Spontaneous and chaperone-assisted metal loading in the active site of protein phosphatase-1.

Protein phosphatase PP1 has two active-site metals (Zn 2+ /Fe 2+ ) that are essential for catalysis. However, when expressed in bacteria, PP1 has two Mn 2+ -ions in its active site, indicating that the incorporation of Zn 2+ /Fe 2+ depends on additional eukaryotic component(s). Here, we used purified, metal-deficient PP1 to study metal incorporation. Fe 2+ was incorporated spontaneously, but Zn 2+ was not. Mn 2+ -incorporation at physiological pH depended on the co-expression of PP1 with PPP1R2 (Inhibitor-2) or PPP1R11 (Inhibitor-3), or a pre-incubation of PP1 at pH 4. We also demonstrate that PPP1R2 and PPP1R11 are Zn 2+ -binding proteins but are, by themselves, not able to load PP1 with Zn 2+ . Our data suggest that PPP1R2 and PPP1R11 function as metal chaperones for PP1 but depend on co-chaperone(s) and/or specific modification(s) for the transfer of associated Zn 2+ to PP1.