Figure 7.
Isoform-specific dephosphorylation of furin by PP2A. (A) Baculovirus recombinants (m.o.i. = 2) were used to express PP2A subunits, either alone or in combination, in Sf9 cells. At 64–72 h postinfection, cells were harvested and lysates were assayed for phosphatase activity with both 32P-labeled phosphorylase a (open bars) and phosphorylated 32P-labeled GST–Furcd (hatched bars). Noninfected Sf9 cells (data not shown) expressed a detectable level of endogenous furin-directed phosphatase activity which was not increased by expression of the catalytic subunit either alone or in combination with the A subunit (C, and A+C, respectively). The C and A subunits were also expressed with either the α or β isoforms of the B family of regulatory subunits (A+C+Bα and A+C+Bβ, respectively), as well as the α subunit from the unrelated B′ family (A+C+B′α). As a control, lysates from A+C+Bβ–expressing cells were exposed to 100 nM tautomycin and okadaic acid (+Inhib.) during the phosphatase assays. Assays were performed in triplicate with relative error <5%. The data shown are representative of several independent experiments. (B) Western analysis of PP2A subunit expression. Lysates of Sf9 cells infected with baculovirus recombinants encoding the indicated PP2A subunits were resolved by SDS-PAGE and screened by Western blot using antisera specific for the catalytic subunit or the Bα, Bβ, or B′α regulatory subunits.