The receptor for urokinase-type plasminogen activator regulates fibronectin matrix assembly in human skin fibroblasts

Previous studies have indicated that the receptor for urokinase-type plasminogen activator, uPAR, can form functional complexes with integrin receptors thereby modulating integrin activity. In the present study, the role of uPAR in the regulation of alpha5beta1-dependent polymerization of the fibronectin matrix was investigated. Incubation of fibroblast monolayers with the P-25 peptide, a uPAR ligand, resulted in a 12-15-fold increase in the accumulation of exogenous fibronectin in the cell layer. The exogenous fibronectin co-localized in the extracellular matrix with endogenous cell-derived fibronectin, and its deposition into the matrix was inhibited by blocking antibodies against the beta1 integrin receptor. The P-25-dependent increase in fibronectin assembly was associated with a 7-8-fold increase in the expression of matrix assembly sites as well as a 37-fold increase in the rate of transfer of cell surface-bound fibronectin into a detergent-insoluble matrix. The effects of P-25 on the matrix assembly were attenuated by incubating cells with either phospholipase C or with antibodies against uPAR, confirming a role for uPAR in the P-25-dependent increase in matrix assembly. P-25-treated cells exhibited a 10-fold increase in the binding of the 120-kDa cell-binding fragment of fibronectin suggesting an increase in alpha5beta1 affinity for fibronectin. Consistent with this, treatment of cells with P-25 also resulted in a 6-10-fold increase in the binding of two different monoclonal antibodies that recognize the active conformation of the beta1 integrin. These results indicate that P-25 increases matrix assembly by altering the activation state of the alpha5beta1 integrin receptor and suggest that changes in integrin activation affect both the number of matrix assembly sites as well as the rate of transfer of cell-bound fibronectin into a detergent-insoluble matrix. These data provide direct evidence that uPAR and integrin receptors synergistically regulate the levels of fibronectin in the extracellular matrix.     

Src-dependent tyrosine phosphorylation regulates dynamin self-assembly and ligand-induced endocytosis of the epidermal growth factor receptor

Endocytosis of ligand-activated receptors requires dynamin-mediated GTP hydrolysis, which is regulated by dynamin self-assembly. Here, we demonstrate that phosphorylation of dynamin I by c-Src induces its self-assembly and increases its GTPase activity. Electron microscopic analyses reveal that tyrosine-phosphorylated dynamin I spontaneously self-assembles into large stacks of rings. Tyrosine 597 was identified as being phosphorylated both in vitro and in cultured cells following epidermal growth factor receptor stimulation. The replacement of tyrosine 597 with phenylalanine impairs Src kinase-induced dynamin I self-assembly and GTPase activity in vitro. Expression of Y597F dynamin I in cells attenuates agonist-driven epidermal growth factor receptor internalization. Thus, c-Src-mediated tyrosine phosphorylation is required for the function of dynamin in ligand-induced signaling receptor internalization.     

Interferon gamma-dependent transactivation of epidermal growth factor receptor

The present report provides evidence that, in A431 cells, interferon gamma (IFNgamma) induces the rapid (within 5 min), and reversible, tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). IFNgamma-induced EGFR transactivation requires EGFR kinase activity, as well as activity of the Src-family tyrosine kinases and JAK2. Here, we show that IFNgamma-induced STAT1 activation in A431 and HeLa cells partially depends on the kinase activity of both EGFR and Src. Furthermore, in these cells, EGFR kinase activity is essential for IFNgamma-induced ERK1,2 activation. This study is the first to demonstrate that EGFR is implicated in IFNgamma-dependent signaling pathways.     

Membrane-type 1 matrix metalloproteinase stimulates cell migration through epidermal growth factor receptor transactivation

Proteolysis of extracellular matrix proteins by membrane-type 1 matrix metalloproteinase (MT1-MMP) plays a pivotal role in tumor and endothelial cell migration. In addition to its proteolytic activity, several studies indicate that the proinvasive properties of MT1-MMP also involve its short cytoplasmic domain, but the specific mechanisms mediating this function have yet to be fully elucidated. Having previously shown that the serum factor sphingosine 1-phosphate stimulates MT1-MMP promigratory function through a process that involves its cytoplasmic domain, we now extend these findings to show that this cooperative interaction is permissive to cellular migration through MT1-MMP-dependent transactivation of the epidermal growth factor receptor (EGFR). In the presence of sphingosine 1-phosphate, MT1-MMP stimulates EGFR transactivation through a process that is dependent upon the cytoplasmic domain of the enzyme but not its catalytic activity. The MT1-MMP-induced EGFR transactivation also involves G(i) protein signaling and Src activities and leads to enhanced cellular migration through downstream extracellular signal-regulated kinase activation. The present study, thus, elucidates a novel role of MT1-MMP in signaling events mediating EGFR transactivation and provides the first evidence of a crucial role of this receptor activity in MT1-MMP promigratory function. Taken together, our results suggest that the inhibition of EGFR may represent a novel target to inhibit MT1-MMP-dependent processes associated with tumor cell invasion and angiogenesis.     

Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells

We have recently reported that osteopontin (OPN) stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase-type plasminogen activator (uPA) through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells (Das, R., Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 28593-28606). However, the role(s) of OPN on AP-1-mediated uPA secretion and cell motility and the involvement of c-Src/epidermal growth factor receptor (EGFR) in these processes in breast cancer cells are not well defined. In this study we report that OPN induces alpha(v)beta(3) integrin-mediated c-Src kinase activity in both highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. Ligation of OPN with alpha(v)beta(3) integrin induces kinase activity and tyrosine phosphorylation of EGFR in MDA-MB-231 and wild type EGFR-transfected MCF-7 cells, and this was inhibited by the dominant negative form of c-Src (dn c-Src) indicating that c-Src kinase plays a crucial role in this process. OPN induces association between alpha(v)beta(3) integrin and EGFR on the cell membrane in a macromolecular form with c-Src. Furthermore, OPN induces alpha(v)beta(3) integrin/EGFR-mediated ERK1/2 phosphorylation and AP-1 activation. Moreover, dn c-Src also suppressed the OPN-induced phosphatidylinositol (PI) 3-kinase activity in these cells indicating that c-Src acts as master switch in regulating MEK/ERK1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways. OPN-induced ERK phosphorylation, AP-1 activation, uPA secretion, and cell motility were suppressed when cells were transfected with dn c-Src or pretreated with alpha(v)beta(3) integrin antibody, c-Src kinase inhibitor (pp2), EGFR tyrosine kinase inhibitor (PD153035), and MEK-1 inhibitor (PD98059). To our knowledge, this is the first report that OPN induces alpha(v)beta(3) integrin-mediated AP-1 activity and uPA secretion by activating c-Src/EGFR/ERK signaling pathways and further demonstrates a functional molecular link between OPN-induced integrin/c-Src-dependent EGFR phosphorylation and ERK/AP-1-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.     

Urokinase-type plasminogen activator receptor regulates apoptotic sensitivity of colon cancer HCT116 cell line to TRAIL via JNK-p53 pathway

The urokinase-type plasminogen activator receptor (uPAR) serves not only as an anchor for urokinase-type plasminogen activator but also participates in intracellular signal transduction events. In this study, we investigated whether uPAR could modulate TRAIL-induced apoptosis in human colon cancer cells HCT116. Using an antisense strategy, we established a stable HCT116 cell line with down-regulated uPAR. The sensitivity to TRAIL-induced apoptosis was evaluated by FACS analysis. Our results show that the inhibition of uPAR could sensitize HCT116 to TRAIL-induced apoptosis. uPAR inhibition changed the expression of mitochondrial apoptotic pathway proteins, including Bcl-2, Bax, Bid and p53, in a pro-apoptotic manner. We also found that the inhibition of uPAR down-regulated the phosphorylation of FAK, ERK and JNK. The inhibition of p53 by RNA interference rescued cells from enhanced apoptosis, thus indicating that p53 is critical for enhancing TRAIL-induced apoptosis. Furthermore, JNK, but not ERK, inhibition involved in the up-regulation of p53. JNK negatively regulated p53 protein level. Overall, our results show that uPAR inhibition can sensitize colon cancer cells HCT116 to TRAIL-induced apoptosis via active p53 and mitochondrial apoptotic pathways that JNK inhibition is involved.     

alpha 2B-adrenergic receptor activates MAPK via a pathway involving arachidonic acid metabolism, matrix metalloproteinases, and epidermal growth factor receptor transactivation

We have investigated the mechanisms whereby alpha(2B)-adrenergic receptor (alpha(2B)-AR) promotes MAPK activation in a clone of the renal tubular cell line, LLC-PK1, transfected with the rat nonglycosylated alpha(2)-AR gene. Treatment of LLC-PK1-alpha(2B) with UK14304 or dexmedetomidine caused arachidonic acid (AA) release and ERK2 phosphorylation. AA release was abolished by prior treatment of the cells with pertussis toxin, quinacrine, or methyl arachidonyl fluorophosphonate but not by the addition of the MEK inhibitor U0126. The effects of alpha(2)-agonists on MAPK phosphorylation were mimicked by cell exposure to exogenous AA. On the other hand, quinacrine abolished the effects of UK14304, but not of AA, suggesting that AA released through PLA2 is responsible for MAPK activation by alpha(2B)-AR. The effects of alpha(2)-agonists or AA were PKC-independent and were attenuated by indomethacin and nordihydroguaiaretic acid. Treatment with batimastat, CRM 197, or tyrphostin AG1478 suppressed MAPK phosphorylation promoted by alpha(2)-agonist or AA. Furthermore, conditioned culture medium from UK14304-treated LLC-PK1-alpha(2B) induced MAPK phosphorylation in wild-type LLC-PK1. Based on these data, we propose a model whereby activation of MAPK by alpha(2B)-AR is mediated through stimulation of PLA2, AA release, generation of AA derivatives, activation of matrix metalloproteinases, release of heparin-binding EGF-like growth factor, transactivation of epidermal growth factor receptor, and recruitment of Shc. Whether this pathway is particular to alpha(2B)-AR and LLC-PK1 or whether it can be extended to other cell types and/or other G-protein-coupled receptors remains to be established.     

Urokinase-type plasminogen activator up-regulates the expression of its cellular receptor

The expression of the receptor for the urokinase-type plasminogen activator (uPAR) can be regulated by several hormones, cytokines, tumor promoters, etc. Recently, it has been reported that uPAR is capable of transducing signals, even though it is lacking a transmembrane domain and a cytoplasmatic tail. We now report that uPAR cell surface expression can be positively regulated by its ligand, uPA, in thyroid cells. The effect of uPA is independent of its proteolytic activity, since inactivated uPA or its aminoterminal fragment have the same effects of the active enzyme. The increase of uPAR on the cell surface correlates with an increase of specific uPAR mRNA. Finally, uPA up-regulates uPAR expression also in other cell lines of different type and origin, thus suggesting that the regulatory role of uPA on uPAR expression is not restricted to thyroid cells, but it occurs in different tissues, both normal and tumoral.     

Urokinase-type plasminogen activator receptor signaling is critical in nasopharyngeal carcinoma cell growth and metastasis

Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in southern China and Southeast Asia, with the highest metastasis rate among head and neck cancers. The mechanisms underlying NPC progression remain poorly understood. Genome-wide expression profiling on 18 NPC vs. 18 noncancerous nasopharyngeal tissues together with GeneGo pathway analysis and expression verification in NPC cells and tissues revealed a potential role of urokinase-type plasminogen activator receptor (uPAR) in NPC progression, which has not been investigated in NPC. We then observed that uPAR expression is increased in poorly differentiated, highly metastatic NPC cells compared with lowly metastatic cells or differentiated NPC cells. In vitro studies demonstrated that uPAR regulates NPC cell growth, colony formation, migration, and invasion and promotes the epithelial–mesenchymal transition (EMT). Additional tumor xenograft and spontaneous metastasis experiments revealed that uPAR promotes NPC cell growth and metastasis in vivo. The JAK–STAT pathway is involved in uPAR-regulated signaling in NPC cells as determined by immunoblotting. Moreover, uPAR-mediated growth and motility is partially abolished upon treatment with the Jak1/Jak2 inhibitor INCB018424. We suppressed uPA expression in uPAR-overexpressing NPC cells and found that uPAR-mediated cellular growth and motility is not exclusively dependent on uPA. In summary, uPAR is a significant regulator of NPC progression and could serve as a promising therapeutic target.     

Neuropeptide-induced transactivation of a neuronal epidermal growth factor receptor is mediated by metalloprotease-dependent formation of heparin-binding epidermal growth factor

Numerous external stimuli, including G protein-coupled receptor agonists, cytokines, growth factors, and steroids activate mitogen-activated protein kinases (MAPKs) through phosphorylation of the epidermal growth factor receptor (EGF-R). In immortalized hypothalamic neurons (GT1-7 cells), agonist binding to the gonadotropin-releasing hormone receptor (GnRH-R) causes phosphorylation of MAPKs that is mediated by protein kinase C (PKC)-dependent transactivation of the EGF-R. An analysis of the mechanisms involved in this process showed that GnRH stimulation of GT1-7 cells causes release/shedding of the soluble ligand, heparin binding epidermal growth factor (HB-EGF), as a consequence of metalloprotease activation. GnRH-induced phosphorylation of the EGF-R and, subsequently, of Shc, ERK1/2, and its dependent protein, p90RSK-1 (p90 ribosomal S6 kinase 1 or RSK-1), was abolished by metalloprotease inhibition. Similarly, blockade of the effect of HB-EGF with the selective inhibitor CRM197 or a neutralizing antibody attenuated signals generated by GnRH and phorbol 12-myristate 13-acetate, but not those stimulated by EGF. In contrast, phosphorylation of the EGF-R, Shc, and ERK1/2 by EGF and HB-EGF was independent of PKC and metalloprotease activity. The signaling characteristics of HB-EGF closely resembled those of GnRH and EGF in terms of the phosphorylation of EGF-R, Shc, ERK1/2, and RSK-1 as well as the nuclear translocation of RSK-1. However, neither the selective Src kinase inhibitor PP2 nor the overexpression of negative regulatory Src kinase and dominant negative Pyk2 had any effect on HB-EGF-induced responses. In contrast to GT1-7 cells, human embryonic kidney 293 cells expressing the GnRH-R did not exhibit metalloprotease induction and EGF-R transactivation during GnRH stimulation. These data indicate that the GnRH-induced transactivation of the EGF-R and the subsequent ERK1/2 phosphorylation result from ectodomain shedding of HBEGF through PKC-dependent activation of metalloprotease(s) in neuronal GT1-7 cells.     

A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly

Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.     

Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13-mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.     

Presenilin 1 regulates epidermal growth factor receptor turnover and signaling in the endosomal-lysosomal pathway

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.     

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.     

Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor

Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.     

Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.     

Coordinate regulation of fibronectin matrix assembly by the plasminogen activator system and vitronectin in human osteosarcoma cells

Background  

Plasminogen activators are known to play a key role in the remodeling of bone matrix which occurs during tumor progression, bone metastasis and bone growth. Dysfunctional remodeling of bone matrix gives rise to the osteoblastic and osteolytic lesions seen in association with metastatic cancers. The molecular mechanisms responsible for the development of these lesions are not well understood. Studies were undertaken to address the role of the plasminogen activator system in the regulation of fibronectin matrix assembly in the osteoblast-like cell line, MG-63.     

Receptor-type protein-tyrosine phosphatase-kappa regulates epidermal growth factor receptor function

Epidermal growth factor receptor (EGFR), the prototypic receptor protein tyrosine kinase, is a major regulator of growth and survival for many epithelial cell types. We report here that receptor-type protein-tyrosine phosphatase-kappa (RPTP-kappa) dephosphorylates EGFR and thereby regulates its function in human keratinocytes. Protein-tyrosine phosphatase (PTP) inhibitors induced EGFR tyrosine phosphorylation in intact primary human keratinocytes and cell-free membrane preparations. Five highly expressed RPTPs (RPTP-beta, delta, kappa, mu, and xi) were functionally analyzed in a Chinese hamster ovary (CHO) cell-based expression system. Full-length human EGFR expressed in CHO cells, which lack endogenous EGFR, displayed high basal (i.e. in the absence of ligand) tyrosine phosphorylation. Co-expression of RPTP-kappa, but not other RPTPs, specifically reduced basal EGFR tyrosine phosphorylation. RPTP-kappa also reduced epidermal growth factor-dependent EGFR tyrosine phosphorylation in CHO cells. Purified RPTP-kappa preferentially dephosphorylated EGFR tyrosines 1068 and 1173 in vitro. Overexpression of wild-type or catalytically inactive RPTP-kappa reduced or enhanced, respectively, basal and EGF-induced EGFR tyrosine phosphorylation in human keratinocytes. Furthermore, siRNA-mediated knockdown of RPTP-kappa increased basal and EGF-stimulated EGFR tyrosine phosphorylation and augmented downstream Erk activation in human keratinocytes. RPTP-kappa levels increased in keratinocytes as cells reached confluency, and overexpression of RPTP-kappa in subconfluent keratinocytes reduced keratinocyte proliferation. Taken together, the above data indicate that RPTP-kappa is a key regulator of EGFR tyrosine phosphorylation and function in human keratinocytes.