Increased levels of both the cysteine protease, cathepsin L, and the serinc protease, uPA (urokinase-typc plasminogen activator), are present in solid tumors and are correlated with malignancy, uPA is released by tumor cells as an inactive single.chain proenzyme (pro-uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protcase, cathepsin L, on recombinant human pro-uPA. Enzymatic assays, SDS-PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro.uPA, As determined by N-terminal amino acid sequence analysis, activation of pro-uPA by cathepsin L is achieved by cleavage of the Lys~K-lle~9 peptidc bond, a common activation site of serine proteases such as plasmin and kallikrcin. Similar to cathepsin B (Kobayashi et al,, J. Biol, Chem, (1991) 266, 5147-5152) cleavage of pro-uPA by cathepsin L was most efl'~ctive at acidic pH (molar ratio ofcathepsin L to pro-uPA of 1:2,000), Nevertheless, even at pH 7,0, pro-uPA was activated by cathepsin L, althougla a 10-fold higher concentration of cathep~in L was required, As tumor cells may produce both pro-uPA and cathepsin L, implications for the activation of tumor cell-derived pro-uPA by cathepsin L may be considered. Different pathways of activation of pro-uPA in tumor tissues may coexist: (i) autoeatalytic intrinsic activation of pro-uPA; (it) activation by serine protcases (plhsmin, kallikrein, Factor Xlla); and (iii) activation by cysteine proteases (cathepsin B and L), Pro-uPA; Urokiuase: Cysteine protease; Cathepsin B; Cathepsin L; N-Terminal amino acid sequence analysis
