Extensions Of Plasma Membrane Research Articles

AEE788 potentiates celecoxib-induced growth inhibition and apoptosis in human colon cancer cells

AimsCombinatorial therapies that target multiple signaling pathways may provide improved therapeutic responses over monotherapies. In the present study, we evaluated the effect of celecoxib and AEE788 alone and in combination on cell proliferation, invasion, migration, angiogenesis, morphological changes, actin filament organization and apoptosis induction in the human colon cancer cell lines. Main methodsEffect of celecoxib and AEE788 alone and in combination on colon cancer cell lines was evaluated by cell proliferation assay, morphological analysis, cell cycle analysis, scratch-wound healing and chorioallantoic membrane assays, zymography, nuclear fragmentation and western blot analyses. Key findingsEither drug alone or in combination inhibited human colonic adenocarcinoma cell lines HCT 15 and HT 29 in a dose-dependent manner. Microscopic analysis revealed inhibition of cell membrane extensions, cell shrinkage, and disorganization of actin filaments. Additionally, either drug alone or in combination inhibited HCT 15 migration, invasion and angiogenesis by suppressing matrix metalloproteinase-2 and -9 activities. Increased reactive oxygen generation, loss of mitochondrial membrane potential, cleavage of PARP, caspase-3 activation and DNA ladder formation characterized the induction of apoptosis by celecoxib and/or AEE788 treatment. Either drug individually induced apoptosis via down-regulation of the anti-apoptotic proteins Bcl2 and Bcl-xL, and up-regulation of pro-apoptotic protein Bax, cleavage of PARP, activation of caspase-3 and inhibition of vascular endothelial growth factor receptor signaling pathways. SignificanceResults indicate that AEE788 potentiates celecoxib-mediated inhibition of proliferation and angiogenesis in HCT 15 colon cancer cells and may prove useful for developing a combinatorial therapy for colon cancer.

Duffy Antigen Receptor for Chemokines Mediates Chemokine Endocytosis through a Macropinocytosis-Like Process in Endothelial Cells

BackgroundThe Duffy antigen receptor for chemokines (DARC) shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear.Methodology/Principal FindingsWe investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated 125I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. 125I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression. 125I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF) enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells.Conclusions/SignificanceThese results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.

Protein 4.1R regulates cell migration and IQGAP1 recruitment to the leading edge

In red blood cells, multifunctional protein 4.1R stabilizes the spectrin-actin network and anchors it to the plasma membrane. To contribute to the characterization of functional roles of 4.1R in nonerythroid cells, we have analyzed the participation of protein 4.1R in cell migration. The distribution of endogenous 4.1R is polarized towards the leading edge of migrating cells. Exogenous 4.1R isoforms containing a complete membrane-binding domain consistently localized to plasma membrane extensions enriched in F-actin. Silencing of 4.1R caused the loss of persistence of migration in subconfluent cells and of directional migration in cells moving into a wound. Coimmunoprecipitation and pull-down assays identified the scaffold protein IQGAP1 as a partner for protein 4.1R and showed that the 4.1R membrane-binding domain is involved in binding IQGAP1. Importantly, we show that protein 4.1R is necessary for the localization of IQGAP1 to the leading edge of cells migrating into a wound, whereas IQGAP1 is not required for protein 4.1R localization. Collectively, our results indicate a crucial role for protein 4.1R in cell migration and in the recruitment of the scaffold protein IQGAP1 to the cell front.

The role of the recycling endosome in regulating lamellipodia formation and macrophage migration

Cell migration is a highly complex process that requires the extension of cell membrane in the direction of travel. This membrane is continuously remodelled to expand the leading edge and alter its membrane properties. For a long time it has been known that there is a continual flow of polarised membrane traffic towards the leading edge during migration and that this trafficking is essential for cell migration. However, there is little information on how the cell co-ordinates exocytosis at the leading edge. It is also unclear whether these internal membranes are incorporated into the leading edge or are just delivering the necessary proteins for migration to occur. We have shown that recycling endosome membrane is incorporated into the plasma membrane at the leading edge to expand the membrane and at the same time delivers receptors to the leading edge to mediate migration. In order for this to happen the surface Q-SNARE complex Stx4/SNAP23 translocates to the leading edge where it binds to the R-SNARE VAMP3 on the recycling endosome allowing incorporation into the plasma membrane. Loss of any one of the components of this complex reduces efficient lamellipodia formation and restrains cell migration.

Uncivil Engineers: Chlamydia , Salmonella and Shigella Alter Cytoskeleton Architecture to Invade Epithelial Cells

The obligate intracellular bacterial pathogen Chlamydia trachomatis is a major cause of blindness and sexually transmitted diseases. Like the enteric pathogens Salmonella and Shigella, Chlamydia injects effector proteins into epithelial cells to initiate extensive remodeling of the actin cytoskeleton at the bacterial attachment site, which culminates in the engulfment of the bacterium by plasma membrane extensions. Numerous Salmonella and Shigella effectors promote this remodeling by activating Rho GTPases and tyrosine kinase signaling cascades and by directly manipulating actin dynamics. Recent studies indicate that similar host-cell alterations occur during Chlamydia invasion, but few effectors are known. The identification of additional Chlamydia effectors and the elucidation of their modes of function are critical steps towards an understanding of how this clinically important pathogen breaches epithelial surfaces and causes infection.

MADD-2, a Homolog of the Opitz Syndrome Protein MID1, Regulates Guidance to the Midline through UNC-40 in Caenorhabditis elegans

The body muscles of Caenorhabditis elegans extend plasma membrane extensions called muscle arms to the midline motor axons to form the postsynaptic membrane of the neuromuscular junction. Through a screen for muscle arm development defective (Madd) mutants, we previously discovered that the UNC-40/DCC guidance receptor directs muscle arm extension through the Rho-GEF UNC-73. Here, we describe a gene identified through our mutant screen called madd-2, and show that it functions in an UNC-40 pathway. MADD-2 is a C1-TRIM protein and a homolog of human MID1, mutations in which cause Opitz Syndrome. We demonstrate that MADD-2 functions cell autonomously to direct muscle and axon extensions to the ventral midline of worms. Our results suggest that MADD-2 may enhance UNC-40 pathway activity by facilitating an interaction between UNC-40 and UNC-73. The analogous phenotypes that result from MADD-2 and MID1 mutations suggest that C1-TRIM proteins may have a conserved biological role in midline-oriented developmental events.

Cholangiocyte Myosin IIB Is Required for Localized Aggregation of Sodium Glucose Cotransporter 1 to Sites ofCryptosporidium parvumCellular Invasion and Facilitates Parasite Internalization

Internalization of the obligate intracellular apicomplexan parasite, Cryptosporidium parvum, results in the formation of a unique intramembranous yet extracytoplasmic niche on the apical surfaces of host epithelial cells, a process that depends on host cell membrane extension. We previously demonstrated that efficient C. parvum invasion of biliary epithelial cells (cholangiocytes) requires host cell actin polymerization and localized membrane translocation/insertion of Na(+)/glucose cotransporter 1 (SGLT1) and of aquaporin 1 (Aqp1), a water channel, at the attachment site. The resultant localized water influx facilitates parasite cellular invasion by promoting host-cell membrane protrusion. However, the molecular mechanisms by which C. parvum induces membrane translocation/insertion of SGLT1/Aqp1 are obscure. We report here that cultured human cholangiocytes express several nonmuscle myosins, including myosins IIA and IIB. Moreover, C. parvum infection of cultured cholangiocytes results in the localized selective aggregation of myosin IIB but not myosin IIA at the region of parasite attachment, as assessed by dual-label immunofluorescence confocal microscopy. Concordantly, treatment of cells with the myosin light chain kinase inhibitor ML-7 or the myosin II-specific inhibitor blebbistatin or selective RNA-mediated repression of myosin IIB significantly inhibits (P < 0.05) C. parvum cellular invasion (by 60 to 80%). Furthermore ML-7 and blebbistatin significantly decrease (P < 0.02) C. parvum-induced accumulation of SGLT1 at infection sites (by approximately 80%). Thus, localized actomyosin-dependent membrane translocation of transporters/channels initiated by C. parvum is essential for membrane extension and parasite internalization, a phenomenon that may also be relevant to the mechanisms of cell membrane protrusion in general.

Cell membrane extensions, generated by mechanical constraint, are associated with a sustained lipid raft patching and an increased cell signaling

Platelet activation triggers an imbalance in plasma membrane phospholipids by a specific aminophospholipid outflux, resulting in filopodia formation. Similarly, the addition of a phospholipid excess in the outer leaflet of the plasma membrane induces cellular extensions and actin polymerization. The implication of membrane microdomains in sustaining these mechanical constraints remains, however, unknown and was investigated in human platelets and mouse fibroblasts. The disruption of lipid rafts by cholesterol depletion prevents actin polymerization and formation of cellular extensions. Phospholipid excess triggers raft patching underneath the cell extensions, recruitment of protein raft markers and increase of tyrosine phosphorylation of raft proteins. Using a mass spectrometric analysis of isolated platelet rafts, we identified tyrosine kinases and proteins implicated in the formation of cell membrane extensions, cell adhesion and motility. They are recruited to rafts in response to a mechanical constraint. Taken together, our results demonstrate that exogenous phospholipid addition causes a modulation of the lateral plasma membrane organization and an activation of the cell signaling triggering actin remodeling and the formation of cellular protrusions. Raft disruption abolishes these processes, demonstrating that their integrity is crucial for cell shape changes in response to a mechanical constraint on plasma membrane.

Actomyosin contractility controls cell surface area of oligodendrocytes

BackgroundTo form myelin oligodendrocytes expand and wrap their plasma membrane multiple times around an axon. How is this expansion controlled?ResultsHere we show that cell surface area depends on actomyosin contractility and is regulated by physical properties of the supporting matrix. Moreover, we find that chondroitin sulfate proteoglycans (CSPG), molecules associated with non-permissive growth properties within the central nervous system (CNS), block cell surface spreading. Most importantly, the inhibitory effects of CSPG on plasma membrane extension were completely prevented by treatment with inhibitors of actomyosin contractility and by RNAi mediated knockdown of myosin II. In addition, we found that reductions of plasma membrane area were accompanied by changes in the rate of fluid-phase endocytosis.ConclusionIn summary, our results establish a novel connection between endocytosis, cell surface extension and actomyosin contractility. These findings open up new possibilities of how to promote the morphological differentiation of oligodendrocytes in a non-permissive growth environment.See related minireview by Bauer and ffrench-Constant:

Characterization of a myristoylated, monomeric HIV Gag protein

The process of HIV assembly requires extensive homomultimerization of the Gag polyprotein on cellular membranes to generate the nascent particle bud. Here we generated a full-length, monomeric Gag polyprotein bearing mutations that eliminated multimerization in living cells as indicated by fluorescence resonance energy transfer (FRET). Monomeric Gag resembled non-myristoylated Gag in its weak membrane binding characteristics and lack of association with detergent-resistant membranes (DRMs or lipid rafts). Monomeric Gag failed to assemble virus-like particles, but was inefficiently rescued into particles by wildtype Gag through the influence of the matrix domain. The subcellular distribution of monomeric Gag was remarkably different than either non-myristoylated Gag or wildtype Gag. Monomeric Gag was found on intracellular membranes and at the plasma membrane, where it highlighted plasma membrane extensions and ruffles. This study indicates that monomeric Gag can traffic to assembly sites in the cell, where it interacts weakly with membranes.

An UNC-40 pathway directs postsynaptic membrane extension inCaenorhabditis elegans

The postsynaptic membrane of the embryonic neuromuscular junction undergoes a dramatic expansion during later development to facilitate the depolarization of larger muscles. In C. elegans, the postsynaptic membrane resides at the termini of plasma membrane extensions called muscle arms. Membrane extension to the motor axons during larval development doubles the number of muscle arms, making them a tractable model to investigate both postsynaptic membrane expansion and guided membrane extension. To identify genes required for muscle arm extension, we performed a forward screen for mutants with fewer muscle arms. We isolated 23 mutations in 14 genes, including unc-40/Dcc, which encodes a transmembrane receptor that guides the migration of cells and extending axons in response to the secreted UNC-6/Netrin spatial cue. We discovered that UNC-40 is enriched at muscle arm termini and functions cell-autonomously to direct arm extension to the motor axons. Surprisingly, UNC-6 is dispensable for muscle arm extension, suggesting that UNC-40 relies on other spatial cues to direct arm extension. We provide the first evidence that the guanine-nucleotide exchange factor UNC-73/Trio, members of the WAVE actin-polymerization complex, and a homolog of the focal adhesion complex can function downstream of UNC-40 to direct membrane extension. Our work is the first to define a pathway for directed muscle membrane extension and illustrates that axon guidance components can play key roles in postsynaptic membrane expansion.

Membrane Ruffles Capture C3bi-opsonized Particles in Activated Macrophages

A widespread belief in phagocyte biology is that FcgammaR-mediated phagocytosis utilizes membrane pseudopods, whereas Mac-1-mediated phagocytosis does not involve elaborate plasma membrane extensions. Here we report that dynamic membrane ruffles in activated macrophages promote binding of C3bi-opsonized particles. We identify these ruffles as components of the macropinocytosis machinery in both PMA- and LPS-stimulated macrophages. C3bi-particle capture is facilitated by enrichment of high-affinity Mac-1 and the integrin-regulating protein talin in membrane ruffles. Membrane ruffle formation and C3bi-particle binding are cytoskeleton dependent events, having a strong requirement for F-actin and microtubules (MTs). MT disruption blunts ruffle formation and PMA- and LPS-induced up-regulation of surface Mac-1 expression. Furthermore, the MT motor, kinesin participates in ruffle formation implicating a requirement for intracellular membrane delivery to active membrane regions during Mac-1-mediated phagocytosis. We observed colocalization of Rab11-positive vesicles with CLIP-170, a MT plus-end binding protein, at sites of particle adherence using TIRF imaging. Rab11 has been implicated in recycling endosome dynamics and mutant Rab11 expression inhibits both membrane ruffle formation and C3bi-sRBC adherence to macrophages. Collectively these findings represent a novel membrane ruffle "capture" mechanism for C3bi-particle binding during Mac-1-mediated phagocytosis. Importantly, this work also demonstrates a strong functional link between integrin activation, macropinocytosis and phagocytosis in macrophages.

T1α/podoplanin is essential for capillary morphogenesis in lymphatic endothelial cells

The lymphatic vasculature functions to maintain tissue perfusion homeostasis. Defects in its formation or disruption of the vessels result in lymphedema, the effective treatment of which is hampered by limited understanding of factors regulating lymph vessel formation. Mice lacking T1alpha/podoplanin, a lymphatic endothelial cell transmembrane protein, have malformed lymphatic vasculature with lymphedema at birth, but the molecular mechanism for this phenotype is unknown. Here, we show, using primary human lung microvascular lymphatic endothelial cells (HMVEC-LLy), that small interfering RNA-mediated silence of podoplanin gene expression has the dramatic effect of blocking capillary tube formation in Matrigel. In addition, localization of phosphorylated ezrin/radixin/moesin proteins to plasma membrane extensions, an early event in the capillary morphogenic program in lymphatic endothelial cells, is impaired. We find that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA early (by 30 min) after plating on Matrigel, and Rac1 shows a delay in its activation. Further indication that podoplanin action is linked to RhoA activation is that use of a cell-permeable inhibitor of Rho inhibited lymphatic endothelial capillary tube formation in the same manner as did podoplanin gene silencing, which was not mimicked by treatment with a Rac1 inhibitor. These data clearly demonstrate that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression. To our knowledge, this is the first time that a mechanism has been suggested to explain the role of podoplanin in lymphangiogenesis.

Docosahexaenoic acid accumulation in thraustochytrids: search for the rationale

Thraustochytrid protists characteristically accumulate high amounts of lipids, much of which is comprised of the polyunsaturated fatty acid, docosahexaenoic acid (DHA). Since DHA is important in human health, thraustochytrids have attracted much attention from the point of view of biotechnology. However, the biological rationale of DHA production in the storage lipids of these organisms is not clear. We carried out three experiments to study this. (1) The fate of lipids in thraustochytrids during development was studied by staining lipids of vegetative cells with the fluorescent vital stain for lipids, namely Nile blue, following the growth of the cells. The fluorescent lipid bodies decreased in abundance in freshly formed motile limaciform amoeboid cells and subsequently disappeared when they moved away. When vegetative cells produced vast extensions of plasma membrane, the ectoplasmic net elements (EN), the fluorescent lipids appeared to be transported to them. (2) Changes in lipids and DHA during starvation were examined in cells with enhanced lipid and DHA contents and those in which they were not enhanced. Cells in which lipids and DHA were enhanced by refrigerating them for 48 h survived starvation for a longer period. Compared to cells that had not been refrigerated, total fatty acids, as well as DHA were marginally higher in pre-refrigerated cells, while palmitic acid levels were lower. Starvation of cells resulted in a gradual decrease of absolute concentrations and percentage levels of DHA after 8 days, while percentage of palmitic acid levels increased. (3) The relationship between DHA and specific gravity of cells was studied by comparing cells pre-refrigerated as above with those which were not pre-refrigerated. Refrigerated cells with increased lipids and DHA showed higher specific gravities than non-refrigerated cells. We suggest the following roles for storage lipids and DHA in thraustochytrids: (1) lipids serve as energy sources during movement of cells and for production of EN; (2) DHA in storage lipids become distributed in the plasma membrane when EN are formed; (3) DHA is preferably utilized as a fatty acid energy reserve during starvation and (4) an unknown mechanism results in negative buoyancy of cells when total lipids and DHA are marginally enhanced. DHA in storage lipids might be crucial to the survival of thraustochytrid cells.

A novel apical midpiece defect in the spermatozoa of a bull without an apparent decrease in motility and fertility: A case study

Despite some limitations as predictors of fertility, evaluation of sperm morphology and progressive motility is the commonest method to assess viability of frozen/thawed semen. In this article we describe by light and transmission electron microscopy a novel midpiece structural defect observed in 24–36% of frozen/thawed sperm cells from a Charolais bull, used in artificial insemination programs without any apparent ill effect to the fertility. After thawing, the sperm progressive motility ranged from 65 to 80% and the pregnancy rate for all artificial inseminations performed (43%) did not differ (p>0.05) from results obtained with insemination with semen of other bulls (40%). The defect consisted in mitochondrial aplasia at the neck region, mitochondrial segmental elongation and gaps and thickening of the outer dense fibers at the apical region of the midpiece, and loss of the cementing substance and development of plasma membrane extensions in the entire midpiece. No structural abnormalities were found in the capitulum, proximal centriole, striated columns, axoneme, annulus and fibrous sheath. The thickness of the outer fibers returned to normal at the distal region of the midpiece. Based on the examination it is suggested that the alterations might be originally caused by loss of the cementing substance that links mitochondria to the plasma membrane in association with mitochondrial aplasia at the neck region of the midpiece. The abnormality appeared not related to other described sperm defect syndromes, although it shared particular characteristics with the dag defect, segmental aplasia of the mitochondrial sheath, corkscrew defect and pseudodroplet defect.

FGF negatively regulates muscle membrane extension inCaenorhabditis elegans

Striated muscles from Drosophila and several vertebrates extend plasma membrane to facilitate the formation of the neuromuscular junction (NMJ) during development. However, the regulation of these membrane extensions is poorly understood. In C. elegans, the body wall muscles (BWMs) also have plasma membrane extensions called muscle arms that are guided to the motor axons where they form the postsynaptic element of the NMJ. To investigate the regulation of muscle membrane extension, we screened 871 genes by RNAi for ectopic muscle membrane extensions (EMEs) in C. elegans. We discovered that an FGF pathway, including let-756(FGF), egl-15(FGF receptor), sem-5(GRB2) and other genes negatively regulates plasma membrane extension from muscles. Although compromised FGF pathway activity results in EMEs, hyperactivity of the pathway disrupts larval muscle arm extension, a phenotype we call muscle arm extension defective or MAD. We show that expression of egl-15 and sem-5 in the BWMs are each necessary and sufficient to prevent EMEs. Furthermore, we demonstrate that let-756 expression from any one of several tissues can rescue the EMEs of let-756 mutants, suggesting that LET-756 does not guide muscle membrane extensions. Our screen also revealed that loss-of-function in laminin and integrin components results in both MADs and EMEs, the latter of which are suppressed by hyperactive FGF signaling. Our data are consistent with a model in which integrins and laminins are needed for directed muscle arm extension to the nerve cords, while FGF signaling provides a general mechanism to regulate muscle membrane extension.

Tracking of migrating cells under phase-contrast video microscopy with combined mean-shift processes

In this paper, we propose a combination of mean-shift-based tracking processes to establish migrating cell trajectories through in vitro phase-contrast video microscopy. After a recapitulation on how the mean-shift algorithm permits efficient object tracking we describe the proposed extension and apply it to the in vitro cell tracking problem. In this application, the cells are unmarked (i.e., no fluorescent probe is used) and are observed under classical phase-contrast microscopy. By introducing an adaptive combination of several kernels, we address several problems such as variations in size and shape of the tracked objects (e.g., those occurring in the case of cell membrane extensions), the presence of incomplete (or noncontrasted) object boundaries, partially overlapping objects and object splitting (in the case of cell divisions or mitoses). Comparing the tracking results automatically obtained to those generated manually by a human expert, we tested the stability of the different algorithm parameters and their effects on the tracking results. We also show how the method is resistant to a decrease in image resolution and accidental defocusing (which may occur during long experiments, e.g., dozens of hours). Finally, we applied our methodology on cancer cell tracking and showed that cytochalasin-D significantly inhibits cell motility.

Actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virus-induced cell fusion.

The membrane fusion events which initiate human immunodeficiency virus type 1 (HIV-1) infection and promote cytopathic syncytium formation in infected cells commence with the binding of the HIV envelope glycoprotein (Env) to CD4 and an appropriate coreceptor. Here, we show that HIV Env-coreceptor interactions activate Rac-1 GTPase and stimulate the actin filament network reorganizations that are requisite components of the cell fusion process. Disrupting actin filament dynamics with jasplakinolide or latrunculin A arrested fusion at a late step in the formation of Env-CD4-coreceptor complexes. Time-lapse confocal microscopy of living cells revealed vigorous activity of actin-based, target cell membrane extensions at the target cell-Env-expressing cell interface. The expression of dominant-negative forms of actin-regulating Rho-family GTPases established that HIV Env-mediated syncytium formation relies on Rac-1 but not on Cdc42 or Rho activation in target cells. Similar dependencies were found when cell fusion was induced by Env expressed on viral or cellular membranes. Additionally, Rac activity was specifically upregulated in a coreceptor-dependent manner in fusion reaction cell lysates. These results define a role for HIV Env-coreceptor interactions in activating the cellular factors essential for virus-cell and cell-cell fusion and provide evidence for the participation of pertussis toxin-insensitive signaling pathways in HIV-induced membrane fusion.

Actin Binding of Human LIM and SH3 Protein Is Regulated by cGMP- and cAMP-dependent Protein Kinase Phosphorylation on Serine 146

Various drugs that elevate cGMP levels and activate cGMP-dependent protein kinase (cGK) inhibit agonist-induced platelet activation. In the present study we identified the LIM and SH3 domain protein (LASP) that was recently cloned from human breast cancer cells (Tomasetto, C., Regnier, C., Moog-Lutz, C., Mattei, M. G., Chenard, M. P., Liderau, R., Basset, P., and Rio, M. C. (1995) Genomics 28, 367-376) as a novel substrate of cGK in human platelets. Recombinant human LASP was phosphorylated by cGMP- and cAMP-dependent protein kinase (cAK) in vitro. Cotransfection of PtK-2 cells with LASP and cGK confirmed phosphorylation of LASP in vivo. Studies with human LASP mutants identified serine 146 as a specific phosphorylation site for cGK and cAK in vivo. LASP is an actin-binding protein, and the phospho-LASP-mimicking mutant S146D showed reduced binding affinity for F-actin in cosedimentation experiments. Immunofluorescence of transfected PtK2 cells demonstrated the localization of LASP in the tips of cell membrane extensions and at cell-cell contacts. Expression of the human LASP mutant S146D resulted in nearly complete relocalization to the cytosol and reduced migration of the cells. Taken together, these data suggest that phosphorylation of LASP by cGK and cAK may be involved in cytoskeletal organization and cell motility.

Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism.

The selective RNA-binding protein QKI is essential for myelination in the central nervous system (CNS). QKI belongs to the family of signal transduction activators of RNA (STARs), characteristic of binding RNA and signaling molecules, therefore is postulated to regulate RNA homeostasis in response to developmental signals. Here we report that QKI acts downstream of the Src family protein tyrosine kinases (Src-PTKs) during CNS myelination. QKI selectively interacted with the mRNA encoding the myelin basic protein (MBP). Such interaction stabilized MBP mRNA and was required for the rapid accumulation of MBP mRNA during active myelinogenesis. We found that the interaction between QKI and MBP mRNA was negatively regulated by Src-PTK-dependent phosphorylation of QKI. During early myelin development, tyrosine phosphorylation of QKI in the developing myelin drastically declined, presumably leading to enhanced interactions between QKI and MBP mRNA, which was associated with the rapid accumulation of MBP mRNA and accelerated myelin production. Therefore, developmental regulation of Src-PTK-dependent tyrosine phosphorylation of QKI suggests a novel mechanism for accelerating CNS myelinogenesis via regulating mRNA metabolism.