We have investigated cellular signalling events induced by urokinase-type plasminogen activator (uPA) independent of its proteolytic activity. Treatment of the human fibrosarcoma cell line HT 1080 with diisopropylphosphorofluoridate-inactivated uPA (Dip-FϪuPA) triggers a cascade of intracellular signals which are mediated by the specific cell surface receptor for uPA (uPAR). We have found that antiuPAR Ig precipitate the src-type protein tyrosine kinases fyn, hck and lck, which belong to a family of structurally and functionally related effectors participating in signalling from antigen and cytokine receptors. Of the three uPAR-associated kinases, only hck is activated by uPA, whereas no changes in the activities of either fyn or lck could be detected by an in vitro immune complex kinase assay. We identified p38 and extracellular-signal-regulated kinase 2 from the mitogen-activated protein kinase family as downstream components of a set of consecutive signalling molecules which teleologically alter the program of gene expression. Exposure of cells to uPA results in a significant increase in c-fos mRNA that is partially due to an elevated rate of gene transcription. Presumably, the activation of the c-fos gene leads to the subsequent formation of the transcription factor activator protein-1 (AP-1), since accumulation of c-fos mRNA is followed by induction of target genes sensitive to AP-1 such as plasminogen activator inhibitor type 2 (PAI-2). These results provide new insights into proteolysis-independent cytokine-like effects of uPA.Keywords : plasminogen activator ; urokinase-type-plasminogen-activator receptor; Src-type kinase ; mitogen-activated protein kinase ; signal transduction.Urokinase-type plasminogen activator (uPA) and its receptor (Danø et al., 1994; Ellis and Danø, 1992; Fazioli and Blasi, 1994). (uPAR) are components of a proteolytic system that regulates There is growing evidence that in addition to its well-characmatrix remodelling during processes such as wound healing, cell terized regulatory proteolytic activity, uPA also acts as a cytomigration, developmental tissue remodelling, inflammation and kine-type cellular-response modifier. These biological activities tumor invasion (Danø et al., 1994;Vassalli et al., 1991; Pöllänen of uPA include a mitogenic effect (Rabbani et al., 1990(Rabbani et al., , 1992(Rabbani et al., ), et al., 1991. uPAR is a glycoprotein with an apparent molecular stimulation of chemotaxis of monocytes (Gyetko et al., 1994), mass of 45Ϫ60 kDa attached to the plasma membrane via a glyneutrophils (Gudewicz and Gilboa, 1987) and epidermal cells cosyl phosphatidylinositol (glycosyl-PtdIns) anchor (Vassalli, (Del Rosso et al., 1993), as well as regulation of adhesion of 1994; Plough et al., 1991). One of the proposed roles of uPAR myeloid cells (Nusrat and Chapman, 1991; Waltz et al., 1993). is the organization of proteolysis in space and time by concenWhereas these activities are well documented, very little infortrating plasmin generation to specific sites on the cell s...

Torsin A and torsin B have similar distribution in the central nervous system, although their subcellular localization is not identical. Strong expression in neuronal processes points to a potential role for torsin proteins in synaptic functioning.

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Early-onset dystonia is caused by mutations in the torsinA protein, a putative member of the AAA+ class of ATPases. In this study we have evaluated the ATPase activity of bacterially expressed wild-type torsinA and its disease-associated mutant forms. Upon overexpression in Escherichia coli, recombinant torsinA proteins were accumulated as insoluble inclusion bodies and required refolding to become soluble and catalytically active. The refolded wild-type and mutant torsinA proteins were capable of hydrolyzing ATP, but their specific ATPase activities differed significantly. Deletions of the amino acid residues E302/303 and F323-Y328 resulted in a decrease of ATPase activity to approximately 35% and approximately 75% of the wild-type level, respectively. ATPase activity of wild-type and mutant torsinA proteins was influenced by factors that varied with cell stress, such as temperature, pH, and ionic strength, and was inhibited by sodium vanadate. Our results provide the first direct evidence for a role of torsinA as an active ATPase and suggest that the mutations in torsinA might affect normal functions of the protein by reducing its enzymatic activity.