Substrate-specificity determinants for a membrane-bound casein kinase of lactating mammary gland. A study with...

A tissue-specific casein kinase, purified from the Golgi-enriched-membrane fraction of guinea-pig lactating mammary gland (GEF-CK), readily phosphorylates the synthetic peptide Ser-Glu5, a good substrate of casein kinase-2, and several derivatives varying for the number and position of acidic residues on the C-terminal side of serine, except those lacking an acidic side chain at position +2. The least acidic peptide, still significantly affected by GEF-CK, is Ser-Ala-Glu-Ala3 which is not a substrate for CK-2. Conversely, the peptides Ser-Ala2-Glu-Ala2, Ser-Ala2-Glu3, Ser-Ala2-Glu5 and Ser-Glu-Ala-Glu3, all of which are more or less readily phosphorylated by CK-2, are not appreciably affected by GEF-CK. On the other hand the presence of additional glutamyl residues, besides the one in the second position, improves the affinity of the peptide substrate for GEF-CK, as indicated by the Km values of Ser-Glu5, Ser-Glu2-Ala3 and Ser-Ala-Glu-Ala3 which are 80, 950 and 3950 microM respectively. It is concluded that although both CK-2 and GEF-CK require, for optimal activity, rather extended acidic clusters on the C-terminal side of the target serine, the most critical residue in the case of GEF-CK is not the one at position +3, which is required for CK-2 catalyzed phosphorylation [Marin, O. et al. (1986) Eur. J. Biochem. 160, 239-244], but the one lying at position +2. Additional differences, concerning the site specificities of these enzymes, have been outlined using the threonyl derivative of Ser-Glu5 and the peptide Arg-Ser-Glu3-Val-Glu. The former is still phosphorylated by CK-2 but not to any appreciable extent by GEF-CK, which apparently is strictly specific for seryl residues. On the contrary, the presence of an N-terminal basic residue, which greatly reduces phosphorylation by CK-2, is tolerated rather well by GEF-CK. On the other hand a C-terminal basic residue, interrupting the acidic cluster, compromises phosphorylation by GEF-CK, as indicated by the extremely high Km value of Ser-Glu3-Lys-Glu vs Ser-Glu3-Val-Glu (13,000 and 170 microM, respectively).