Phosphorylation of the phosphatase modulator subunit (inhibitor‐2) by casein kinase‐1 Identification of the phosphorylation sites

Abstract

The isolated modulator subunit of the inactive protein phosphatase-I is phosphorylat~ in vitro by casein kinase.l at two different sims: Ser.86 and Ser-174. The Set-S6 site is a common target for casein kinaze-1 and casein kinase-2, but is preferentially ~hosphorylated by the former enzyme. The Ser-174 site seems to be specific for casein kinase-l, aud is phosphorylated at a slower rate. Th¢~ rcsult~ give a n~w insisht into the in vitro phosphorylation pattern of the modulator subanit of the phosphatase and providez additional data on the specificity of cabin kiaas¢-l.

Phosphorylation of synthetic fragments of inhibitor‐2 of protein phosphatase‐1 by casein kinase‐1 and ‐2

“…A pentadecapeptide reproducing such a site is in fact a substrate as good as the parent protein both in terms of K,, which is nearly identical, and of V,,,, which is actually slightly higher with the peptide substrate. The superiority of the I-2(78 -93) peptide substrate over I-2(112-128) (which is accounted for by a fivefold lower K,) is in agreement with the observation that Ser86 is phosphorylated more rapidly than Serl20/ Serl21 in the parent protein as well (Agostinis et al, 1992). The structural basis for the preferential phosphorylation of Ser86 therefore probably resides in the different sequence of amino acids surrounding Ser86 as opposed to Ser120/121: in particular, an acidic residue at position + 1, whose absence causes a 10-fold increase in K , (Pinna, 1990), is present only in the former site; Serl20-Serl21 moreover are preceded by a basic stretch upstream (KYRIR), which is likely to play a detrimental role, by analogy with basic residues at other positions (Pinna, 1990).…”

“…2; their concentration was 1 mM. As expected, the peptides I-2(67-93) and I-2(166-180) are readily phosphorylated while the peptide I-2(112-128) which includes a site affected by CK2 but not by CKI (Agostinis et al, 1992), is not appreciably affected. The phosphorylation of I-2(67-93) is mostly accounted for by Serf', though traces of ThrP are also detectable (not shown).…”

“…This result was unexpected since Ser174 neither fulfils the canonical consensus sequence for CK2 phosphorylation, requiring an acidic residue at position +3 (see Fig. 1), nor is it appreciably affected by CK2 in intact 1-2 (Holmes et al, 1986;Agostinis et al, 1992). It is likely therefore that in this case CK2 recognizes an atypical consensus sequence, similar to those recently outlined with a series of phosphopeptides (Perich et al, 1992), crucially grounded on acidic residues flanking the target Ser.…”

“…CK2 affects two sites of 1-2, Ser86 and Serl20/Ser121 (Holmes et al, 1986); the former alone, however, is responsible for the synergistic effect leading to the phosphorylation of Thr72 by GSK-3 and consequently to the activation of PP-1, (De Paoli-Roach et al, 1989). 1-2 is also a good target for casein kinase-1 (CK1) (Agostinis et al, 1987) and we have recently reported that two sites are affected in this case: Ser86, which is also a target for CK2, and Ser174 which is not appreciably phosphorylated by CK2 (Agostinis et al, 1992). This finding disclosed the possibility that CK1 could be responsible for the hierarchal phosphorylation of 1-2 leading to the activation of PP-l,, instead of, or in addition to, CK2.…”

Phosphorylation of Yeast Plasma Membrane H+-ATPase by Casein Kinase I

“…The wild-type strain LRB341 which has a genetic background different from that of ⌺1278b is the parental strain of the yck mutants (21,22) used in this study (see below). Fig.…”