Activation of Easter by Snake when all three zymogens are coexpressed.
Western blots with anti-Easter antibody to detect Easter in cell pellet
(A and B) and culture medium
(A and C) after transfection of S2 cells.
(A) EAS-A is cleaved from zymogen form (z) to 35-kDa
C-terminal product (c) when expressed with GD + SNK zymogens (lane 4 or
8), but not when expressed with only GD (lane 2 or 6) or SNK (lane 3 or
7). (B) When expressed with GD + SNK zymogens, EAS-A is
cleaved to generate a 35-kDa product (c, lane 4) that migrates
similarly to EAΔN, the functional Easter catalytic domain (lane 2);
wild-type EA is cleaved to generate predominantly a smaller product
(c′, lane 3). Double catalytic and zymogen-activation mutant EAR-V,S-A
is not cleaved (lane 5). (C) EAS-A is cleaved when
expressed with preactivated SNK (SNKΔN; lane 2) but not when
expressed with preactivated GD (GDΔN1; lane 1).