A Function for Phosphatidylinositol 3-Kinase β (p85α-p110β) in Fibroblasts during Mitogenesis: Requirement for Insulin- and Lysophosphatidic Acid-Mediated Signal Transduction

We have previously shown that phosphatidylinositol 3-kinase α (PI 3-Kα) (p85α-p110α) is required for DNA synthesis induced by various growth factors (S. Roche, M. Koegl, and S. A. Courtneidge, Proc. Natl. Acad. Sci. USA 91:9185–9189, 1994) in fibroblasts. In the present study, we have investigated the function of PI 3-Kβ (p85α-p110β) during mitogenesis. By using antibodies specific to p110β we showed that PI 3-Kβ is expressed in NIH 3T3 cells. PI 3-Kβ and PI 3-Kα have common features: PI 3-Kβ is tightly associated with a protein serine kinase that phosphorylates p85α, it interacts with the Src-middle T antigen complex and the activated platelet-derived growth factor (PDGF) receptor in fibroblasts in vivo, and it becomes tyrosine phosphorylated after PDGF stimulation. PI 3-Kβ was also activated in Swiss 3T3 and Cos7 cells stimulated with lysophosphatidic acid (LPA), a mitogen that interacts with a heterotrimeric G protein-coupled receptor. In contrast PI 3-Kα was activated to a lesser extent in these cells. Microinjection of neutralizing antibodies specific for p110β into quiescent fibroblasts inhibited DNA synthesis induced by both insulin and LPA but poorly affected PDGF receptor signaling. Therefore, PI 3-Kβ plays an important role in transmitting the mitogenic response induced by some, but not all, growth factors. Finally, we show that while oncogenic V12Ras interacts with type I PI 3-Ks, it could induce DNA synthesis in the absence of active PI 3-Kα and PI 3-Kβ, suggesting that Ras uses other effectors for DNA synthesis.     

Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus

Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNα/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNα/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.     

Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

Interleukin (IL)-12 and IL-23 are composite cytokines consisting of p35/p40 and p19/p40, respectively, which signal via the common IL-12 receptor β1 (IL-12Rβ1) and the cytokine-specific receptors IL-12Rβ2 and IL-23R. Previous data showed that the p40 component interacts with IL-12Rβ1, whereas p19 and p35 subunits solely bind to IL-23R and IL-12Rβ2, resulting in tetrameric signaling complexes. In the absence of p19 and p35, p40 forms homodimers and may induce signaling via IL-12Rβ1 homodimers. The critical amino acids of p19 and p35 required for binding to IL-23R and IL-12Rβ2 are known, and two regions of p40 critical for binding to IL-12Rβ1 have recently been identified. In order to characterize the involvement of the N-terminal region of p40 in binding to IL-12Rβ1, we generated deletion variants of the p40-p19 fusion cytokine. We found that an N-terminal deletion variant missing amino acids M23 to P39 failed to induce IL-23-dependent signaling and did not bind to IL-12Rβ1, whereas binding to IL-23R was maintained. Amino acid replacements showed that p40W37K largely abolished IL-23-induced signal transduction and binding to IL-12Rβ1, but not binding to IL-23R. Combining p40W37K with D36K and T38K mutations eliminated the biological activity of IL-23. Finally, homodimeric p40D36K/W37K/T38K did not interact with IL-12Rβ1, indicating binding of homodimeric p40 to IL-12Rβ1 is comparable to the interaction of IL-23/IL-12 and IL-12Rβ1. In summary, we have defined D36, W37, and T38 as hotspot amino acids for the interaction of IL-12/IL-23 p40 with IL-12Rβ1. Structural insights into cytokine–cytokine receptor binding are important to develop novel therapeutic strategies.     

Gq-coupled Purinergic Receptors Inhibit Insulin-like Growth Factor-I/Phosphoinositide 3-Kinase Pathway-dependent Keratinocyte Migration

Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Gα_(q/11)-coupled-P2Y₂ purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y₂ receptors by extracellular UTP inhibits the IGF-I–induced p110α-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Gα_(q/11)—and not G_(i/o)—independently of phospholipase Cβ. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110α mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I–induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110α mutant, in a Gα(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Gα_(q/11)-coupled receptors, which mediate opposite effects on p110α-PI3K activity and keratinocyte migration.     

Stimulation of β1-Integrin Function by Epidermal Growth Factor and Heregulin-β Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

Integrins and growth factor receptors are important participants in cellular adhesion and migration. The EGF receptor (EGFR) family of tyrosine kinases and the β1-integrin adhesion receptors are of particular interest, given the implication for their involvement in the initiation and progression of tumorigenesis. We used adhesion and chemotaxis assays to further elucidate the relationship between these two families of transmembrane signaling molecules. Specifically, we examined integrin-mediated adhesive and migratory characteristics of the metastatic breast carcinoma cell line MDA-MB-435 in response to stimulation with growth factors that bind to and activate the EGFR or erbB3 in these cells. Although ligand engagement of the EGFR stimulated modest β1-dependent increases in cell adhesion and motility, heregulin-β (HRGβ) binding to the erbB3 receptor initiated rapid and potent induction of breast carcinoma cell adhesion and migration and required dimerization of erbB3 with erbB2. Pharmacologic inhibitors of phosphoinositide 3-OH kinase (PI 3-K) or transient expression of dominant negative forms of PI 3-K inhibited both EGF- and HRGβ-mediated adhesion and potently blocked HRGβ- and EGF-induced cell motility. Our results illustrate the critical role of PI 3-K activity in signaling pathways initiated by the EGFR or erbB3 to up-regulate β1-integrin function.     

Forskolin-inducible cAMP Pathway Negatively Regulates T-cell Proliferation by Uncoupling the Interleukin-2 Receptor Complex

Cytokine-mediated regulation of T-cell activity involves a complex interplay between key signal transduction pathways. Determining how these signaling pathways cross-talk is essential to understanding T-cell function and dysfunction. In this work, we provide evidence that cross-talk exists between at least two signaling pathways: the Jak3/Stat5 and cAMP-mediated cascades. The adenylate cyclase activator forskolin (Fsk) significantly increased intracellular cAMP levels and reduced proliferation of the human T-cells via inhibition of cell cycle regulatory genes but did not induce apoptosis. To determine this inhibitory mechanism, effects of Fsk on IL-2 signaling was investigated. Fsk treatment of MT-2 and Kit 225 T-cells inhibited IL-2-induced Stat5a/b tyrosine and serine phosphorylation, nuclear translocation, and DNA binding activity. Fsk treatment also uncoupled IL-2 induced association of the IL-2Rβ and γ_c chain, consequently blocking Jak3 activation. Interestingly, phosphoamino acid analysis revealed that Fsk-treated cells resulted in elevated serine phosphorylation of Jak3 but not Stat5, suggesting that Fsk can negatively regulate Jak3 activity possibly mediated through PKA. Indeed, in vitro kinase assays and small molecule inhibition studies indicated that PKA can directly serine phosphorylate and functionally inactivate Jak3. Taken together, these findings suggest that Fsk activation of adenylate cyclase and PKA can negatively regulate IL-2 signaling at multiple levels that include IL-2R complex formation and Jak3/Stat5 activation.     

Identification of a Proline-Rich Sequence in the CD2 Cytoplasmic Domain Critical for Regulation of Integrin-Mediated Adhesion and Activation of Phosphoinositide 3-Kinase

The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in β1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of β1 integrin activity. CD2-induced increases in β1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of β1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.     

IL-4 Receptor-Alpha-Dependent Control of Cryptococcus neoformans in the Early Phase of Pulmonary Infection

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα–dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα^−/− mice unexpectedly show decreased fungal control early upon infection with C. neoformans, whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα^−/− mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα^−/− mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase.     

Syk-Mediated Translocation of PI3Kδ to the Leading Edge Controls Lamellipodium Formation and Migration of Leukocytes

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in β₂ integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during β₂ integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr³²³) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I_A. Syk-Tyr³²³ was found to be critical for the enrichment of the catalytic subunit p110δ of PI3K class I_A as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110δ to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of β₂ integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110δ signaling for β₂ integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.     

Non-Canonical Interleukin 23 Receptor Complex Assembly: P40 PROTEIN RECRUITS INTERLEUKIN 12 RECEPTOR β1 VIA SITE II AND INDUCES P19/INTERLEUKIN 23 RECEPTOR INTERACTION VIA SITE III*

IL-23, composed of the cytokine subunit p19 and the soluble α receptor subunit p40, binds to a receptor complex consisting of the IL-23 receptor (IL-23R) and the IL-12 receptor β1 (IL-12Rβ1). Complex formation was hypothesized to follow the “site I-II-III” architectural paradigm, with site I of p19 being required for binding to p40, whereas sites II and III of p19 mediate binding to IL-12Rβ1 and IL-23R, respectively. Here we show that the binding mode of p19 to p40 and of p19 to IL-23R follow the canonical site I and III paradigm but that interaction of IL-23 to IL-12Rβ1 is independent of site II in p19. Instead, binding of IL-23 to the cytokine binding module of IL-12Rβ1 is mediated by domains 1 and 2 of p40 via corresponding site II amino acids of IL-12Rβ1. Moreover, domains 2 and 3 of p40 were sufficient for complex formation with p19 and to induce binding of p19 to IL-23R. The Fc-tagged fusion protein of p40_D2D3/p19 did, however, not act as a competitive IL-23 antagonist but, at higher concentrations, induced proliferation via IL-23R but independent of IL-12Rβ1. On the basis of our experimental validation, we propose a non-canonical topology of the IL-23·IL-23R·IL-12Rβ1 complex. Furthermore, our data help to explain why p40 is an antagonist of IL-23 and IL-12 signaling and show that site II of p19 is dispensable for IL-23 signaling.     

Functionally distinct PI 3-kinase pathways regulate myelination in the peripheral nervous system

The PI 3-kinase (PI 3-K) signaling pathway is essential for Schwann cell myelination. Here we have characterized PI 3-K effectors activated during myelination by probing myelinating cultures and developing nerves with an antibody that recognizes phosphorylated substrates for this pathway. We identified a discrete number of phospho-proteins including the S6 ribosomal protein (S6rp), which is down-regulated at the onset of myelination, and N-myc downstream-regulated gene-1 (NDRG1), which is up-regulated strikingly with myelination. We show that type III Neuregulin1 on the axon is the primary activator of S6rp, an effector of mTORC1. In contrast, laminin-2 in the extracellular matrix (ECM), signaling through the α6β4 integrin and Sgk1 (serum and glucocorticoid-induced kinase 1), drives phosphorylation of NDRG1 in the Cajal bands of the abaxonal compartment. Unexpectedly, mice deficient in α6β4 integrin signaling or Sgk1 exhibit hypermyelination during development. These results identify functionally and spatially distinct PI 3-K pathways: an early, pro-myelinating pathway driven by axonal Neuregulin1 and a later-acting, laminin–integrin-dependent pathway that negatively regulates myelination.     

The PI3K p110α isoform regulates endothelial adherens junctions via Pyk2 and Rac1

Endothelial cell–cell junctions control efflux of small molecules and leukocyte transendothelial migration (TEM) between blood and tissues. Inhibitors of phosphoinositide 3-kinases (PI3Ks) increase endothelial barrier function, but the roles of different PI3K isoforms have not been addressed. In this study, we determine the contribution of each of the four class I PI3K isoforms (p110α, -β, -γ, and -δ) to endothelial permeability and leukocyte TEM. We find that depletion of p110α but not other p110 isoforms decreases TNF-induced endothelial permeability, Tyr phosphorylation of the adherens junction protein vascular endothelial cadherin (VE-cadherin), and leukocyte TEM. p110α selectively mediates activation of the Tyr kinase Pyk2 and GTPase Rac1 to regulate barrier function. Additionally, p110α mediates the association of VE-cadherin with Pyk2, the Rac guanine nucleotide exchange factor Tiam-1 and the p85 regulatory subunit of PI3K. We propose that p110α regulates endothelial barrier function by inducing the formation of a VE-cadherin–associated protein complex that coordinates changes to adherens junctions with the actin cytoskeleton.     

Stoichiometric Structure-Function Analysis of the Prolactin Receptor Signaling Domain by Receptor Chimeras

The intracellular domain of the prolactin (PRL) receptor (PRLr) is required for PRL-induced signaling and proliferation. To identify and test the functional stoichiometry of those PRLr motifs required for transduction and growth, chimeras consisting of the extracellular domain of either the α or β subunit of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-CSFr) and the intracellular domain of the rat PRLr were synthesized. Because the high-affinity binding of GM-CSF results from the specific pairing of one α- and one β-GM-CSFr, use of GM-CSFr/PRLr chimera enabled targeted dimerization of the PRLr intracellular domain. To that end, the extracellular domains of the α- and β-GM-CSFr were conjugated to one of the following mutations: (i) PRLr C-terminal truncations, termed α278, α294, α300, α322, or β322; (ii) PRLr tyrosine replacements, termed Y309F, Y382F, or Y309+382F; or, (iii) PRLr wild-type short, intermediate, or long isoforms. These chimeras were cotransfected into the cytokine-responsive Ba/F3 line, and their expression was confirmed by ligand binding and Northern and Western blot analyses. Data from these studies revealed that heterodimeric complexes of the wild type with C-terminal truncation mutants of the PRLr intracellular domain were incapable of ligand-induced signaling or proliferation. Replacement of any single tyrosine residue (Y309F or Y382F) in the dimerized PRLr complex resulted in a moderate reduction of receptor-associated Jak2 activation and proliferation. In contrast, trans replacement of these residues (i.e., αY309F and βY382F) markedly reduced ligand-driven Jak2 activation and proliferation, while cis replacement of both tyrosine residues in a single intracellular domain (i.e., αY309+382F) produced an inactive signaling complex. Analysis of these GM-CSFr–PRLr complexes revealed equivalent levels of Jak2 in association with the mutant receptor chains, suggesting that the tyrosine residues at 309 and 382 do not contribute to Jak association, but instead to its activation. Heterodimeric pairings of the intracellular domains from the known PRLr receptor isoforms (short-intermediate, short-long, and intermediate-long) also yielded inactive receptor complexes. These data demonstrate that the tyrosine residues at 309 and 382, as well as additional residues within the C terminus of the dimerized PRLr complex, contribute to PRL-driven signaling and proliferation. Furthermore, these findings indicate a functional requirement for the pairing of Y309 and Y382 in trans within the dimerized receptor complex.     

Signaling through CD5 Activates a Pathway Involving Phosphatidylinositol 3-Kinase, Vav, and Rac1 in Human Mature T Lymphocytes

CD5 acts as a coreceptor on T lymphocytes and plays an important role in T-cell signaling and T-cell–B-cell interactions. Costimulation of T lymphocytes with anti-CD5 antibodies results in an increase of the intracellular Ca²⁺ levels, and subsequently in the activation of Ca²⁺/calmodulin-dependent (CaM) kinase type IV. In the present study, we have characterized the initial signaling pathway induced by anti-CD5 costimulation. The activation of phosphatidylinositol (PI) 3-kinase through tyrosine phosphorylation of its p85 subunit is a proximal event in the CD5-signaling pathway and leads to the activation of the lipid kinase activity of the p110 subunit. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit the CD5-induced response as assessed in interleukin-2 (IL-2) secretion experiments. The expression of an inactivated Rac1 mutant (Rac1 · N17) in T lymphocytes transfected with an IL-2 promoter-driven reporter construct also abrogates the response to CD5 costimulation, while the expression of a constitutively active Rac1 mutant (Rac1-V12) completely replaces the CD5 costimulatory signal. The Rac1-specific guanine nucleotide exchange factor Vav is heavily phosphorylated on tyrosine residues upon CD5 costimulation, which is a prerequisite for its activation. A role for Vav in the CD5-induced signaling pathway is further supported by the findings that the expression of a dominant negative Vav mutant (Vav-C) completely abolishes the response to CD5 costimulation while the expression of a constitutively active Vav mutant [Vav(Δ1–65)] makes the CD5 costimulation signal superfluous. Wortmannin is unable to block the Vav(Δ1–65)- or Rac1 · V12-induced signals, indicating that both Vav and Rac1 function downstream from PI 3-kinase. Vav and Rac1 both act upstream from the CD5-induced activation of CaM kinase IV, since KN-62, an inhibitor of CaM kinases, and a dominant negative CaM kinase IV mutant block the Vav(Δ1–65)-and Rac1 · V12-mediated signals. We propose a model for the CD5-induced signaling pathway in which the PI 3-kinase lipid products, together with tyrosine phosphorylation, activate Vav, resulting in the activation of Rac1 by the Vav-mediated exchange of GDP for GTP.     

Essential Role of Class II Phosphatidylinositol-3-kinase-C2α in Sphingosine 1-Phosphate Receptor-1-mediated Signaling and Migration in Endothelial Cells

The phosphatidylinositol (PtdIns) 3-kinase (PI3K) family regulates diverse cellular processes, including cell proliferation, migration, and vesicular trafficking, through catalyzing 3′-phosphorylation of phosphoinositides. In contrast to class I PI3Ks, including p110α and p110β, functional roles of class II PI3Ks, comprising PI3K-C2α, PI3K-C2β, and PI3K-C2γ, are little understood. The lysophospholipid mediator sphingosine 1-phosphate (S1P) plays the important roles in regulating vascular functions, including vascular formation and barrier integrity, via the G-protein-coupled receptors S1P_1–3. We studied the roles of PI3K-C2α in S1P-induced endothelial cell (EC) migration and tube formation. S1P stimulated cell migration and activation of Akt, ERK, and Rac1, the latter of which acts as a signaling molecule essential for cell migration and tube formation, via S1P₁ in ECs. Knockdown of either PI3K-C2α or class I p110β markedly inhibited S1P-induced migration, lamellipodium formation, and tube formation, whereas that of p110α or Vps34 did not. Only p110β was necessary for S1P-iduced Akt activation, but both PI3K-C2α and p110β were required for Rac1 activation. FRET imaging showed that S1P induced Rac1 activation in both the plasma membrane and PtdIns 3-phosphate (PtdIns(3)P)-enriched endosomes. Knockdown of PI3K-C2α but not p110β markedly reduced PtdIns(3)P-enriched endosomes and suppressed endosomal Rac1 activation. Also, knockdown of PI3K-C2α but not p110β suppressed S1P-induced S1P₁ internalization into PtdIns(3)P-enriched endosomes. Finally, pharmacological inhibition of endocytosis suppressed S1P-induced S1P₁ internalization, Rac1 activation, migration, and tube formation. These observations indicate that PI3K-C2α plays the crucial role in S1P₁ internalization into the intracellular vesicular compartment, Rac1 activation on endosomes, and thereby migration through regulating vesicular trafficking in ECs.     

Release of cAMP Gating by the α6β4 Integrin Stimulates Lamellae Formation and the Chemotactic Migration of Invasive Carcinoma Cells

The α6β4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz, H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91: 949–960). We demonstrate here using MDA-MB-435 breast carcinoma cells that α6β4 stimulates chemotactic migration, a key component of invasion, but that it has no influence on haptotaxis. Stimulation of chemotaxis by α6β4 expression was observed in response to either lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of lamellae in these cells is dependent upon α6β4 expression. Both lamellae formation and chemotactic migration are inhibited or “gated” by cAMP and our results reveal that a critical function of α6β4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways. The essence of our findings is that α6β4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion.     

The Role of Interchain Heterodisulfide Formation in Activation of the Human Common β and Mouse βIL-3 Receptors

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common β (βc) receptor is shared by the three cytokines and functions together with cytokine-specific α subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human βc (hβc) and unidentified cysteine residues in the N-terminal domains of the α receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hβc is part of the cytokine binding epitope of this receptor and that an IL-3Rα isoform lacking the N-terminal Ig-like domain (the “SP2” isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hβc and the related mouse receptor, β_IL-3, could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hβc led to both a complete loss of high affinity binding with the human IL-3Rα SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, β_IL-3, not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Rα SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hβc and β_IL-3 in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.