PHOSPHORYLATION OF SYNTHETIC FRAGMENTS OF INHIBITOR-2 OF PROTEIN PHOSPHATASE-1 BY CASEIN KINASE-1 AND KINASE-2 - EVIDENCE THAT PHOSPHORYLATED RESIDUES ARE NOT STRICTLY REQUIRED FOR EFFICIENT TARGETING BY CASEIN KINASE-1

The major phosphorylation site for both casein kinase-2 (CK2) and casein kinase-1 (CK1) in protein phosphatase-1 (PP-1) inhibitor-2 (I-2) is Ser86. Minor phosphorylation sites affected by either CK2 or CK1 are Ser120/Ser121 and Ser174, respectively. A synthetic peptide of 25 amino acids encompassing residues 67-93 of I-2 is phosphorylated by either CK2 or CK1 at its seryl residue corresponding to Ser86 with higher V-max and K-m values similar to those of the intact protein (9 vs 7.2 mu M and 14.2 vs 5.3 mu M with CK2 and CK1, respectively). No detectable phosphorylation of this peptide which also includes the glycogen synthase kinase-3 (GSK-3) site (Thr72), could be observed with either GSK-3 or p34(cdc2) kinase whether or not its seryl residue equivalent to Ser86 had been previously phosphorylated by CK2. Shorter derivatives of I-2(67-93), encompassing residues 72-93 and 78-93, are also readily phosphorylated by both CK1 and CK2, with phosphorylation efficiencies similar to those of the parent peptide. A synthetic heptadecapeptide reproducing the phosphoacceptor site around Ser120/Ser121 is phosphorylated by CK2, but not to any detectable extent by CK1, with a K-m value fivefold higher than that of the corresponding pentadecapeptide including Ser86 (78-93). A synthetic pentadecapeptide (166-180) reproducing the phosphoacceptor site around Ser174 is phosphorylated by CK1 less efficiently than the pentadecapeptide including its main phosphorylation site (78-93) (K-m 280 mu M vs 33 mu M). This peptide is readily phosphorylated by CK2 as well, although it lacks the canonical consensus sequence for CK2 and its Ser174 is almost unaffected by CK2 in intact I-2. These data provide the clear-cut demonstration that the consensus sequence with N-tenminal prephosphorylated residue(s), SerP/ThrP-Xaa-Xaa-Ser/Thr, [Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W & Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269; Meggio, F., Perich, J. W, Reynolds, E. C. & Pinna, L. A, (1991) FEBS Lett. 283, 303-306] is not always required to achieve efficient and high-affinity phosphorylation by CK1. They also show that the specificity determinants for I-2 phosphorylation by either CK2 or CK1, but not by GSK3, are entirely grounded on local structural features of the phosphoacceptor site, being only marginally affected by the overall structure of I-2.