NERVE GROWTH FACTOR-GAMMA ACTIVATES SOLUBLE AND RECEPTOR-BOUND SINGLE-CHAIN UROKINASE-TYPE PLASMINOGEN-ACTIVATOR

Nerve growth factor-gamma (NGF-gamma) is a serine proteinase which rev ersibly associates with the well characterized neurotrophin NGF-beta. In this study, we demonstrated that NGF-gamma cleaves recombinant sing le chain urokinase-type plasminogen activator (scu-PA), converting the zymogen into a two-chain form (tcu-PA). The apparent masses of the tw o u-PA chains were 33 and 22 kDa, as determined by SDS-polyacrylamide gel electrophoresis (PAGE). There was no evidence for secondary cleava ge sites or further digestion of tcu-PA by NGF-gamma, even when conver sion of scu-PA was complete. The NH2-terminal sequence of the 33-kDa b and was Ile-Ile-Gly-Gly-Glu, indicating that NGF-gamma cleaved scu-PA at Lys158-Ile159, the plasmin cleavage site. Cleavage of scu-PA by NGF -gamma resulted in scu-PA activation. The k(cat) and K(m) for this rea ction, as determined in a continuous assay with the tcu-PA-specific su bstrate L-pyroglutamyl-glycyl-arginine-p-nitroanilide hydrochloride (S -2444), were (4.1 +/- 0.6) x 10(-2) s-1 and 2.3 +/- 0.4 muM, respectiv ely. The catalytic efficiency (k(cat)/K(m)) for scu-PA activation by N GF-gamma was 1.3 x 10(4) M-1 s-1, compared with 6.2 x 10(5) M-1 s-1 fo r the activation of scu-PA by plasmin. NGF-gamma-cleaved scu-PA which was bound to receptors on U937 monocytoid cells. The apparent masses o f the resulting u-PA cleavage products were identical to those generat ed in solution as determined by SDS-PAGE. Cell-associated scu-PA was a ctivated by NGF-gamma, as determined by the generation of activity aga inst the tcu-PA-specific fluorogenic substrate, glutamyl-glycyl-argini ne-7-amino-4-methyl coumarin. By activating scu-PA, NGF-gamma may init iate the u-PA-dependent cell-surface proteinase cascade and support NG F-beta activities which involve cellular migration and/or extracellula r matrix remodeling.