Protein phosphatase 1 regulation by inhibitors and targeting subunits

Regulation of protein phosphatase 1 (PP1) by protein inhibitors and targeti ng subunits has been previously studied through the use of recombinant prot ein expressed in Escherichia coli. This preparation is limited by several k ey differences in its properties compared with native PP1. In the present s tudy, we have analyzed recombinant PP1 expressed in Sf9 insect cells using baculovirus. Sf9 PP1 exhibited properties identical to those of native PP1, with respect to regulation by metals, inhibitor proteins, and targeting su bunits, and failure to dephosphorylate a phosphotyrosine-containing substra te or phospho-DARPP-32 (Dopamine and cAMP-regulated phosphoprotein, M-r 32, 000). Mutations at Y272 in the beta 12/beta 13 loop resulted in a loss of a ctivity and reduced the sensitivity to thiophospho-DARPP-32 and inhibitor-2 . Mutations of Y272 also increased the relative activity toward a phosphoty rosine-containing substrate or phospho-DARPP-32. Mutation of acidic groove residues caused no change in sensitivity to thiophospho-DARPP-32 or inhibit or-2, but one mutant (E252A:D253A:E256R) exhibited an increased K-m for pho sphorylase a. Several PP1/PP2A chimeras were prepared in which C-terminal s equences of PP2A were substituted into PP1. Replacement of residues 274-330 of PP1 with the corresponding region of PP2A resulted in a large loss of s ensitivity to thiophospho-DARPP-32 and inhibitor-2, and also resulted in a loss of interaction with the targeting subunits, spinophilin and PP1 nuclea r targeting subunit (PNUTS). More limited alterations in residues in beta 1 2, beta 13, and beta 14 strands highlighted a key role for M290 and C291 in the interaction of PP1 with thiophospho-DARPP-32, but not inhibitor-2.