Binding of uPA to nucleolin. (A) Domain organization of uPA and its derivatives. GFD indicates growth factor-like domain; K, kringle-domain; and PD, protease domain. (B) Mapping of the nucleolin-binding domain in scuPA. Lysates from human CASMCs (designated as h-SMC on the figure) and mouse embryonic fibroblasts (MEF-1) were incubated with affinity matrices containing immobilized WT-scuPA, LMW-uPA, ATF, ΔGFD-scuPA, ΔK-scuPA, or BSA as a negative control. Bound proteins were detected by WB using α-nucleolin pAb (top panels), α-human uPAR pAb (lower left panel), and α-mouse uPAR pAb (bottom right panel). GFD-containing uPA mutants bound uPAR, whereas those containing the KD but not GFD bound nucleolin in uPAR-independent manner. (C) Pull-down of nucleolin with uPA deletion variants. ¹²⁵I-WT-scuPA, ¹²⁵I-ΔGFD-scuPA, or ¹²⁵I-ΔK-scuPA (5 nM) was added to 293HEK lysates, and nucleolin was immunoprecipitated with an α-nucleolin pAb (lanes “α-nucleolin”). Rabbit Ig was used as a negative control (lanes “IgG”). The immune complexes were analyzed by WB. Nucleolin was detected using an α-nucleolin MAb (top panel). Co-IP: Bound scuPA deletion variants were detected by autoradiography (bottom panel). (D) Domain organization of nucleolin. AS indicates acidic stretch; GAR, glycine- and arginine-rich domain; RBD, RNA-binding domain; and NLS, nuclear localization signal. (E) Mapping the uPA-binding region in nucleolin. 293 HEK cells were transfected with pcDNA3.1 vectors encoding FLAG-tagged nucleolin mutants. Mutants are as described in panel D. The corresponding lysates were incubated with ¹²⁵I-WT-scuPA for 1 hour at 20°C, and nucleolin mutants were immunoprecipitated with agarose-conjugated α-FLAG MAb. The immunoprecipitates were analyzed by WB, FLAG-nucleolin variants were visualized with an HRP-α-FLAG MAb (α-FLAG, top panel), and ¹²⁵I-WT-scuPA was imaged by autoradiography (¹²⁵I-scuPA, bottom panel). Vertical lines have been inserted to indicate repositioned gel lanes (“N-FLAG-WT” and “ΔGAR”). (F) Coimmunoprecipitation of nucleolin with ¹²⁵I-WT-scuPA. HeLa cells were preincubated with 50 nM ¹²⁵I-WT-scuPA for 1 hour at 37°C and then lysed. Nucleolin or scuPA was immunoprecipitated with rabbit polyclonal α-nucleolin Ab (lane “α-nucleolin”) or with rabbit polyclonal α-uPA Ab (lane “α-uPA”), respectively. Rabbit Ig was used as a negative control (lanes “IgG”). The immunoprecipitates were analyzed by WB using mouse α-nucleolin MAb (panel “WB: α-nucleolin”), and autoradiography to detect ¹²⁵I-WT-scuPA (panel “Autograph” ¹²⁵I-scuPA). An experiment representative of 3 so performed is shown.