Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated Src-related intracellular tyrosine kinase. Although not expressed in the normal mammary gland, PTK6 is expressed in a majority of human breast tumors examined, and it has been linked to ErbB receptor signaling and AKT activation. Here we demonstrate that AKT is a direct substrate of PTK6 and that AKT tyrosine residues 315 and 326 are phosphorylated by PTK6. Association of PTK6 with AKT occurs through the SH3 domain of PTK6 and is enhanced through SH2 domain-mediated interactions following tyrosine phosphorylation of AKT. Using Src, Yes, and Fyn null mouse embryonic fibroblasts (SYF cells), we show that PTK6 phosphorylates AKT in a Src family kinase-independent manner. Introduction of PTK6 into SYF cells sensitized these cells to physiological levels of epidermal growth factor (EGF) and increased AKT activation. Stable introduction of active PTK6 into SYF cells also resulted in increased proliferation. Knockdown of PTK6 in the BPH-1 human prostate epithelial cell line led to decreased AKT activation in response to EGF. Our data indicate that in addition to promoting growth factor receptor-mediated activation of AKT, PTK6 can directly activate AKT to promote oncogenic signaling.Protein tyrosine kinase 6 (PTK6; also known as the breast tumor kinase BRK) is an intracellular Src-related tyrosine kinase (9, 48). Human PTK6 was identified in cultured human melanocytes (32) and breast tumor cells (39), while its mouse orthologue was cloned from normal small intestinal epithelial cell RNA (50). Although PTK6 shares overall structural similarity with Src family tyrosine kinases, it lacks an N-terminal myristoylation consensus sequence for membrane targeting (39, 51). As a consequence, PTK6 is localized to different cellular compartments, including the nucleus (14, 15). PTK6 is expressed in normal differentiated epithelial cells of the gastrointestinal tract (34, 42, 51), prostate (14), and skin (51-53). Expression of PTK6 is upregulated in different types of cancers, including breast carcinomas (6, 39, 54), colon cancer (34), ovarian cancer (47), head and neck cancers (33), and metastatic melanoma cells (16). The significance of apparent opposing signaling roles for PTK6 in normal differentiation and cancer is still poorly understood.In human breast tumor cells, PTK6 enhances signaling from members of the ErbB receptor family (10, 29, 30, 36, 40, 49, 54). In the HB4a immortalized human mammary gland luminal epithelial cell line, PTK6 promoted epidermal growth factor (EGF)-induced ErbB3 tyrosine phosphorylation and AKT activation (29). In response to EGF stimulation, PTK6 promoted phosphorylation of the focal adhesion protein paxillin and Rac1-mediated cell migration (10). PTK6 can be activated by the ErbB3 ligand heregulin and promotes activation of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein kinase (MAPK) in breast cancer cells (40). PTK6 can also phosphorylate p190RhoGAP-A and stimulate its activity, leading to RhoA inactivation and Ras activation and thereby promoting EGF-dependent breast cancer cell migration and proliferation (49). Expression of PTK6 has been correlated with ErbB2 expression in human breast cancers (4, 5, 54).AKT (also called protein kinase B) is a serine-threonine kinase that is activated downstream of growth factor receptors (38). It is a key player in signaling pathways that regulate energy metabolism, proliferation, and cell survival (7, 45). Aberrant activation of AKT through diverse mechanisms has been discovered in different cancers (2). AKT activation requires phosphorylation of AKT on threonine residue 308 and serine residue 473. The significance of phosphorylation of AKT on tyrosine residues is less well understood. Src has been shown to phosphorylate AKT on conserved tyrosine residues 315 and 326 near the activation loop (11). Substitution of these two tyrosine residues with phenylalanine abolished AKT kinase activity stimulated by EGF (11). Use of the Src family inhibitor PP2 impaired AKT activation following IGF-1 stimulation of oligodendrocytes (13). The RET/PTC receptor tyrosine kinase that responds to glial cell-line-derived neurotrophic factor also phosphorylated AKT tyrosine residue 315 promoting activation of AKT (28). AKT tyrosine residue 474 was phosphorylated when cells were treated with the tyrosine phosphatase inhibitor pervanadate, and phosphorylation of tyrosine 474 contributed to full activation of AKT (12). Recently, the nonreceptor tyrosine kinase Ack1 was shown to regulate AKT tyrosine phosphorylation and activation (37).Here we show that AKT is a cytoplasmic substrate of the intracellular tyrosine kinase PTK6. We identify the tyrosine residues on AKT that are targeted by PTK6, and we demonstrate that tyrosine phosphorylation plays a role in regulating association between PTK6 and AKT. In addition, we show that PTK6 promotes AKT activation and cell proliferation in a Src-independent manner.     

c-Src Associates with ErbB2 through an Interaction between Catalytic Domains and Confers Enhanced Transforming Potential

Previous studies have demonstrated that c-Src tyrosine kinase interacts specifically with ErbB2, but not with other members of the epidermal growth factor receptor (EGFR) family. To identify the site of interaction, we recently used a chimeric EGFR/ErbB2 receptor approach to show that c-Src requires the kinase region of ErbB2 for binding. Here, we demonstrate that retention of a conserved amino acid motif surrounding tyrosine 877 (referred to here as EGFR^YHAD) is sufficient to confer binding to c-Src. Surprisingly the association of c-Src was not dependent on its SH2 or SH3 domain or on the phosphorylation or kinase activity of the receptor. We further show that the chimeric EGFRs that contain the Y877 motif are transforming in vitro and in vivo following ligand stimulation. Transformation was also partially dependent on sustained activation of Stat3. Finally, we demonstrate that EGFRs with mutations in the catalytic domain, originally identified in lung cancer and conferring increased sensitivity to gefitinib and erlotinib, two EGFR kinase inhibitors, gained the capacity to bind c-Src. Moreover, transformation by these EGFR mutants was inhibited by Src inhibitors regardless of their sensitivities to gefitinib and erlotinib. These observations have important implications for understanding the molecular basis for resistance to EGFR inhibitors and implicate c-Src as a critical signaling molecule in EGFR mutant-induced transformation.The epidermal growth factor receptor (EGFR) family is comprised of four members, EGFR, ErbB2, ErbB3, and ErbB4, with distinct ligand specificities, which, upon homo- or heterodimerization after ligand binding, autophosphorylate and recruit different effector proteins to specific tyrosine residues located in their cytoplasmic tails. These signaling molecules, which are either adapter molecules that recruit other kinases or kinases themselves, mediate diverse functions, such as proliferation, growth, and survival (27). There are now several pieces of evidence demonstrating that these growth factor receptors are mutated or overexpressed in a variety of different cancers, including salivary gland adenocarcinoma (44), breast cancer (47), esophageal squamous carcinoma (22), bladder cancer (58), and lung cancer (57). Accordingly, ErbB2 is overexpressed in 20 to 30% of all human breast cancer, which correlates with poor prognosis, and in 40 to 60% of ductal carcinoma in situ (19). ErbB2 is 100-fold more potent in its transforming ability than ErbB1/EGFR, although the two receptors are 85% homologous (14, 15). Breast carcinoma cells devoid of ErbB2, but not other ErbB receptor family members, are defective in cell invasion upon EGF ligand stimulation (49). In fact, ErbB2 could induce cell migration when overexpressed in cells devoid of any other ErbB receptors. In a three-dimensional cell culture system, overexpression of ErbB2, but not EGFR, disrupts mammary acinus structure by reinitiating cell proliferation, leading to an absence of lumen and disruption of tight junctions and of cell polarity, although the cells still lack invasive properties (31).Src is a nonreceptor tyrosine kinase implicated in signal transduction pathways downstream of multiple receptors, such as platelet-derived growth factor, insulin receptor, G-coupled receptors, and ErbB family receptors, where it regulates a wide variety of cellular functions that include proliferation, migration, and apoptosis (17). Src tyrosine kinase activity is sporadically increased in many cases of human cancer, including colon and breast cancer (10, 38, 52). Moreover, Src kinase activity is elevated in ErbB2-induced mammary tumors (33). Direct evidence supporting a role in mammary tumor progression derives from observations made in transgenic mice. Constitutive activation of c-Src in mammary epithelia led to frequent mammary epithelial hyperplasias, which occasionally developed into solid tumors (54). Conversely, deletion of c-Src in a mouse mammary tumor virus/polyomavirus middle T-antigen (PyMT) transgenic strain abrogates mammary tumor formation (21).c-Src is also an important player downstream of the EGFR family. Phosphorylation of several tyrosine residues within the EGFR has been demonstrated to be increased following c-Src overexpression both in vitro and in vivo, suggesting that c-Src is required for full biological response following EGF stimulation (29, 51). In addition to EGFR, c-Src specifically interacts with tyrosine-phosphorylated ErbB2 in ErbB2-induced mammary tumors. This association was further demonstrated to result in enhanced c-Src kinase activity (3, 28, 34, 35). More recently, using chimeric EGF/ErbB2 receptors, we demonstrated that c-Src specifically associates with ErbB2, but not with other ErbB family members. c-Src was demonstrated to specifically associate with the ErbB2 kinase domain (24). Moreover, the chimeric EGFR that contained the c-Src binding site was able to disrupt cell polarity and cell-cell junctions to induce epithelial cell scattering in a three-dimensional cell culture system in a MAPK-dependent manner (24).Here, we demonstrate that c-Src association with ErbB2 is conformation dependent and that the residues necessary for interaction are centered around Y877 in the kinase domain of ErbB2, an association that is further strengthened by residues located in the amino-terminal part of the kinase domain. This association was not dependent on the SH2 or SH3 domain or the kinase activity of c-Src or ErbB2. We further show that mammary epithelial cells expressing the EGFR/ErbB2 chimeric receptors that have regained the capacity to associate with c-Src have disrupted epithelial polarity that is correlated with enhanced transforming potential, an effect dependent on c-Src kinase activity and Stat3 activation. Finally, we show that mutant EGFRs isolated from lung adenocarcinomas have the capacity to associate with c-Src and that these EGFR mutants require Src kinase activity for transformation.     

Semaphorin 4D Signaling Requires the Recruitment of Phospholipase Cγ into the Plexin-B1 Receptor Complex

The semaphorin 4D (Sema4D) receptor plexin-B1 constitutively interacts with particular Rho guanine nucleotide exchange factors (RhoGEFs) and thereby mediates Sema4D-induced RhoA activation, a process which involves the tyrosine phosphorylation of plexin-B1 by ErbB-2. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGEF activity. We show here that activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation creates docking sites for the SH2 domains of phospholipase Cγ (PLCγ). PLCγ is thereby recruited into the plexin-B1 receptor complex and via its SH3 domain activates the Rho guanine nucleotide exchange factor PDZ-RhoGEF. PLCγ-dependent RhoGEF activation is independent of its lipase activity. The recruitment of PLCγ has no effect on the R-Ras GTPase-activating protein activity of plexin-B1 but is required for Sema4D-induced axonal growth cone collapse as well as for the promigratory effects of Sema4D on cancer cells. These data demonstrate a novel nonenzymatic function of PLCγ as an important mechanism of plexin-mediated signaling which links tyrosine phosphorylation of plexin-B1 to the regulation of a RhoGEF protein and downstream cellular processes.Mammalian semaphorins were originally identified as axon guidance factors but are now recognized also as important regulators of morphogenesis and homeostasis in various organ systems, including the immune, cardiovascular, and renal systems (3-5, 7, 19, 23, 30, 35, 40, 56, 64, 76). Most effects of semaphorins are mediated by a group of large transmembrane proteins called plexins, of which four families exist in the mammalian system: plexin-A1 to -4, plexin-B1 to -3, plexin-C1, and plexin-D1 (60, 61). The four members of the plexin-A family in most cases require neuropilins as ligand binding partners to respond to semaphorins, whereas the three members of the plexin-B family are directly activated by semaphorins. While plexin-B1 binds Sema4D, plexin-B2 can be activated by Sema4C and Sema4D, and plexin-B3 has been shown to respond to Sema5A (31, 35).The activation of plexins by semaphorins initiates a variety of signaling processes, which involve several small GTPases of the Ras and Rho families (31, 34, 43). All plexin family members possess an R-Ras GTPase-activating protein (GAP) domain (36). Activated plexin-B1 and -A1 have been shown to also interact with other small GTPases, including GTP-bound Rac1 and RhoD as well as Rnd1, Rnd2, and Rnd3 (14, 37, 48, 63, 67, 68, 74). Different from other plexin families, the C terminus of B-family plexins contains a PDZ domain-binding motif which mediates a stable interaction with the guanine nucleotide exchange factors PDZ-RhoGEF and LARG (1, 15, 26, 39, 57). Activation of the plexin-B1/PDZ-RhoGEF complex by semaphorin 4D (Sema4D) results in RhoA activation downstream of plexin-B1 (15, 39, 57). Members of the plexin-B family also interact with and are phosphorylated by the receptor tyrosine kinases ErbB-2 and c-Met (12, 22, 58). ErbB-2-mediated phosphorylation of plexin-B1 is required for plexin-mediated RhoA activation and downstream cellular effects, including the promigratory effects of Sema4D on cancer cells and the induction of axonal growth cone collapse by Sema4D (58, 59). However, the molecular mechanisms linking ErbB-2-mediated phosphorylation of plexin-B1 to the regulation of RhoA activity and subsequent cellular effects are unknown.Here we report that upon activation by Sema4D, plexin-B1 becomes phosphorylated by ErbB-2 at particular tyrosine residues on its intracellular portion. These phosphorylated tyrosine residues serve as docking sites for the SH2 domains of PLCγ. PLCγ is thereby recruited into the plexin-B1 receptor complex and through its SH3 domain mediates RhoA activation and downstream cellular effects.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Proteorhodopsin-Bearing Bacteria in Antarctic Sea Ice

Proteorhodopsins (PRs) are widespread bacterial integral membrane proteins that function as light-driven proton pumps. Antarctic sea ice supports a complex community of autotrophic algae, heterotrophic bacteria, viruses, and protists that are an important food source for higher trophic levels in ice-covered regions of the Southern Ocean. Here, we present the first report of PR-bearing bacteria, both dormant and active, in Antarctic sea ice from a series of sites in the Ross Sea using gene-specific primers. Positive PR sequences were generated from genomic DNA at all depths in sea ice, and these sequences aligned with the classes Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria. The sequences showed some similarity to previously reported PR sequences, although most of the sequences were generally distinct. Positive PR sequences were also observed from cDNA reverse transcribed from RNA isolated from sea ice samples. This finding indicates that these sequences were generated from metabolically active cells and suggests that the PR gene is functional within sea ice. Both blue-absorbing and green-absorbing forms of PRs were detected, and only a limited number of blue-absorbing forms were found and were in the midsection of the sea ice profile in this study. Questions still remain regarding the protein''s ecological functions, and ultimately, field experiments will be needed to establish the ecological and functional role of PRs in the sea ice ecosystem.Proteorhodopsins (PRs) are retinal binding bacterial integral membrane proteins that function as light-driven proton pumps (9, 10) and belong to the microbial rhodopsin superfamily of proteins (54). Since the first reported PR sequence from members of SAR86 clade marine (class Gammaproteobacteria) in 2000 (9), many other PR-bearing bacteria have been identified in a range of marine habitats (5, 18, 20, 24, 25, 46, 62). In the recent Global Ocean Sampling (GOS) expedition, almost 4,000 PR sequences from 41 distinct surface marine environments were acquired, demonstrating that these PR genes are extremely abundant in the genomes of ocean bacterioplankton (46). In fact, PR-containing bacteria account for 13% of the community in the Mediterranean Sea and Red Sea and 70% of the community in the Sargasso Sea (18, 46, 49, 60). These light-harvesting bacteria are present in three major marine classes of bacteria: the Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria. In addition, two distinct PR genes encode pigments with “blue-absorbing” and “green-absorbing” properties, which is achieved by a substitution at a single amino acid position, which thereby functions as a spectral tuning switch (10, 37, 48).Sea ice represents a complex physicochemical environment in polar regions and covers up to 13% of the Earth''s surface (59). Although extreme gradients of temperature, salinity, nutrient availability, and light stratify the ice matrix from the surface to the ice-water interface (41), the sea ice habitat nevertheless supports a diverse microbial community of phytoplankton, Bacteria, Archaea, viruses, and protists that grow in liquid brine channels within the ice (14, 35, 56). This sea ice microbial community (SIMCO) is highly metabolically active despite being unable to avoid the extreme environmental conditions that they experience (39). In fact, very-high-standing stocks of the SIMCO exist in many regions of the Southern Ocean. For example, the concentration of chlorophyll a, a proxy for microalgal biomass, typically reaches 200 mg m⁻² in the Ross Sea, while the concentration of chlorophyll a in the water column below is approximately 2 orders of magnitude less (47), and the percentage of metabolically active bacteria (32% [39]) is significantly higher than the 10% observed for temperate marine systems (36). The SIMCO is thus a major source of biomass in ice-covered regions of the Southern Ocean (59), providing a critical food source for grazing zooplankton (and, consequently, also for higher trophic levels) for much of the year (3, 59). This biomass is of particular importance during the darkness of the polar winter, where the bottom-ice community is the only available food source for juvenile krill. These grazers absolutely rely on the sea ice microbial community to survive, as the water lacks other food sources (6, 28).In the past decade, reports of the widespread occurrence of bacteriochlorophyll and PR pigments in planktonic marine bacteria have challenged the assumption that chlorophyll a is the only principal light-capturing pigment in ocean surface waters. These alternative pigments may in fact play a critical role in light energy harvesting for microbial metabolism in various aquatic ecosystems (5, 10, 25, 40, 49). It has been proposed that energy, rather than nutrient conservation, is important for the regulation of productivity (7). PR-containing phototrophic eubacteria could play a significant role in the energy budget of cells in the photic zone in marine environments (15). PR sequences have been detected in the Southern Ocean (9), but to our knowledge, there have been no reports of PR-bearing bacteria within the sea ice matrix.The majority of the microbial rhodopsin genes found in oceanic samples have been detected by environmental sequencing (30, 46, 48, 60). We have used degenerate PR gene primers (5) in this study to positively identify PR-bearing operational taxonomic units (OTUs) from sea ice. Also, specific bacterial mRNA can now be detected from extracted nucleic acids and used to examine gene expression and, thus, infer metabolic activity (8). With this in mind, we have generated cDNA from RNA extracted from sea ice samples. From these observations, we deduce that PR-bearing bacteria are present in sea ice and may be actively contributing to the ecosystem within this extreme microenvironment.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.Angiogenesis, the formation of new blood vessels, involves several well-coordinated cellular processes, including endothelial cell (EC) migration, synthesis and deposition of extracellular matrix proteins, such as fibronectin, cell-cell adhesion, and formation of branching point structures (1-3, 19, 33); however, less is known about the underlying mechanisms of these processes (6, 8, 12, 14, 16, 17). For example, adherens junctions (AJs), which mediate cell-cell adhesion between ECs, may be involved in limiting the extent of cell migration (2, 14, 38, 40). VE-cadherin, a protein found in AJs, is a single-pass transmembrane polypeptide responsible for calcium-dependent homophilic interactions through its extracellular domains (2, 38, 40). The VE-cadherin cytoplasmic domain interacts with the Armadillo domain-containing proteins, β-catenin, γ-catenin (plakoglobin), and p120-catenin (p120ctn) (2, 15, 38, 40, 43). Genetic and biochemical evidence documents a crucial role of β-catenin in regulating cell adhesion as well as proliferation secondary to the central position of β-catenin in the Wnt signaling pathway (13, 16, 25, 31, 44). In addition, the juxtamembrane protein p120ctn regulates AJ stability via binding to VE-cadherin (2, 7, 9, 15, 21, 28, 32, 43). The absence of regulation or inappropriate regulation of β-catenin and VE-cadherin functions is linked to cardiovascular disease and tumor progression (2, 6).We previously identified lipid phosphate phosphatase 3 (LPP3), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis (18, 19, 41, 42). LPP3 not only exhibits lipid phosphatase activity but also functions as a cell-associated integrin ligand (18, 19, 35, 41, 42). The known LPPs (LPP1, LPP2, and LPP3) (20-23) are six transmembrane domain-containing plasma membrane-bound enzymes that dephosphorylate sphingosine-1-phosphate (S1P) and its structural homologues, and thus, these phosphatases generate lipid mediators (4, 5, 23, 35, 39). All LPPs, which contain a single N-glycosylation site and a putative lipid phosphatase motif, are situated such that their N and C termini are within the cell (4, 5, 22, 23, 35, 39). Only the LPP3 isoform contains an Arg-Gly-Asp (RGD) sequence in the second extracellular loop, and this RGD sequence enables LPP3 to bind integrins (18, 19, 22). Transfection experiments with green fluorescent protein (GFP)-tagged LPP1 and LPP3 showed that LPP1 is apically sorted, whereas LPP3 colocalized with E-cadherin at cell-cell contact sites with other Madin-Darby canine kidney (MDCK) cells (22). Mutagenesis and domain swapping experiments established that LPP1 contains an apical targeting signal sequence (FDKTRL) in its N-terminal segment. In contrast, LPP3 contains a dityrosine (109Y/110Y) basolateral sorting motif (22). Interestingly, conventional deletion of Lpp3 is embryonic lethal, since the Lpp3 gene plays a critical role in extraembryonic vasculogenesis independent of its lipid phosphatase activity (11). In addition, an LPP3-neutralizing antibody was shown to prevent cell-cell interactions (19, 42) and angiogenesis (42). Here, we addressed the hypothesis that LPP3 plays a key role in EC migration, cell-cell adhesion, and formation of branching point structures by stimulating β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) signaling.