CHARACTERIZATION OF THE INTERACTION BETWEEN DARPP-32 AND PROTEIN PHOSPHATASE-1 (PP-1) - DARPP-32 PEPTIDES ANTAGONIZE THE INTERACTION OF PP-1 WITH BINDING-PROTEINS

The catalytic subunit of PP-1 (PP-1C) is potently inhibited (IC50, app roximate to 1 nM) by DARPP-32 ((d) under bar opamine- and c (A) under bar MP-(r) under bar egulated (p) under bar hos (p) under bar hoprotei n, M-r<(32)under bar>,000), inhibitor-1, and inhibitor-2. The NH2-term inal 50 amino acid residues of DARPP-32 and inhibitor-1 are similar, a nd phosphorylation of a common threonine residue (Thr-34/Thr-35) is ne cessary for inhibition of PP-1C. We have characterized further the int eraction between DARPP-32 and PP-1C. Using synthetic peptides derived from the NH2-terminal region of DARPP-32, residues 6-11, RKKIQF, have been shown to be required for inhibition of PP-1C. Peptides containing this motif were able to antagonize the inhibition of PP-1C by phospho -DARPP-32 and phosphoinhibitor-1. The inhibition of PP-1C by inhibitor -2, but not by okadaic acid, microcystin, or calyculin A, was also att entuated by these antagonist peptides. These results together with res ults from other studies support a model in which two subdomains of pho spho-DARPP-32 interact with PP-1C. The region encompassing phospho-Thr -34 appears to interact with the active site of the enzyme blocking en zyme activity. The region encompassing the RKKIQF motif binds to a dom ain of PP-1C removed from the active site. Amino acid sequence analysi s indicates that basic and hydrophobic features of the RKKIQF motif ar e conserved in the binding domains of certain PP-1C targeting proteins , suggesting that interaction of inhibitor proteins and targeting prot eins may be mutually exclusive.