The proteolytic enzyme, urokinase-type plasminogen activator (uPA) is involved directly in the invasive and metastatic behavior of a number of malignant cell systems. The regulation and control of uPA's expression, activation, catalytic specificity and inhibition by natural inhibitors is not well understood. As a structurally complex, multi- domain serine protease it appears that part of uPA's regulation and specificity is inherent in its complex structure. Chicken uPA (chuPA), although structurally and catalytically a true member of the family of PA proteins, appears to be and unusual family member. When it is expressed at elevated levels by transformed and cytokine-treated chicken cells, chuPA manifests its activity in an uncontrolled, unregulated manner: It is insensitive to the known uPA-specific serpins, it accumulates in plasminogen-free cultures as active enzyme, and most unusually it is activated by an autocatalytic mechanism. No other uPA or tPA is known to exhibit these characteristics. The overall aim of this proposal is to conduct a structure-function analysis of the catalytic and non-catalytic domains of chuPA. The objective is to relate specific domain or sub-structures in the chuPA protein to the regulation and control of the enzyme and to determine how the presence or absence of these sub-structures affects cell and tissue phenotype.
The specific aims are: 1) To analyze the regulatory role of the specific serpin binding motif, RRHR, present in human uPA and total absent in chicken uPA. Mutagenesis and motif swapping of the chuPA and huPA cDNAs followed by transfection, expression and phenotype analysis will be carried out: 2) To identify through chimeric constructs and site-directed mutagenesis the structural motifs that allow chuPA to autoactivate and human uPA to maintained as a stable zymogen: 3) To search for, detect, clone and characterize the missing natural inhibitor of chuPA. Both biochemical detection and oligonucleotide probing approaches will be utilized in the search and cloning attempts.
| Ramos-DeSimone, N; Hahn-Dantona, E; Sipley, J et al. (1999) Activation of matrix metalloproteinase-9 (MMP-9) via a converging plasmin/stromelysin-1 cascade enhances tumor cell invasion. J Biol Chem 274:13066-76 |
| Alexander, D S; Sipley, J D; Quigley, J P (1998) Autoactivation of avian urokinase-type plasminogen activator (uPA). A novel mode of initiation of the uPA/plasmin cascade. J Biol Chem 273:7457-61 |
