Nerve growth factor-γ activates soluble and receptor-bound single chain urokinase-type plasminogen activator

Beni B. Wolf, Jayanand Vasudevan, Jack Henkin, Steven L. Gonias

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35 Scopus citations

Abstract

Nerve growth factor-γ (NGF-γ) is a serine proteinase which reversibly associates with the well characterized neurotrophin NGF-β. In this study, we demonstrated that NGF-γ cleaves recombinant single chain urokinase-type plasminogen activator (scu-PA), converting the zymogen into a two-chain form (tcu-PA). The apparent masses of the two u-PA chains were 33 and 22 kDa, as determined by SDS-polyacrylamide gel electrophoresis (PAGE). There was no evidence for secondary cleavage sites or further digestion of tcu-PA by NGF-γ, even when conversion of scu-PA was complete. The NH2-terminal sequence of the 33-kDa band was Ile-Ile-Gly-Gly-Glu, indicating that NGF-γ cleaved scu-PA at Lys158-Ile159, the plasmin cleavage site. Cleavage of scu-PA by NGF-γ resulted in scu-PA activation. The kcat and Km for this reaction, as determined in a continuous assay with the tcu-PA-specific substrate L-pyroglutamyl-glycyl-arginine-p-nitroanilide hydrochloride (S-2444), were (4.1 ± 0.6) × 10-2 s-1 and 2.3 ± 0.4 μM, respectively. The catalytic efficiency (kcat/Km) for scu-PA activation by NGF-γ was 1.3 × 104 M-1 s-1, compared with 6.2 × 105 M-1 s-1 for the activation of scu-PA by plasmin. NGF-γ-cleaved scu-PA which was bound to receptors on U937 monocytoid cells. The apparent masses of the resulting u-PA cleavage products were identical to those generated in solution as determined by SDS-PAGE. Cell-associated scu-PA was activated by NGF-γ, as determined by the generation of activity against the tcu-PA-specific fluorogenic substrate, glutamyl-glycyl-arginine-7-amino-4-methyl coumarin. By activating scu-PA, NGF-γ may initiate the u-PA-dependent cell-surface proteinase cascade and support NGF-β activities which involve cellular migration and/or extracellular matrix remodeling.

Original languageEnglish (US)
Pages (from-to)16327-16331
Number of pages5
JournalJournal of Biological Chemistry
Volume268
Issue number22
StatePublished - Aug 5 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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