds to and regulates multiple signaling molecules, including the Rho family GTPases Rac1 and Cdc42. There are conflicting data in the published literature regarding interactions between IQGAP and Ras proteins. Initial reports showed no binding, but subsequent studies claim associations of IQGAP1 and IQGAP3 with K-Ras and H-Ras, respectively. Therefore, we set out to resolve this controversy. Here we demonstrate that neither endogenous IQGAP1 nor endogenous IQGAP3 binds to the major Ras isoforms, namely H-, K-, and N-Ras. Importantly, Ras activation by epidermal growth factor is not altered when IQGAP1 or IQGAP3 proteins are depleted from cells. These data strongly suggest that IQGAP proteins are not functional interactors of H-, K-, or N-Ras and challenge the rationale for targeting the interaction of Ras with IQGAP for the development of therapeutic agents.

Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation by scaffolded phosphoinositide kinases.

Generation of the lipid messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) is crucial for development, cell growth and survival, and motility, and it becomes dysfunctional in many diseases including cancers. Here we reveal a mechanism for PtdIns(3,4,5)P3 generation by scaffolded phosphoinositide kinases. In this pathway, class I phosphatidylinositol-3-OH kinase (PI(3)K) is assembled by IQGAP1 with PI(4)KIIIα and PIPKIα, which sequentially generate PtdIns(3,4,5)P3 from phosphatidylinositol. By scaffolding these kinases into functional proximity, the PtdIns(4,5)P2 generated is selectively used by PI(3)K for PtdIns(3,4,5)P3 generation, which then signals to PDK1 and Akt that are also in the complex. Moreover, multiple receptor types stimulate the assembly of this IQGAP1-PI(3)K signalling complex. Blockade of IQGAP1 interaction with PIPKIα or PI(3)K inhibited PtdIns(3,4,5)P3 generation and signalling, and selectively diminished cancer cell survival, revealing a target for cancer chemotherapy.

IQGAPs choreograph cellular signaling from the membrane to the nucleus.

Since its discovery in 1994, recognized cellular functions for the scaffold protein IQGAP1 have expanded immensely. Over 100 unique IQGAP1-interacting proteins have been identified, implicating IQGAP1 as a critical integrator of cellular signaling pathways. Initial research established functions for IQGAP1 in cell-cell adhesion, cell migration, and cell signaling. Recent studies have revealed additional IQGAP1 binding partners, expanding the biological roles of IQGAP1. These include crosstalk between signaling cascades, regulation of nuclear function, and Wnt pathway potentiation. Investigation of the IQGAP2 and IQGAP3 homologs demonstrates unique functions, some of which differ from those of IQGAP1. Summarized here are recent observations that enhance our understanding of IQGAP proteins in the integration of diverse signaling pathways.

Endosomal type Iγ PIP 5-kinase controls EGF receptor lysosomal sorting.

Endosomal trafficking and degradation of epidermal growth factor receptor (EGFR) play an essential role in the control of its signaling. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)) is an established regulator of endocytosis, whereas PtdIns3P modulates endosomal trafficking. However, we demonstrate here that type I gamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme that synthesizes PtdIns4,5P(2), controls endosome-to-lysosome sorting of EGFR. In this pathway, PIPKIγi5 interacts with sorting nexin 5 (SNX5), a protein that binds PtdIns4,5P(2) and other phosphoinositides. PIPKIγi5 and SNX5 localize to endosomes, and loss of either protein blocks EGFR sorting into intraluminal vesicles (ILVs) of the multivesicular body. Loss of ILV sorting greatly enhances and prolongs EGFR signaling. PIPKIγi5 and SNX5 prevent Hrs ubiquitination, and this facilitates the Hrs association with EGFR that is required for ILV sorting. These findings reveal that PIPKIγi5 and SNX5 form a signaling nexus that controls EGFR endosomal sorting, degradation, and signaling.