Vitronectin is a multifunctional glycoprotein present in blood and in the extracellular matrix. It binds glycosaminoglycans, collagen, plasminogen and the urokinase-receptor, and also stabilizes the inhibitory conformation of plasminogen activation inhibitor-1. By its localization in the extracellular matrix and its binding to plasminogen activation inhibitor-1, vitronectin can potentially regulate the proteolytic degradation of this matrix. In addition, vitronectin binds to complement, to heparin and to thrombin-antithrombin III complexes, implicating its participation in the immune response and in the regulation of clot formation. The biological functions of vitronectin can be modulated by proteolytic enzymes, and by exo- and ecto-protein kinases present in blood. Vitronectin contains an RGD sequence, through which it binds to the integrin receptor alpha v beta 3, and is involved in the cell attachment, spreading and migration. Antibodies against alpha v beta 3 or synthetic peptides containing an RGD sequence are now being tested as therapeutic agents in the treatment of human cancers, bone diseases (e.g. osteoporosis) and in pathological disorders which involve angiogenesis.

A set of 45 mutants of the carboxyl terminal tail of the PKA catalytic subunit was prepared and used to assess the contribution of this tail to the structure and function of the kinase. Ala substitutions of Asp 323, Phe 327, Glu 333, and Phe 350 resulted in a complete loss of enzymatic activity. Other replacements by Ala (Phe 314, Tyr 330, Glu 332, and Phe 347) brought about either a drop in activity to less than 10% of the wild-type enzyme or a reduction of affinity toward ATP (Lys 317, Lys 319, Tyr 330, and Glu 332) or toward Kemptide (Ile 315, Tyr 330, Val 337, Ile 339, Lys 345, and Glu 346). Mutations of Ser 338, a major autophosphorylation site of PKA, by Ala, Glu, Asp, Gln, and Asn showed that the kinetic parameters of these mutants are similar to those of the wild-type. The contribution of each of these tail mutations to the structure and stability of the kinase was assessed by monitoring its effect on the heat stability (when measurable) or by determining the susceptibility of the mutant kinase to cleavage by the Kinase Splitting Membranal Proteinase/Meprin beta. Here we show that the tail of PKA has a key role in creating the active conformation of the kinase. It does so by means of specific amino acid residues, which act as "snapping points" to embrace the two lobes of the kinase and orient them in the correct juxtaposition for substrate docking, biorecognition, and catalysis.

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Morphogenesis and tissue repair require appropriate cross-talk between the cells and their surrounding milieu, which includes extracellular components and soluble factors, e.g., cytokines and growth factors. The present work deals with this communication needed for recovery after axotomy in the central nervous system (CNS). The failure of CNS axons to regenerate after axonal injury has been attributed, in part, to astrocyte failure to repopulate the injury site. The goal of this work was to provide an in vitro model to mimic the in vivo response of astrocytes to nerve injury and to find ways to modulate this response and create a milieu that favors astrocyte migration and repopulation of the injury site. In an astrocyte scratch wound model, we blocked astrocyte migration by tumor necrosis factor alpha (TNF-alpha). This effect could not be reversed by astrocyte migration-inducing factors such as transforming growth factor beta 1 (TGF-beta 1) or by any of the tested extracellular matrix (ECM) components (laminin and fibronectin) except for vitronectin (Vn). Vn, added together with TNF-alpha, counteracted the TNF-alpha blockage and allowed a massive migration of astrocytes (not due to cell proliferation) beyond that allowed by Vn only. Heparan sulfate proteoglycans (HSPG) were shown to be involved in the migration. The results may be relevant to regeneration of CNS axons, and may also provide an example that an extracellular component (Vn) can overcome and neutralize a negative effect of a growth factor/cytokine (TNF-alpha) and can act in synergy with other features of this cytokine to promote a necessary function (e.g., cell migration) that is otherwise inhibited.

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Pigment epithelium-derived factor (PEDF), a neurotrophic and antiangiogenic protein, is an extracellular component of the retinal interphotoreceptor matrix which has been shown to be secreted by human fetal retinal pigment epithelial cells. It belongs to the serpin superfamily and contains the typical exposed reactive center loop. The function of this loop is still unknown. In this study we used site-directed mutagenesis of the cDNA encoding PEDF to show that (a) truncation of the C-terminal tail (Pro415-Pro418) of PEDF, (b) deletion of the Pro373-Ala380 segment that resides within the reactive center loop of the protein, and (c) alanine substitution of amino-acid residues Asn391-Thr403 located within its hydrophobic core inhibit PEDF secretion, but not its transcription, by cells transfected with the various PEDF cDNAs. On the basis of the crystal structure of PEDF, these mutations are presumed to alter the protein conformation, suggesting that conservation of the 3D structure of PEDF is essential for its secretion. In addition, we show that replacement of Gly376 and Leu377 with alanine prevents PEDF secretion. As these two residues are located within the highly exposed segment of the reactive center loop, we propose a novel function for this loop in PEDF. Our results imply that the reactive center loop, specifically Gly376 and Leu377, is involved in the interaction of PEDF with components of the quality control system in the endoplasmic reticulum, thus ensuring its efficient secretion.

We have studied the association between protein tyrosine phosphorylation and the mitogenic effect induced by platelet-derived growth factor (PDGF) in the osteoblast-like cell line MC3T3-E1. PDGF caused a dose-dependent increase in [3H]thymidine incorporation in MC3T3-E1 cells, reaching a plateau at 10 ng/ml. Vanadate, a potent phosphatase inhibitor, induced a twofold increase in thymidine incorporation. The combination of vanadate and PDGF resulted in a dose-dependent synergistic effect on thymidine incorporation. Genistein, a tyrosine kinase inhibitor, inhibited in a dose-related manner (2-20 microM) the mitogenic effect induced by either PDGF or the combination of vanadate and PDGF. These observations suggest that tyrosine kinases are involved in mediating the mitogenic effect of PDGF in these cells. PDGF treatment of MC3T3-E1 cells and subsequent immunoblotting with antiphosphotyrosine antibodies resulted in a marked phosphorylation of the PDGF receptor. Vanadate had a lesser effect on PDGF receptor phosphorylation, but given together with PDGF it induced a significant increase in the intensity of receptor phosphorylation. Preincubation with genistein abrogated these effects. Taken together, these findings indicate a direct correlation between thymidine incorporation and tyrosine phosphorylation in MC3T3-E1 cells and suggest that tyrosine phosphorylation plays a role in PDGF-induced mitogenic activity in osteoblast-like cells.

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