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 Vmax and Km values similar to those 
of the intact protein (9 vs 7.2 microM and 14.2 vs 5.3 microM 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 p34cdc2 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 Km 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) (Km 280 microM vs 33 microM). 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-terminal 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.