Golgi apparatus mammary gland casein kinase: monitoring by a specific peptide substrate and definition of specificity determinants

Abstract

The casein kinase from the Golgi apparatus of lactating mammary gland (GEF‐CK) is distinct from ubiquitous ‘casein kinases’ termed protein kinases CK1 and CK2 and appears to define a family of secretory pathways protein kinases that phosphorylate seryl residues followed by an acidic residue at position +2. In this report we show that a new synthetic peptide substrate derived from β‐casein (β[28–40]) is suitable for the fast, efficient and selective monitoring of GEF‐CK, being unaffected by CK1 and CK2, and we define the consensus sequence of this protein kinase as being Ser‐Xaa‐Glu/SerP, distinct from that of CK2 (Ser/Thr‐X‐X‐Glu/Asp/SerP/TyrP). In particular, the failure to recognize Asp as crucial specificity determinant prevents the phosphorylation of the specific CK2 peptide substrate RRRADDSDDDD by GEF‐CK. Thus, peptide substrates are now available for the fast and specific monitoring of all the three classes of ‘casein kinases’, CK1, CK2 and GEF‐CK.