Substrate‐specificity determinants for a membrane‐bound casein kinase of lactating mammary gland.

Abstract

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 K_m values of Ser‐Glu₅, Ser‐Glu₂‐Ala₃ and Ser‐Ala‐Glu‐Ala₃ which are 80, 950 and 3950 μM 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 K_m value of Ser‐Glu₃‐Lys‐Glu vs Ser‐Glu₃‐Val‐Glu (13 000 and 170 μM, respectively).