Endosomal accumulation of the activated epidermal growth factor receptor (EGFR) induces apoptosis

Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.     

Distinct involvement of the Gab1 and Grb2 adaptor proteins in signal transduction by the related receptor tyrosine kinases RON and MET

Although the signal transduction mechanisms of the receptor tyrosine kinase MET are well defined, less is known about its close relative RON. MET initiates intracellular signaling by autophosphorylation on specific cytoplasmic tyrosines that form docking sites for the adaptor proteins Grb2 and Gab1. Grb2 binds directly and is essential for all of the biological activities of MET. Gab1 docks either directly or indirectly via Grb2 and controls only a subset of MET functions. Because MET and RON possess similar adaptor binding sites, it was anticipated that their adaptor interactions would be conserved. Here we show that in contrast to MET, RON relies primarily on Gab1 for signal transmission. Surprisingly, disruption of the Grb2 docking site of RON or Grb2 depletion augments activity, whereas enhancement of Grb2 binding attenuates Gab1 recruitment and signaling. Hence, RON and MET differ in their adaptor interactions; furthermore, Grb2 performs a novel antagonistic role in the context of RON signaling.     

Membrane-bound trafficking regulates nuclear transport of integral epidermal growth factor receptor (EGFR) and ErbB-2

Nuclear localization of multiple receptor-tyrosine kinases (RTKs), such as EGF receptor (EGFR), ErbB-2, FGF receptor (FGFR), and many others, has been reported by several groups. We previously showed that cell surface EGFR is trafficked to the nucleus through a retrograde pathway from the Golgi to the endoplasmic reticulum (ER) and that EGFR is then translocated to the inner nuclear membrane (INM) through the INTERNET (integral trafficking from the ER to the nuclear envelope transport) pathway. However, the nuclear trafficking mechanisms of other membrane RTKs, apart from EGFR, remain unclear. The purpose of this study was to compare the nuclear transport of EGFR family proteins with that of FGFR-1. Interestingly, we found that digitonin permeabilization, which selectively releases soluble nuclear transporters from the cytoplasm and has been shown to inhibit nuclear transport of FGFR-1, had no effects on EGFR nuclear transport, raising the possibility that EGFR and FGFR-1 use different pathways to be translocated into the nucleus. Using the subnuclear fractionation assay, we further demonstrated that biotinylated cell surface ErbB-2, but not FGFR-1, is targeted to the INM, associating with Sec61β in the INM, similar to the nuclear trafficking of EGFR. Thus, ErbB-2, but not FGFR-1, shows a similar trafficking pathway to EGFR for translocation to the nucleus, indicating that at least two different pathways of nuclear transport exist for cell surface receptors. This finding provides a new direction for investigating the trafficking mechanisms of various nuclear RTKs.     

Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin

Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca(2+)/CaM complexes, which interact with and activate target proteins. In the present study the role of Ca(2+)/CaM in the regulation of the ligand-dependent activation of the epidermal growth factor receptor (EGFR) has been examined in living cells. We show that addition of different cell permeable CaM antagonists to cultured cells or loading cells with a Ca(2+) chelator inhibited ligand-dependent EGFR auto(trans)phosphorylation. This occurred also in the presence of inhibitors of protein kinase C, CaM-dependent protein kinase II and calcineurin, which are known Ca(2+)- and/or Ca(2+)/CaM-dependent EGFR regulators, pointing to a direct effect of Ca(2+)/CaM on the receptor. Furthermore, we demonstrate that down-regulation of CaM in conditional CaM knock out cells stably transfected with the human EGFR decreased its ligand-dependent phosphorylation. Substitution of six basic amino acid residues within the CaM-binding domain (CaM-BD) of the EGFR by alanine resulted in a decreased phosphorylation of the receptor and of its downstream substrate phospholipase Cγ1. These results support the hypothesis that Ca(2+)/CaM regulates the EGFR activity by directly interacting with the CaM-BD of the receptor located at its cytosolic juxtamembrane region.     

Conservation and diversification of dileucine signal recognition by adaptor protein (AP) complex variants

The clathrin-associated, heterotetrameric adaptor protein (AP) complexes, AP-1, AP-2, and AP-3, recognize signals in the cytosolic domains of transmembrane proteins, leading to their sorting to endosomes, lysosomes, lysosome-related organelles, and/or the basolateral membrane of polarized epithelial cells. One type of signal, referred to as "dileucine-based," fits the consensus motif (D/E)XXXL(L/I). Previous biochemical analyses showed that (D/E)XXXL(L/I) signals bind to a combination of two subunits of each AP complex, namely the AP-1 γ-σ1, AP-2 α-σ2, and AP-3 δ-σ3 hemicomplexes, and structural studies revealed that an imperfect variant of this motif lacking the (D/E) residue binds to a site straddling the interface of α and σ2. Herein, we report mutational and binding analyses showing that canonical (D/E)XXXL(L/I) signals bind to this same site on AP-2, and to similar sites on AP-1 and AP-3. The strength and amino acid requirements of different interactions depend on the specific signals and AP complexes involved. We also demonstrate the occurrence of diverse AP-1 heterotetramers by combinatorial assembly of various γ and σ1 subunit isoforms encoded by different genes. These AP-1 variants bind (D/E)XXXL(L/I) signals with marked preferences for certain sequences, implying that they are not functionally equivalent. Our results thus demonstrate that different AP complexes share a conserved binding site for (D/E)XXXL(L/I) signals. However, the characteristics of the binding site on each complex vary, providing for the specific recognition of a diverse repertoire of (D/E)XXXL(L/I) signals.     

Transactivation of the epidermal growth factor receptor by heat shock protein 90 via Toll-like receptor 4 contributes to the migration of glioblastoma cells

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl β-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.     

The translocon Sec61beta localized in the inner nuclear membrane transports membrane-embedded EGF receptor to the nucleus

Accumulating evidence indicates that endocytosis plays an essential role in the nuclear transport of the ErbB family members, such as epidermal growth factor receptor (EGFR) and ErbB-2. Nevertheless, how full-length receptors embedded in the endosomal membrane pass through the nuclear pore complexes and function as non-membrane-bound receptors in the nucleus remains unclear. Here we show that upon EGF treatment, the biotinylated cell surface EGFR is trafficked to the inner nuclear membrane (INM) through the nuclear pore complexes, remaining in a membrane-bound environment. We further find that importin β regulates EGFR nuclear transport to the INM in addition to the nucleus/nucleoplasm. Unexpectedly, the well known endoplasmic reticulum associated translocon Sec61β is found to reside in the INM and associate with EGFR. Knocking down Sec61β expression reduces EGFR level in the nucleoplasm portion and accumulates it in the INM portion. Thus, the Sec61β translocon plays an unrecognized role in the release of the membrane-anchored EGFR from the lipid bilayer of the INM to the nucleus. The newly identified Sec61β function provides an alternative pathway for nuclear transport that can be utilized by membrane-embedded proteins such as full-length EGFR.     

Vascular endothelial growth factor receptor 2 direct interaction with nephrin links VEGF-A signals to actin in kidney podocytes

The transmembrane protein nephrin is an essential component of slit diaphragms, the specialized cell junctions that link podocyte foot processes. Podocytes are epithelial cells that surround the glomerular capillaries in the kidney and are necessary for the organ-filtering function. Nephrin signaling complex transduces extracellular cues to the podocyte cytoskeleton and regulates podocyte shape and function. Vascular endothelial growth factor A (VEGF-A) is a required growth factor produced and secreted by podocytes. Accumulating evidence suggests a cross-talk between VEGF-A and nephrin signaling pathways. We previously showed that in vivo nephrin associates with VEGF receptor-2 (VEGFR2), the signaling receptor for VEGF-A. In the present work, we characterized the interaction between nephrin and VEGFR2 in cultured cells and in vitro. We demonstrate that nephrin-VEGFR2 interaction is direct using mass spectrometry, immunoprecipitation, GST-binding assays, and blot overlay experiments. This interaction occurs through VEGFR2 and nephrin cytoplasmic domains. Nephrin-VEGFR2 interaction is modulated by tyrosine phosphorylation of both cytoplasmic domains. Furthermore, the nephrin-VEGFR2 complex involves Nck and actin. VEGF-A signaling via this complex results in decreased cell size. We provide evidence that this multiprotein interaction occurs in cultured podocytes. We propose that the nephrin-VEGFR2 complex acts as a key mediator to transduce local VEGF-A signals to the podocyte actin cytoskeleton, regulating the foot process structure and glomerular filter integrity.     

The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor

Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.     

Bispecific designed ankyrin repeat proteins (DARPins) targeting epidermal growth factor receptor inhibit A431 cell proliferation and receptor recycling

The EGF receptor (EGFR) has been implicated in the development and progression of many tumors. Although monoclonal antibodies directed against EGFR have been approved for the treatment of cancer in combination with chemotherapy, there are limitations in their clinical efficacy, necessitating the search for robust targeting molecules that can be equipped with new effector functions or show a new mechanism of action. Designed ankyrin repeat proteins (DARPins) may provide the targeting component for such novel reagents. Previously, four DARPins were selected against EGFR with (sub)nanomolar affinity. As any targeting module should preferably be able to inhibit EGFR-mediated signaling, their effect on A431 cells overexpressing EGFR was examined: three of them were shown to inhibit proliferation by inducing G(1) arrest, as seen for the Food and Drug Administration-approved antibody cetuximab. To understand this inhibitory mechanism, we mapped the epitopes of the DARPins using yeast surface display. The epitopes for the biologically active DARPins overlapped with the EGF-binding site, whereas the fourth DARPin bound to a different domain, explaining the lack of a biological effect. To optimize the biological activity of the DARPins, we combined two DARPins binding to different epitopes with a flexible linker or with a leucine zipper, leading to a homodimer. The latter DARPin was able to reduce surface EGFR by inhibiting receptor recycling, leading to a dramatic decrease in cell viability. These results indicate that multispecific EGFR-specific DARPins are superior to cetuximab and may form the basis of new opportunities in tumor targeting and tumor therapy.     

The Importance of LAT in the Activation,Homeostasis, and Regulatory Function of T Cells

LAT (linker for activation of T cells) is a transmembrane adaptor protein that plays an essential role in TCR-mediated signaling and thymocyte development. Because LAT-deficient mice have an early block in thymocyte development, we utilized an inducible system to delete LAT in primary T cells to study LAT function in T cell activation, homeostasis, and survival. Deletion of LAT caused primary T cells to become unresponsive to stimulation from the TCR and impaired T cell homeostatic proliferation and long term survival. Furthermore, deletion of LAT led to reduced expression of Foxp3, CTLA-4, and CD25 in T_reg cells and impaired their function. Consequently, mice with LAT deleted developed a lymphoproliferative syndrome similar to that in LATY136F mice, although less severe. Our data implicate that LAT has positive and negative roles in the regulation of mature T cells.     

The membrane-proximal intracellular domain of the epidermal growth factor receptor underlies negative cooperativity in ligand binding

The binding of EGF induces dimerization of its receptor, leading to the stimulation of its intracellular tyrosine kinase activity. Kinase activation occurs within the context of an asymmetric dimer in which one kinase domain serves as the activator for the other kinase domain but is not itself activated. How ligand binding is related to the formation and dynamics of this asymmetric dimer is not known. The binding of EGF to its receptor is negatively cooperative--that is, EGF binds with lower affinity to the second site on the dimer than to the first site on the dimer. In this study, we analyzed the binding of (125)I-EGF to a series of EGF receptor mutants in the intracellular juxtamembrane domain and demonstrate that the most membrane-proximal portion of this region plays a significant role in the genesis of negative cooperativity in the EGF receptor. The data are consistent with a model in which the binding of EGF to the first site on the dimer induces the formation of one asymmetric kinase dimer. The binding of EGF to the second site is required to disrupt the initial asymmetric dimer and allow the formation of the reciprocal asymmetric dimer. Thus, some of the energy of binding to the second site is used to reorient the first asymmetric dimer, leading to a lower binding affinity and the observed negative cooperativity.     

Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 ⁴²⁰AKKAA⁴²⁴ mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gα_q association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.     

Growth suppression of human hepatocellular carcinoma xenografts by a monoclonal antibody CH12 directed to epidermal growth factor receptor variant III

Human hepatocellular carcinoma (HCC) is considered difficult to cure because it is resistant to radio- and chemotherapy and has a high recurrence rate after curative liver resection. Epidermal growth factor receptor variant III (EGFRvIII) has been reported to express in HCC tissues and cell lines. This article describes the efficacy of an anti-EGFRvIII monoclonal antibody (mAb CH12) in the treatment of HCC xenografts with EGFRvIII expression and the underlying mechanism of EGFRvIII as an oncogene in HCC. The results demonstrated that CH12 bound preferentially to EGFRvIII with a dissociation constant (K(d)) of 1.346 nm/liter. In addition, CH12 induces strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in Huh7-EGFRvIII (with exogenous expression of EGFRvIII) and SMMC-7721 (with endogenous expression of EGFRvIII) cells. Notably, CH12 significantly inhibited the growth of Huh7-EGFRvIII and SMMC-7721 xenografts in vivo with a growth inhibition ratio much higher than C225, a U. S. Food and Drug Administration-approved anti-EGFR antibody. Treatment of the two HCC xenografts with CH12 significantly suppressed tumor proliferation and angiogenesis. Mechanistically, in vivo treatment with CH12 reduced the phosphorylation of constitutively active EGFRvIII, Akt, and ERK. Down-regulation of the apoptotic protectors Bcl-x(L), Bcl-2, and the cell cycle regulator cyclin D1, as well as up-regulation of the cell-cycle inhibitor p27, were also observed after in vivo CH12 treatment. Collectively, these results indicate that the monoclonal antibody CH12 is a promising therapeutic agent for HCC with EGFRvIII expression.     

Internalization of proprotein convertase PC7 from plasma membrane is mediated by a novel motif

Proprotein convertase 7 (PC7) is a member of the subtilisin-like proprotein convertase family, which is involved in the endoproteolysis of a variety of precursor proteins. Under steady state conditions, PC7 is mainly localized in the trans-Golgi network, but a small fraction is found at the cell surface. So far, no sorting signals for membrane trafficking have been identified in PC7. In this study, we have examined the internalization of PC7 from the plasma membrane. Our results show that internalization of PC7 is mediated by clathrin-coated vesicles. After inhibition of clathrin-mediated endocytosis using hypertonic conditions or the small molecule inhibitor, Pitstop 2, PC7 accumulated at the plasma membrane. Furthermore, PC7 was present in isolated clathrin-coated vesicles. To determine the internalization motif, constructs were generated in which parts of the N and C terminus of the cytoplasmic tail of PC7 were deleted, and chimeric proteins were constructed consisting of the luminal and transmembrane domains of Tac (CD25) and parts of the cytoplasmic domain of PC7. Antibody uptake experiments as well as surface biotinylation experiments demonstrated that the region between Ala(713) and Cys(726) in the cytoplasmic domain of PC7 is essential and sufficient for the internalization of PC7 but not for trans-Golgi network localization. Individual amino acids in this region were substituted with alanine, which identified Pro, Leu, and Cys as the essential amino acids. In conclusion, internalization of PC7 depends on a short transferable sequence in the cytoplasmic tail, which contains the three crucial amino acids PLC.     

Characterization of RIN3 as a guanine nucleotide exchange factor for the Rab5 subfamily GTPase Rab31

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.     

Fibroblast growth factor receptor 3 (FGFR3) is a strong heat shock protein 90 (Hsp90) client: implications for therapeutic manipulation

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of growth and differentiation, whose aberrant activation causes a number of genetic diseases including achondroplasia and cancer. Hsp90 is a specialized molecular chaperone involved in stabilizing a select set of proteins termed clients. Here, we delineate the relationship of Hsp90 and co-chaperone Cdc37 with FGFR3 and the FGFR family. FGFR3 strongly associates with these chaperone complexes and depends on them for stability and function. Inhibition of Hsp90 function using the geldanamycin analog 17-AAG induces the ubiquitination and degradation of FGFR3 and reduces the signaling capacity of FGFR3. Other FGFRs weakly interact with these chaperones and are differentially influenced by Hsp90 inhibition. The Hsp90-related ubiquitin ligase CHIP is able to interact and destabilize FGFR3. Our results establish FGFR3 as a strong Hsp90 client and suggest that modulating Hsp90 chaperone complexes may beneficially influence the stability and function of FGFR3 in disease.     

A novel mode of protein kinase inhibition exploiting hydrophobic motifs of autoinhibited kinases: discovery of ATP-independent inhibitors of fibroblast growth factor receptor

Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases.