Na,K-ATPase β-Subunit Is Required for Epithelial Polarization, Suppression of Invasion, and Cell Motility

The cell adhesion molecule E-cadherin has been implicated in maintaining the polarized phenotype of epithelial cells and suppression of invasiveness and motility of carcinoma cells. Na,K-ATPase, consisting of an α- and β-subunit, maintains the sodium gradient across the plasma membrane. A functional relationship between E-cadherin and Na,K-ATPase has not previously been described. We present evidence that the Na,K-ATPase plays a crucial role in E-cadherin–mediated development of epithelial polarity, and suppression of invasiveness and motility of carcinoma cells. Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK) have highly reduced levels of E-cadherin and β1-subunit of Na,K-ATPase. Forced expression of E-cadherin in MSV-MDCK cells did not reestablish epithelial polarity or inhibit the invasiveness and motility of these cells. In contrast, expression of E-cadherin and Na,K-ATPase β1-subunit induced epithelial polarization, including the formation of tight junctions and desmosomes, abolished invasiveness, and reduced cell motility in MSV-MDCK cells. Our results suggest that E-cadherin–mediated cell-cell adhesion requires the Na,K-ATPase β-subunit's function to induce epithelial polarization and suppress invasiveness and motility of carcinoma cells. Involvement of the β1-subunit of Na,K-ATPase in the polarized phenotype of epithelial cells reveals a novel link between the structural organization and vectorial ion transport function of epithelial cells.

Phosphorylation of a Conserved Integrin α3 QPSXXE Motif Regulates Signaling, Motility, and Cytoskeletal EngagementV⃞

Integrin α3A cytoplasmic tail phosphorylation was mapped to amino acid S1042, as determined by mass spectrometry, and confirmed by mutagenesis. This residue occurs within a “QPSXXE” motif conserved in multiple α chains (α3A, α6A, α7A), from multiple species. Phosphorylation of α3A and α6A did not appear to be directly mediated by protein kinase C (PKC) α, β, γ, δ, ε, ζ, or μ, or by any of several other known serine kinases, although PKC has an indirect role in promoting phosphorylation. A S1042A mutation did not affect α3-Chinese hamster ovary (CHO) cell adhesion to laminin-5, but did alter 1) α3-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin (in the presence or absence of phorbol 12-myristate 13 acetate stimulation), and p130CAS (in the absence of phorbol 12-myristate 13 acetate stimulation), 2) the shape of cells spread on laminin-5, and 3) α3-dependent random CHO cell migration on laminin-5. In addition, S1042A mutation altered the PKC-dependent, ligand-dependent subcellular distribution of α3 and F-actin in CHO cells. Together, the results demonstrate clearly that α3A phosphorylation is functionally relevant. In addition, the results strongly suggest that α3 phosphorylation may regulate α3 integrin interaction with the cytoskeleton.

Novel Nuclear Signaling Pathway Mediates Activation of Fibroblast Growth Factor-2 Gene by Type 1 and Type 2 Angiotensin II Receptors

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1′,3′-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.

The Chlamydomonas PF6 Locus Encodes a Large Alanine/Proline-Rich Polypeptide That Is Required for Assembly of a Central Pair Projection and Regulates Flagellar Motility

Efficient motility of the eukaryotic flagellum requires precise temporal and spatial control of its constituent dynein motors. The central pair and its associated structures have been implicated as important members of a signal transduction cascade that ultimately regulates dynein arm activity. To identify central pair components involved in this process, we characterized a Chlamydomonas motility mutant (pf6-2) obtained by insertional mutagenesis. pf6-2 flagella twitch ineffectively and lack the 1a projection on the C1 microtubule of the central pair. Transformation with constructs containing a full-length, wild-type copy of the PF6 gene rescues the functional, structural, and biochemical defects associated with the pf6 mutation. Sequence analysis indicates that the PF6 gene encodes a large polypeptide that contains numerous alanine-rich, proline-rich, and basic domains and has limited homology to an expressed sequence tag derived from a human testis cDNA library. Biochemical analysis of an epitope-tagged PF6 construct demonstrates that the PF6 polypeptide is an axonemal component that cosediments at 12.6S with several other polypeptides. The PF6 protein appears to be an essential component required for assembly of some of these polypeptides into the C1-1a projection.

Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3.

Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins

The docking protein FRS2 is a major downstream effector that links fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein kinase signaling cascade. In this report, we demonstrate that FRS2 also plays a pivotal role in FGF-induced recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase). We demonstrate that tyrosine phosphorylation of FRS2α leads to Grb2-mediated complex formation with the docking protein Gab1 and its tyrosine phosphorylation, resulting in the recruitment and activation of PI3-kinase. Furthermore, Grb2 bound to tyrosine-phosphorylated FRS2 through its SH2 domain interacts primarily via its carboxyl-terminal SH3 domain with a proline-rich region in Gab1 and via its amino-terminal SH3 domain with the nucleotide exchange factor Sos1. Assembly of FRS2α:Grb2:Gab1 complex induced by FGF stimulation results in activation of PI3-kinase and downstream effector proteins such as the S/T kinase Akt, whose cellular localization and activity are regulated by products of PI3-kinase. These experiments reveal a unique mechanism for generation of signal diversity by growth factor-induced coordinated assembly of a multidocking protein complex that can activate the Ras/mitogen-activated protein kinase cascade to induce cell proliferation and differentiation, and PI3-kinase to activate a mediator of a cell survival pathway.

Symmetries in bacterial motility

Descriptions are given of three kinds of symmetries encountered in studies of bacterial locomotion, and of the ways in which they are circumvented or broken. A bacterium swims at very low Reynolds number: it cannot propel itself using reciprocal motion (by moving through a sequence of shapes, first forward and then in reverse); cyclic motion is required. A common solution is rotation of a helical filament, either right- or left-handed. The flagellar rotary motor that drives each filament generates the same torque whether spinning clockwise or counterclockwise. This symmetry is broken by coupling to the filament. Finally, bacterial populations, grown in a nutrient medium from an inoculum placed at a single point, usually move outward in symmetric circular rings. Under certain conditions, the cells excrete a chemoattractant, and the rings break up into discrete aggregates that can display remarkable geometric order.

The physiological significance of β-actin mRNA localization in determining cell polarity and directional motility

β-actin mRNA is localized near the leading edge in several cell types, where actin polymerization is actively promoting forward protrusion. The localization of the β-actin mRNA near the leading edge is facilitated by a short sequence in the 3′ untranslated region, the “zip code.” Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zip code) in the 3′ untranslated region leads to delocalization of β-actin mRNA, alteration of cell phenotype, and a decrease in cell motility. To determine the components of this process responsible for the change in cell behavior after β-actin mRNA delocalization, the Dynamic Image Analysis System was used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zip code. It was found that net path length and average speed of antisense-treated cells were significantly lower than in sense-treated cells. Total path length and the velocity of protrusion of antisense-treated cells were not affected compared with those of control cells. These results suggest that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zip code-directed antisense oligonucleotides. To test this, direct analysis of directionality was performed on antisense-treated cells and showed a decrease in directionality (net path/total path) and persistence of movement. Less directional movement of antisense-treated cells correlated with a unpolarized and discontinuous distribution of free barbed ends of actin filaments and of β-actin protein. These results indicate that delocalization of β-actin mRNA results in delocalization of nucleation sites and β-actin protein from the leading edge followed by loss of cell polarity and directional movement.

Structural basis for fibroblast growth factor receptor 2 activation in Apert syndrome

Apert syndrome (AS) is characterized by craniosynostosis (premature fusion of cranial sutures) and severe syndactyly of the hands and feet. Two activating mutations, Ser-252 → Trp and Pro-253 → Arg, in fibroblast growth factor receptor 2 (FGFR2) account for nearly all known cases of AS. To elucidate the mechanism by which these substitutions cause AS, we determined the crystal structures of these two FGFR2 mutants in complex with fibroblast growth factor 2 (FGF2) . These structures demonstrate that both mutations introduce additional interactions between FGFR2 and FGF2, thereby augmenting FGFR2–FGF2 affinity. Moreover, based on these structures and sequence alignment of the FGF family, we propose that the Pro-253 → Arg mutation will indiscriminately increase the affinity of FGFR2 toward any FGF. In contrast, the Ser-252 → Trp mutation will selectively enhance the affinity of FGFR2 toward a limited subset of FGFs. These predictions are consistent with previous biochemical data describing the effects of AS mutations on FGF binding. Alterations in FGFR2 ligand affinity and specificity may allow inappropriate autocrine or paracrine activation of FGFR2. Furthermore, the distinct gain-of-function interactions observed in each crystal structure provide a model to explain the phenotypic variability among AS patients.

Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803

To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an “inverted motility response” (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis. Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis. Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for taxAY1 was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the Tax3 locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.

Hyaluronan Activates Cell Motility of v-Src-transformed Cells via Ras-Mitogen–activated Protein Kinase and Phosphoinositide 3-Kinase-Akt in a Tumor-specific Manner

We investigated the production of hyaluronan (HA) and its effect on cell motility in cells expressing the v-src mutants. Transformation of 3Y1 by v-src virtually activated HA secretion, whereas G2A v-src, a nonmyristoylated form of v-src defective in cell transformation, had no effect. In cells expressing the temperature-sensitive mutant of v-Src, HA secretion was temperature dependent. In addition, HA as small as 1 nM, on the other side, activated cell motility in a tumor-specific manner. HA treatment strongly activated the motility of v-Src–transformed 3Y1, whereas it showed no effect on 3Y1- and 3Y1-expressing G2A v-src. HA-dependent cell locomotion was strongly blocked by either expression of dominant-negative Ras or treatment with a Ras farnesyltransferase inhibitor. Similarly, both the MEK1 inhibitor and the kinase inhibitor clearly inhibited HA-dependent cell locomotion. In contrast, cells transformed with an active MEK1 did not respond to the HA. Finally, an anti-CD44–neutralizing antibody could block the activation of cell motility by HA as well as the HA-dependent phosphorylation of mitogen-activated protein kinase and Akt. Taken together, these results suggest that simultaneous activation of the Ras-mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase pathway by the HA-CD44 interaction is required for the activation of HA-dependent cell locomotion in v-Src–transformed cells.

Maspin acts at the cell membrane to inhibit invasion and motility of mammary and prostatic cancer cells.

Maspin, a novel serine protease inhibitor (serpin), inhibits tumor invasion and metastasis of mammary carcinoma. We show here that recombinant maspin protein blocks the motility of these carcinoma cells in culture over 12 h, as demonstrated by time-lapse video microscopy. Lamellopodia are withdrawn but ruffling continues. Both exogenous recombinant maspin and maspin expressed by tumor transfectants exhibit inhibitory effects on cell motility and cell invasion as shown in modified Boyden chamber assays. In addition, three prostatic cancer cell lines treated with recombinant maspin exhibited similar inhibition of both invasion and motility, suggesting a similar mode of maspin action in these two glandular epithelial cancers. When mammary carcinoma cells were treated with recombinant maspin, the protein was shown by immunostaining to bind specifically to the cell surface, suggesting that maspin activity is membrane associated. When pretreated with antimaspin antibody, maspin loses its inhibitory effects on both invasion and motility. However, when maspin is added to these cells preceding antibody treatment, the activity of maspin is no longer inhibited by subsequent addition of the antibody. It is concluded therefore that the inhibition of invasion and motility by maspin is initially localized to the cell surface.

Correction of hypertension by normalization of endothelial levels of fibroblast growth factor and nitric oxide synthase in spontaneously hypertensive rats.

Acidic and basic fibroblast growth factors (FGFs) share a wide range of diverse biological activities. To date, low levels of FGF have not been correlated with a pathophysiologic state. We report that blood vessels of spontaneously hypertensive rats are shown to be associated with a marked decrement in endothelial basic FGF content. This decrement correlates both with hypertension and with a decrease in the endothelial content of nitric oxide synthase. Restoration of FGF to physiological levels in the vascular wall, either by systemic administration or by in vivo gene transfer, significantly augmented the number of endothelial cells with positive immunostaining for nitric oxide synthase, corrected hypertension, and ameliorated endothelial-dependent responses to vasoconstrictors. These results suggest an important role for FGFs in blood pressure homeostasis and open new avenues for the understanding of the etiology and treatment of hypertension.