Figure 2.

Nodal precursor cleavage is enhanced by SPC1 and SPC4, or by structural modification of the cleavage site. (A) Native Nodal or a mutant form, termed Nodal-H246L, was analyzed by Western blotting in cell lysates (top) or supernatants (bottom) using anti-Nodal antiserum directed against a peptide in the middle of the COOH-terminal domain. The mutant sequence in Nodal-H246L near the multibasic cleavage site is aligned with that of native Nodal (top). Where indicated, COS cells were cotransfected with 0.5 μg of either SPC1, SPC4, or SPC7 expression vector. Bands corresponding to unprocessed precursor are marked pre. Note the absence of any detectable mature Nodal. An additional, nonspecific band is present in all supernatants, suggesting that comparable amounts of protein were loaded. (B) Cleavage of Flag-tagged Nodal precursor and of FlagNodal-H246L visualized by anti-FLAG immunoblotting. The position of the Flag epitope (open box) relative to the cleavage site (arrow) within Nodal and Nodal-H246L is shown schematically. The Nodal pro domain (pro) accumulates in cell supernatants (bottom), but not in lysates (top). Cotransfection with SPC1 or SPC4, but not SPC7, resulted in enhanced precursor cleavage. (C) The multibasic motif R-Q-R-R present in Nodal-H246L is replaced by SQAG in the Flag-cmNodal-H246L construct, resulting in complete inhibition of processing by either resident COS cell proteases or by SPC1 transfection.