Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases

Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.     

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatase-1B

Receptor tyrosine kinases (RTKs) are key regulators of cellular homeostasis. Based on in vitro and ex vivo studies, protein tyrosine phosphatase-1B (PTP1B) was implicated in the regulation of several RTKs, yet mice lacking PTP1B show defects mainly in insulin and leptin receptor signaling. To address this apparent paradox, we studied RTK signaling in primary and immortalized fibroblasts from PTP1B(-/-) mice. After growth factor treatment, cells lacking PTP1B exhibit increased and sustained phosphorylation of the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). However, Erk activation is enhanced only slightly, and there is no increase in Akt activation in PTP1B-deficient cells. Our results show that PTP1B does play a role in regulating EGFR and PDGFR phosphorylation but that other signaling mechanisms can largely compensate for PTP1B deficiency. In-gel phosphatase experiments suggest that other PTPs may help to regulate the EGFR and PDGFR in PTP1B(-/-) fibroblasts. This and other compensatory mechanisms prevent widespread, uncontrolled activation of RTKs in the absence of PTP1B and probably explain the relatively mild effects of PTP1B deletion in mice.     

An attempt to promote neo-vascularization by employing a newly synthesized inhibitor of protein tyrosine phosphatase

Vascular endothelial growth factor (VEGF) and its receptors play a key role in angiogenesis. VEGF receptor-2 (VEGFR-2) has a tyrosine kinase domain, and, once activated, induces the phosphorylation of cytoplasmic signaling proteins. The phosphorylated VEGFR-2 may be a substrate for intracellular protein tyrosine phosphatases (PTPs) which prevent VEGF signaling. We synthesized a series of alpha,alpha-difluoro(phenyl)methylphosphonic acids (DFPMPAs) which inhibit the action of PTP. In this study, we test their effects on VEGF-induced angiogenesis. DFPMPA-3, the most effective inhibitor of human PTP-1B, promoted tube formation by human umbilical vein endothelial cells (HUVEC) on Matrigel more effectively than any other DFPMPAs. The inhibitor promoted the VEGF-induced proliferation and migration of HUVEC by inhibiting the dephosphorylation of VEGFR-2. Its effectiveness was proven through neo-vascularization in mice. The present findings suggest that targeting PTP to promote therapeutic neo-vascularization may be a potential strategy.     

The biology of VEGF and its receptors

Vascular endothelial growth factor (VEGF) is a key regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. VEGF has also been implicated in pathological angiogenesis associated with tumors, intraocular neovascular disorders and other conditions. The biological effects of VEGF are mediated by two receptor tyrosine kinases (RTKs), VEGFR-1 and VEGFR-2, which differ considerably in signaling properties. Non-signaling co-receptors also modulate VEGF RTK signaling. Currently, several VEGF inhibitors are undergoing clinical testing in several malignancies. VEGF inhibition is also being tested as a strategy for the prevention of angiogenesis, vascular leakage and visual loss in age-related macular degeneration.     

A single amino acid substitution in the activation loop defines the decoy characteristic of VEGFR-1/FLT-1

VEGFR-1 is a kinase-defective receptor tyrosine kinase (RTK) and negatively modulates angiogenesis by acting as a decoy receptor. The decoy characteristic of VEGFR-1 is required for normal development and angiogenesis. To date, there is no molecular explanation for this unusual characteristic of VEGFR-1. Here we show that the molecular mechanisms underlying the decoy characteristic of VEGFR-1 is linked to the replacement of a highly conserved amino acid residue in the activation loop. This amino acid is highly conserved among all the type III RTKs and corresponds to aspartic acid, but in VEGFR-1 it is substituted to asparagine. Mutation of asparagine (Asn(1050)) within the activation loop to aspartic acid promoted enhanced ligand-dependent tyrosine autophosphorylation and kinase activation in vivo and in vitro. The mutant VEGFR-1 (Asp(1050)) promoted endothelial cell proliferation but not tubulogenesis. It also displayed an oncogenic phenotype as its expression in fibroblast cells elicited transformation and colony growth. Furthermore, mutation of the invariable aspartic acid to asparagine in VEGFR-2 lowered the autophosphorylation of activation loop tyrosines 1052 and 1057. We propose that the conserved aspartic acid in the activation loop favors the transphosphorylation of the activation loop tyrosines, and its absence renders RTK to a less potent enzyme by disfavoring transphosphorylation of activation loop tyrosines.     

Tyrosine phosphorylation regulates maturation of receptor tyrosine kinases

Constitutive activation of receptor tyrosine kinases (RTKs) is a frequent event in human cancer cells. Activating mutations in Fms-like tyrosine kinase 3 (FLT-3), notably, internal tandem duplications in the juxtamembrane domain (FLT-3 ITD), have been causally linked to acute myeloid leukemia. As we describe here, FLT-3 ITD exists predominantly in an immature, underglycosylated 130-kDa form, whereas wild-type FLT-3 is expressed predominantly as a mature, complex glycosylated 150-kDa molecule. Endogenous FLT-3 ITD, but little wild-type FLT-3, is detectable in the endoplasmic reticulum (ER) compartment. Conversely, cell surface expression of FLT-3 ITD is less efficient than that of wild-type FLT-3. Inhibition of FLT-3 ITD kinase by small molecules, inactivating point mutations, or coexpression with the protein-tyrosine phosphatases (PTPs) SHP-1, PTP1B, and PTP-PEST but not RPTPalpha promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants.     

Mechanism of insulin sensitization by BMOV (bis maltolato oxo vanadium); unliganded vanadium (VO4) as the active component

Organovanadium compounds have been shown to be insulin sensitizers in vitro and in vivo. One potential biochemical mechanism for insulin sensitization by these compounds is that they inhibit protein tyrosine phosphatases (PTPs) that negatively regulate insulin receptor activation and signaling. In this study, bismaltolato oxovanadium (BMOV), a potent insulin sensitizer, was shown to be a reversible, competitive phosphatase inhibitor that inhibited phosphatase activity in cultured cells and enhanced insulin receptor activation in vivo. NMR and X-ray crystallographic studies of the interaction of BMOV with two different phosphatases, HCPTPA (human low molecular weight cytoplasmic protein tyrosine phosphatase) and PTP1B (protein tyrosine phosphatase 1B), demonstrated uncomplexed vanadium (VO(4)) in the active site. Taken together, these findings support phosphatase inhibition as a mechanism for insulin sensitization by BMOV and other organovanadium compounds and strongly suggest that uncomplexed vanadium is the active component of these compounds.     

Identification of protein tyrosine phosphatases associating with the PDGF receptor

Protein tyrosine phosphatase (PTP) in-gel assays were used to explore association of PTPs with the platelet-derived growth factor beta-receptor (PDGFbetaR). Five PTP activity bands of approximately 120, approximately 70, approximately 60, approximately 53, and approximately 45 kDa could be detected in PDGFbetaR immunoprecipitates and were identified by immunodepletion experiments as PTP-PEST, SHP-2, an active fragment of SHP-2, PTP-1B, and T-cell PTP, respectively. The PTP pattern that was obtained was similar in PDGFbetaR immunoprecipitates from HEK 293 cells overexpressing the human PDGFbetaR and from murine fibroblasts. Association of PTP-1B with the PDGFbetaR was stabilized by pretreatment of the cells with hydrogen peroxide. The epidermal growth factor receptor (EGFR) immunoprecipitated from fibroblasts, and c-Kit isolated from CHRF myeloid cells, were associated with partially overlapping but quantitatively different patterns of PTPs. PTP-PEST was the predominant PTP in EGFR immunoprecipitates, and SHP-1 appeared in c-Kit immunoprecipitates. We propose that the differential association of PTPs with different RTKs is related to their respective contributions to regulation of RTK signaling.     

Blockade of EphA receptor tyrosine kinase activation inhibits vascular endothelial cell growth factor-induced angiogenesis

Angiogenesis is a multistep process involving a diverse array of molecular signals. Ligands for receptor tyrosine kinases (RTKs) have emerged as critical mediators of angiogenesis. Three families of ligands, vascular endothelial cell growth factors (VEGFs), angiopoietins, and ephrins, act via RTKs expressed in endothelial cells. Recent evidence indicates that VEGF cooperates with angiopoietins to regulate vascular remodeling and angiogenesis in both embryogenesis and tumor neovascularization. However, the relationship between VEGF and ephrins remains unclear. Here we show that interaction between EphA RTKs and ephrinA ligands is necessary for induction of maximal neovascularization by VEGF. EphA2 RTK is activated by VEGF through induction of ephrinA1 ligand. A soluble EphA2-Fc receptor inhibits VEGF-, but not basic fibroblast growth factor-induced endothelial cell survival, migration, sprouting, and corneal angiogenesis. As an independent, but complementary approach, EphA2 antisense oligonucleotides inhibited endothelial expression of EphA2 receptor and suppressed ephrinA1- and VEGF-induced cell migration. Taken together, these data indicate an essential role for EphA receptor activation in VEGF-dependent angiogenesis and suggest a potential new target for therapeutic intervention in pathogenic angiogenesis.     

Receptor tyrosine kinases and their activation in melanoma

Receptor tyrosine kinases (RTKs) and their downstream signalling pathways have long been hypothesized to play key roles in melanoma development. A decade ago, evidence was derived largely from animal models, RTK expression studies and detection of activated RAS isoforms in a small fraction of melanomas. Predictions that overexpression of specific RTKs implied increased kinase activity and that some RTKs would show activating mutations in melanoma were largely untested. However, technological advances including rapid gene sequencing, siRNA methods and phospho-RTK arrays now give a more complete picture. Mutated forms of RTK genes including KIT, ERBB4, the EPH and FGFR families and others are known in melanoma. Additional over- or underexpressed RTKs and also protein tyrosine phosphatases (PTPs) have been reported, and activities measured. Complex interactions between RTKs and PTPs are implicated in the abnormal signalling driving aberrant growth and survival in malignant melanocytes, and indeed in normal melanocytic signalling including the response to ultraviolet radiation. Kinases are considered druggable targets, so characterization of global RTK activity in melanoma should assist the rational development of tyrosine kinase inhibitors for clinical use.     

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin,beta-catenin,and the phosphatase DEP-1/CD148

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin-null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin-null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.     

PTP1B Targets the Endosomal Sorting Machinery: DEPHOSPHORYLATION OF REGULATORY SITES ON THE ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT COMPONENT STAM2*

Dephosphorylation and endocytic down-regulation are distinct processes that together control the signaling output of a variety of receptor tyrosine kinases (RTKs). PTP1B can directly dephosphorylate several RTKs, but it can also promote activation of downstream pathways through largely unknown mechanisms. These positive signaling functions likely contribute to the tumor-promoting effect of PTP1B in mouse cancer models. Here, we have identified STAM2, an endosomal protein involved in sorting activated RTKs for lysosomal degradation, as a substrate of PTP1B. PTP1B interacts with STAM2 at defined phosphotyrosine sites, and knockdown of PTP1B expression augments STAM2 phosphorylation. Intriguingly, manipulating the expression and phosphorylation state of STAM2 did not have a general effect on epidermal growth factor (EGF)-induced EGF receptor trafficking, degradation, or signaling. Instead, phosphorylated STAM2 specifically suppressed Akt activation, and a phosphorylation-deficient STAM2 mutant displayed prolonged localization on endosomes following EGF stimulation. These results reveal a novel link between the dephosphorylation and endocytic machinery and suggest that PTP1B can affect RTK signaling in a previously unrecognized manner.     

Receptor Tyrosine Kinase-Mediated Angiogenesis

The endothelial cell is the essential cell type forming the inner layer of the vasculature. Two families of receptor tyrosine kinases (RTKs) are almost completely endothelial cell specific: the vascular endothelial growth factor (VEGF) receptors (VEGFR1-3) and the Tie receptors (Tie1 and Tie2). Both are key players governing the generation of blood and lymphatic vessels during embryonic development. Because the growth of new blood and lymphatic vessels (or the lack thereof) is a central element in many diseases, the VEGF and the Tie receptors provide attractive therapeutic targets in various diseases. Indeed, several drugs directed to these RTK signaling pathways are already on the market, whereas many are in clinical trials. Here we review the VEGFR and Tie families, their involvement in developmental and pathological angiogenesis, and the different possibilities for targeting them to either block or enhance angiogenesis and lymphangiogenesis.     

Leucine motif-dependent tyrosine autophosphorylation of type III receptor tyrosine kinases

Activation loop tyrosine autophosphorylation is an essential requirement for full kinase activation of receptor tyrosine kinases (RTKs). However, mechanisms involved are not fully understood. In general, kinase domains of RTKs are folded into two main lobes, NH2- and COOH-terminal lobes. The COOH-terminal lobe of vascular endothelial growth factor receptor-2 (VEGFR-2) is folded into seven alpha-helices (alphaD-alphaI). In the studies presented here we demonstrate that leucine residues of helix I (alphaI) regulate tyrosine autophosphorylation and phosphotransferase activity of VEGFR-2. The presence of leucines 1158, 1161, and 1162 are essential for tyrosine autophosphorylation and kinase activation of VEGFR-2 and are involved in helix-helix packing via hydrophobic interactions. The presence of leucine 1158 is critical for kinase activation of VEGFR-2 and appears to interact with alphaE, alphaF, alphaH, and beta7. The analogous residue, leucine 957 on platelet-derived growth factor receptor-beta and leucine 910 on colony stimulating factor-1R are also found to be critical for tyrosine autophosphorylation of these receptors. Leucines 1161 and 1162 are also involved in helix-helix packing but they play a less critical role in VEGFR-2 activation. Thus, we conclude that leucine motif-mediated helix-helix interactions are critical for kinase regulation of type III RTKs. This mechanism is likely to be shared with other kinases and might provide a basis for the design of a novel class of tyrosine kinase inhibitors.     

VEGF receptor antagonism blocks arteriogenesis, but only partially inhibits angiogenesis, in skeletal muscle of exercise-trained rats

Both collateral vessel enlargement (arteriogenesis) and capillary growth (angiogenesis) in skeletal muscle occur in response to exercise training. Vascular endothelial growth factor (VEGF) is implicated in both processes. Thus we examined the effect of a VEGF receptor (VEGF-R) inhibitor (ZD4190, AstraZeneca) on collateral-dependent blood flow in vivo and collateral artery size ex vivo (indicators of arteriogenesis) and capillary contacts per fiber (CCF; an index of angiogenesis) in skeletal muscle of both sedentary and exercise-trained rats 14 days after bilateral occlusion of the femoral arteries. Total daily treadmill run time increased appreciably from approximately 70 to approximately 100 min (at 15-20 m/min, twice per day) and produced a large (approximately 75%, P < 0.01) increase in calf muscle blood flow and a greater size of the collateral artery (wall cross-sectional area). ZD4190, which previously has been shown to inhibit the activity of VEGF-R2 and -R1 tyrosine kinase in vitro (IC50 = 30 and 700 nM, respectively), completely blocked the increase in collateral-dependent blood flow and inhibited collateral vessel enlargement. Thus exercise-stimulated collateral arteriogenesis appears to be completely dependent on VEGF-R signaling. Interestingly, enhanced mRNA expression of the VEGF family ligand placental growth factor (2- to 3.5-fold), VEGF-R1 (approximately 2-fold), and endothelial nitric oxide synthase (2- to 3.5-fold) in an isolated collateral artery implicates these factors as important in arteriogenesis. Training of ischemic muscle also induced angiogenesis, as shown by an increase (approximately 25%, P < 0.01) in CCF in white gastrocnemius muscle. VEGF-R inhibition only partially blocked (P < 0.01) but did not eliminate the increase (P < 0.01) in capillarity. Our findings indicate that VEGF-R tyrosine kinase activity is essential for collateral arteriogenesis and important for the angiogenesis induced in ischemic muscle by exercise training; however, other angiogenic stimuli are also important for angiogenesis in flow-limited active muscle.     

(−)-Epigallocatechin gallate inhibits growth and activation of the VEGF/VEGFR axis in human colorectal cancer cells

(?)-Epigallocatechin gallate (EGCG), the major constituent of green tea, inhibits the growth of colorectal cancer cells by inhibiting the activation of various types of receptor tyrosine kinases (RTKs). The RTK vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis induces tumor angiogenesis in colorectal cancer. This study examined the effects of EGCG on the activity of the VEGF/VEGFR axis and the expression of hypoxia-inducible factor (HIF)-1α, which promotes angiogenesis by elevating VEGF levels, in human colorectal cancer cells. Total and phosphorylated (i.e., activated) form (p-VEGFR-2) of VEGFR-2 proteins were overexpressed in a series of human colorectal cancer cell lines. Within 3 h, EGCG caused a decrease in the expression of HIF-1α protein and VEGF, HIF-1α, insulin-like growth factor (IGF)-1, IGF-2, epidermal growth factor (EGF), and heregulin mRNAs in SW837 colorectal cancer cells, which express a constitutively activated VEGF/VEGFR axis. A decrease was also observed in the expression of VEGFR-2, p-VEGFR-2, p-IGF-1 receptor, p-ERK, and p-Akt proteins within 6 h after EGCG treatment. Drinking EGCG significantly inhibited the growth of SW837 xenografts in nude mice, and this was associated with the inhibition of the expression and activation of VEGFR-2. The consumption of EGCG also inhibited activation of ERK and Akt, both of which are downstream signaling molecules of the VEGF/VEGFR axis, and reduced the expression of VEGF mRNA in xenografts. These findings suggest that EGCG may exert, at least in part, growth-inhibitory effects on colorectal cancer cells by inhibiting the activation of the VEGF/VEGFR axis through suppressing the expression of HIF-1α and several major growth factors. EGCG may therefore be useful in the chemoprevention and/or treatment of colorectal cancer.     

A Role for Cadherin-5 in Regulation of Vascular Endothelial Growth Factor Receptor 2 Activity in Endothelial Cells

FLK-1/vascular endothelial growth factor receptor 2 (VEGFR-2) is one of the receptors for VEGF. In this study we examined the effect of cell density on activation of VEGFR-2. VEGF induces only very slight tyrosine phosphorylation of VEGFR-2 in confluent (95-100% confluent) pig aortic endothelial (PAE) cells. In contrast, robust VEGF-dependent tyrosine phosphorylation of VEGFR-2 was observed in cells plated in sparse culture conditions (60-65% confluent). A similar cell density-dependent phenomenon was observed in different endothelial cells but not in NIH-3T3 fibroblast cells expressing VEGFR-2. Stimulating cells with high concentrations of VEGF or replacing the extracellular domain of VEGFR-2 with that of the colony-stimulating factor 1 receptor did not alleviate the sensitivity of VEGFR-2 to cell density, indicating that the confluent cells were probably not secreting an antagonist to VEGF. Furthermore, in PAE cells, ectopically introduced platelet-derived growth factor alpha receptor could be activated at both high and low cell density conditions, indicating that the density effect was not universal for all receptor tyrosine kinases expressed in endothelial cells. In addition to lowering the density of cells, removing divalent cations from the medium of confluent cells potentiated VEGFR-2 phosphorylation in response to VEGF. These findings suggested that cell-cell contact may be playing a role in regulating the activation of VEGFR-2. To this end, pretreatment of confluent PAE cells with a neutralizing anti-cadherin-5 antibody potentiated the response of VEGFR-2 to VEGF. Our data demonstrate that endothelial cell density plays a critical role in regulating VEGFR-2 activity, and that the underlying mechanism appears to involve cadherin-5.     

Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis

Several receptor tyrosine kinases require heparan sulfate proteoglycans (HSPGs) as coreceptors for efficient signal transduction. We have studied the role of HSPGs in the development of blood capillary structures from embryonic stem cells, a process strictly dependent on signaling via vascular endothelial growth factor receptor-2 (VEGFR-2). We show, by using chimeric cultures of embryonic stem cells defective in either HS production or VEGFR-2 synthesis, that VEGF signaling in endothelial cells is fully supported by HS expressed in trans by adjacent perivascular smooth muscle cells. Transactivation of VEGFR-2 leads to prolonged and enhanced signal transduction due to HS-dependent trapping of the active VEGFR-2 signaling complex. Our data imply that direct signaling via HSPG core proteins is dispensable for a functional VEGF response in endothelial cells. We propose that transactivation of tyrosine kinase receptors by HSPGs constitutes a mechanism for crosstalk between adjacent cells.     

Src-mediated phosphorylation of Hsp90 in response to vascular endothelial growth factor (VEGF) is required for VEGF receptor-2 signaling to endothelial NO synthase

Nitric oxide (NO) release from endothelial cells, via endothelial NO synthase (eNOS) activation, is central to the proangiogenic actions of vascular endothelial growth factor (VEGF). VEGF signaling to eNOS is principally mediated by an Akt-dependent phosphorylation of eNOS and by increased association of eNOS to the molecular chaperone, heat-shock protein 90 kDa (Hsp90). Herein, we report that VEGFR-2 activation induces tyrosine phosphorylation of VEGF receptor 2 (VEGFR-2)-associated Hsp90beta. Tyrosine phosphorylation of Hsp90beta in response to VEGF is dependent on internalization of the VEGFR-2 and on Src kinase activation. Furthermore, we demonstrate that c-Src directly phosphorylates Hsp90 on tyrosine 300 residue and that this event is essential for VEGF-stimulated eNOS association to Hsp90 and thus NO release from endothelial cells. Our work identifies Y300 phosphorylation of Hsp90 as a novel regulated posttranslational modification of the chaperone and demonstrates its importance in the proangiogenic actions of VEGF, namely by regulating NO release from endothelial cells.     

Protein-tyrosine phosphatase 1B modulates early endosome fusion and trafficking of Met and epidermal growth factor receptors

The endoplasmic reticulum-localized non-receptor protein-tyrosine phosphatase 1B (PTP1B) is associated with oncogenic, metabolic, and cytokine-related signaling and functionally targets multiple receptor tyrosine kinases (RTKs) for dephosphorylation. Loss of PTP1B activity leads to enhanced ligand-dependent biological activity of the Met RTK among others. Here, we demonstrate that knockdown of PTP1B or expression of a PTP1B trapping aspartic acid-to-alanine substitution (D/A) mutant delayed ligand-induced degradation of the Met and EGF RTKs. Loss of PTP1B function abrogated trafficking of Met and EGF receptor to Rab5- and phosphatidylinositol 3-phosphate (Pl3P)-positive early endosomes and subsequent trafficking through the degradative pathway. Under these conditions, internalization of the Met and EGF receptors was unaltered, suggesting a block at the level of early endosome formation. We show that the N-ethylmaleimide-sensitive factor (NSF), an essential component of the vesicle fusion machinery, was hyperphosphorylated in PTP1B knockdown or PTP1B D/A-expressing cells and was a target for PTP1B. NSF knockdown phenocopied PTP1B knockdown, demonstrating a mechanism through which PTP1B regulates endocytic trafficking. Finally, we show that PTP1B dephosphorylated NSF and that this interaction was required for physiological RTK trafficking and appropriate attenuation of downstream signaling.