Urokinase-induced mitogenesis is mediated by casein kinase 2 and nucleolin.

BACKGROUND: Urokinase (uPA) and the urokinase receptor (uPAR) form a multifunctional system capable of concurrently regulating pericellular proteolysis, cell-surface adhesion, and mitogenesis. The role of uPA and uPAR in directed proteolysis is well established and its function in cellular adhesiveness has recently been clarified by numerous studies. The molecular mechanisms underlying the mitogenic effects of uPA and uPAR are still unclear, however. RESULTS: We identified mechanisms that might participate in uPA-related mitogenesis in human vascular smooth muscle cells and demonstrated that uPA induces activation of a unique signaling complex. This complex contains uPAR and two additional proteins, nucleolin and casein kinase 2, which are implicated in cell proliferation. Both proteins were isolated by affinity chromatography on uPA-conjugated cyanogen-bromide-activated Sepharose 4B and were identified using nano-electrospray mass spectrometry and immunoblotting. We used laser scanning and immunoelectron microscopy studies to further demonstrate that nucleolin and casein kinase 2 are located on the cell surface where they colocalize with the uPAR. Moreover, the proteins were co-internalized into the cell as an entire complex. Immunoprecipitation experiments in combination with an in vitro kinase assay demonstrated a specific association of uPAR with nucleolin and casein kinase 2 and revealed a uPA-induced activation of casein kinase 2, which presumably led to phosphorylation of nucleolin. Blockade of nucleolin and casein kinase 2 with specific modulators led to the inhibition of uPA-induced cell proliferation. CONCLUSIONS: We conclude that in human vascular smooth muscle cells, uPA induces the formation and activation of a newly identified signaling complex comprising uPAR, nucleolin, and casein kinase 2, that is responsible for the uPA-related mitogenic response. The complex is not a unique feature of vascular smooth muscle cells, as it was also found in other uPAR-expressing cell types.

Protein inhibitor of cyclic adenosine 3':5'-monophosphate phosphodiesterase in retina.

A protein acting as inhibitor of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.1.) activity was found in the ox retina tissue. An inhibitor from one tissue (ox retina) effectively cross-inhibited a phosphodiesterase from another tissue (rat brain), indicating a lack of tissue specificity. Kinetic analysis showed that inhibition was independent of the time of preliminary incubation of the inhibitor with enzyme but dependent on its concentration in the reaction mixture. An inhibitor decreased the V of the enzyme and had no effect on its Km for cyclic adenosine-3':5'-monophosphate. The inhibitory effect was more pronounced with cyclic adenosine-3':5'-monophosphate than with cyclic guanosine-3':5'-monophosphate used as substrates of the reaction. The extractable form of the phosphodiesterase of the retina rod outer segments was much more sensitive to the inhibitory action than the membrane-bound one. The binding of labeled cyclic adenosine-3':5'-monophosphate to the inhibitory protein was shown not to occur. The inhibitor was sensitive to trypsin treatment, indicating that it was a proten attempt was mode to purify the inhibitory factor. Gel filtration indicated that the inhibitor had a molecular weight of 38 000.

Induction of c-fos gene expression by urokinase-type plasminogen activator in human ovarian cancer cells.

Binding of urokinase-type plasminogen activator (u-PA) to u-PA receptor (u-PAR) induces the rapid and transient expression of c-fos in OC-7 ovarian carcinoma cells. The pretreatment of the cells with protein tyrosine kinase (PTK) inhibitors, but not the inactivation of the u-PA active site by DFP (diisopropyl fluorophosphate), abrogates this effect. A soluble u-PAR fragment, expressed in baculovirus-infected Sf9 cells and purified by affinity chromatography, competes for binding of u-PA to u-PAR and inhibits c-fos induction. We conclude that activation of u-PAR after interaction with u-PA at the cell surface initiates a transmembrane signal, most likely in conjunction with other still unknown protein(s). This signal generates PTK activity feeding into a signal transduction pathway which activates nuclear transcription factors.

Interaction of urokinase-type plasminogenactivator (u-PA) with its cellular receptor (u-PAR) induces phosphorylation on tyrosine of a 38 kDa protein.

We demonstrate by immunoprecipitation that u-PAR is associated with a 38 kDa protein that is phosphorylated on tyrosine after u-PA treatment of cells. As tyrosine phosphorylation is the hallmark of many signal transduction pathways that promote growth and differentiation, these data suggest that u-PA, besides its role as a regulatory protease, might act as a para- or autocrine hormone.

Active sites of the cyclic GMP phosphodiesterase gamma-subunit of retinal rod outer segments.

Monoclonal antibodies were prepared to the gamma-subunit of the cGMP phosphodiesterase. One of them gamma p-1, suppresses the activation of phosphodiesterase through the alpha-subunit of transducin. The gamma-subunit fragment 24-45 rich in Arg and Lys residues is involved in gamma p-1 binding and is essential for the gamma-subunit interaction with transducin. Carboxypeptidase Y cleaves off seven amino acid residues from the C-terminus of the gamma-subunit resulting in phosphodiesterase activation. Thus, the C-terminal fragment of gamma-subunit participates in phosphodiesterase inhibition.

The detection and characterization of G-proteins in the eyespot of Chlamydomonas reinhardtii.

The presence of G-proteins in the eyespot fraction of Chlamydomonas reinhardtii is shown. This fraction is capable of binding (GTP gamma[35S], possesses the GTPase activity and interacts with antibodies raised against a highly conserved peptide of most G-proteins' alpha-subunit. Cross-reaction with a 24-kDa protein is detected on immunoblots. Using an antiserum prepared from vertebrate beta-subunit peptide, two additional proteins with apparent Mr 21 and 29 kDa could be revealed. The light-dependence of GTPase extraction from eyespot membranes is shown. The results make it possible to suggest the participation of G-proteins in the photosensory transduction chain of Ch. reinhardtii.

Immunochemical study of the cyclic nucleotide system of retinal photoreceptor membranes: antibodies raised to phosphodiesterase, its protein inhibitor and GTP-binding proteins.

Monospecific precipitating antibodies raised to phosphodiesterase, its protein inhibitor and GTP-binding proteins of bovine retina photoreceptor membranes were obtained. The characterization of the antibodies was carried out by immunochemical methods and according to their functional properties as determined by their effect on enzyme activities. The antibodies were used to study the distribution of immunolike proteins in different animal retinas and to purify the inhibitor protein by the method of immunoaffinity chromatography.

Induction of c-fos Gene Expression by Urokinase-Type Plasminogen Activator in Human Ovarian Cancer Cells.

The ability to fractionate nucleic acids and to determine which of them has sequences complementary to an array of DNA or RNA molecules is one of the most powerful tools of molecular biology. The Southern blot, named for its inventor, is a method for transferring size-fractionated DNA from a gel matrix to a solid support followed by hybridization to a labeled probe (1. The identical process for RNA became known as) the Northern blot i2 1i The identical process for RNA became known as the Northern blot (2). Both are, then, often key elements in establishing the identity of nucleic acids of interest. Northern blot analysis was used by us as a tool in order to answer the question whether or not the nuclear transcripitional apparatus of human ovarian cancer cells might be activated in response to the urokinase.

[Synthesis of visual rhodopsin in a cell-free translation system. II. Functional properties of recombinant rhodopsin and its mutant forms]. / Sintez zritel'nogo rodopsina v beskletochnoi sisteme transliatsii. II. Funktsional'nye svoistva rekombinantnogo rodopsina i ego mutantnykh form.

Functional expression of bovine visual rhodopsin in the cell-free translation system with cotranslational insertion of the protein into phosphatidylcholine liposomes is described. The recombinant rhodopsin has spectral and functional properties similar to those of natural rhodopsin from bovine retina. Two mutant rhodopsins with amino acid substitutions in the hydrophilic C-terminal domain were obtained using oligonucleotide-directed mutagenesis. It was found that substitution Cys-316----Ser does not affect rhodopsin's ability to activate the visual amplification cascade, whereas double mutation Asp-330----Asn, Asp-331----Asn dramatically lowers the rhodopsin functional activity.

[Participation of the estradiol receptor in the hormonal inhibition of cyclic nucleotide phosphodiesterase in the uterus]. / Uchastie retseptora éstradiola v ingibirovanii gormonom aktivnosti fosfodiésterazy tsiklicheskikh nukleotidov tkani matki.

The role of estradiol receptor was studied in the inhibitory effect of hormone on the cyclic nucleotide phosphodiesterase from immature Wistar rat uterus. It was shown that the preparative separation of the enzyme and hormone receptor by ultracentrifugation in isokinetic sucrose density gradient results in a 2.5-3-fold decrease of the estradiol effect on phosphodiesterase. This effect is completely restored after adding the separated estradiol receptor to the phosphodiesterase devoid of it. The effect of estradiol on the phosphodiesterase activity depends on a degree of receptor component aggregation: the action of estradiol on the enzyme intensities after transformation of receptor into the dissociated form (4S) and removes in the presence of the receptor component associated form (8S).

[Phosphodiesterase of cyclic nucleotides]. / Fosfodiesteraza tsiklicheskikh nukleotidov.

Problems are considered on form multiplicity, purification and molecular weight of cyclic nucleotides phosphodiesterase. A supposition is made that the molecular weight of the catalytic subunit of the enzyme for most studied objects is about 60000. The catalytic subunit may form di- and trimers and be associated with regulatory proteins of different type. The problem of phosphodiesterase regulation is analyzed on the basis of potentialities of the equilibrium shift between the protein subunits of the enzyme; the role of cyclic nucleotides as well as of triphosphonucleotides are shown to influence the regulation of the enzymic activity. In some cases the mechanism of changes in the activity of phosphodiesterase bound with the receptor is shown to be similar to that for adenylate cyclase. In particular, the role of GTP and one of the protein subunits of phosphodiesterase in this process is stated.

[Effect of estradiol on 3',5'-AMP-phosphodiesterase activity in rat uterus. Participation of hormone receptors, the role of guanyl nucleotides]. / Vliianie éstradiola na 3',5'-AMP-fosfodiésterazu tkani matki krys. Uchastie gormonal'nogo retseptora, rol' guanilovykh nukleotidov.

Estradiol (10(-7)-2 . 10(-5)M) is shown to inhibit the activity of phosphodiesterase of the soluble fraction of the mature rat womb tissue and to have no effect on the activity of adenylate cyclase of this tissue membrane fraction and on the phosphodiesterase of the tissue of the brain, heart and outer segments of the rod-cell. When blocking the estradiol cytosol receptors by clomiphene there is no inhibitory effect of the hormone on the activity of phosphodiesterase of the womb tissue preparations. Specific binding of estradiol by the cytosol receptors increases in the presence of guanylic nucleotides (10(-5)-10(-4)M); ATP does not affect this process. In the presence of guanyl-5-ilimidodiphosphate (Gpp(NH)p), a nonhydrolyzed analog of GTP, clomiphene does not block receptor binding of estradiol. ATP and GTP (10(-6)-10(-5)M), contrary to Gpp (NH)p, inhibit the phosphodiesterase activity. Independent of their effect on the enzymic activity, all the studied nucleotides partially or completely eliminate the inhibitory effect of estradiol on phosphodiesterase.

[Adenyl cyclase and adenosine-3',5'-monophosphate phosphodiesteraze: their nature, properties and regulation]. / Adeniltsiklaza i fosfodiésteraza adenozin-3',5'-monofosfata: priroda, svoistva i reguliatsiia

Modern data on the nature, properties and pathways of regulation enzymes, participated metabolism of cyclic, adenosine-3',5'-monophosphate, are described. Much consideration is being given to the problem of regulation of the phosphodiesterase activity due to the effect of Ca2+-binding proteins.

[The functional similarity of vertebrate rhodopsin and of a photosensitive pigment from the unicellular flagellate alga Chlamydomonas reinhardtii]. / O funktsional'nom podobii rodopsina pozvonochnykh i fotochuvstvitel'nogo pigmenta odnokletochnoi zhgutikovoi vodorosli Chlamydomonas reinhardtii.

Photoreceptor membranes in the bovine retina may be substituted in reconstructed systems by pigment fractions from Chlamydomonas reinhardtii which account for GTP-dependent activation of cGMP-phosphodiesterase. Fractions from carotinoid-less mutant did not exhibit this capacity. The results obtained reveal significant structural and functional similarity between rhodopsin-like pigment of Ch. reinhardtii and rhodopsin from vertebrates.

[Effect of liposomes containing antibodies to cyclic nucleotide phosphodiesterase on the receptor potential of rods and cones of the frog retina]. / Vliianie liposom, soderzhashchikh antitela k fosfodiésteraze tsiklicheskikh nukleotidov, na retseptornyi potentsial palochek i kolbochek setchatki liagushki.

Liposomes loaded with monospecific cGMP-phosphodiesterase (PDE) antibodies act like PDE inhibitors (methylxantins, papaverine) on the aspartate-isolated rod and cone receptor potentials of the isolated perfused rod retina. The response amplitude drastically increases, especially at low stimulus strength, and its time course slows down. Pure lipid liposomes with no antibodies, liposomes loaded with control immunoglobulins, as well as PDE antibodies without liposomes, have no such actions. The effects are readily reversible by wash-out, which implies the liposome + antibodies action without penetrating the cells. The increase in the response amplitude can be only partly explained by the measured increase in the extracellular resistance of the photoreceptor layer under liposome action. It is supposed that the specific adsorption of liposomes on the photoreceptor cell surface changes the function of the intracellular transmission mechanism or (and) the properties of ionic channels of the plasma membrane in yet unknown way.

[Soluble uterine proteins similar to GTP-binding proteins of receptor systems]. / O rastvorimykh belkakh matki, podobnykh GTP-sviazyvaiushchim belkam retseptornykh sistem.

Proteins whose molecular mass, GTPase activity and immunochemical properties are similar to those of transducin, a GTP-binding protein of photoreceptor cells, were isolated from the soluble fraction of calf uterine tissue. The proteins were purified and the possibility of their incorporation into a reconstituted system made up of photoreceptor membranes, of phosphodiesterase extracted from these membranes and of transducin was demonstrated.

[Effect of nucleoside triphosphates on cyclic nucleotide phosphodiesterase: role of protein modulators]. / Vliianie nukleozidtrifosfatov na fosforiésterazu tsiklicheskikh nukleotidov; rol' belkovykh moduliatorov.

The effects of nucleoside triphosphates (ATP and GTP) on phosphodiesterase (PDE) of brain and outer segments of the retina enriched or devoid of protein modulators were studied. In the case of retinal outer segment PDE the enzyme activity was considerably inhibited by both nucleosides only when the enzyme was separated from the inhibitor. In case of brain PDE, on the contrary, the effect of the nucleosides was much more pronounced in the enzyme preparation coupled with the protein activator, calmodulin. The latter when added to brain PDE devoid of the activator in the presence of ATP and GTP considerably reduced the enzyme activity. An addition of the inhibitor simultaneously with GTP to the purified PDE of outer segments increased the PDE activity. The constants for the inhibition of brain PDE coupled with calmodulin and retinal outer segment PDE separated from the inhibitor by ATP and GTP were determined.