Cyclic Nucleotide‐Independent Protein Kinases from Rabbit Reticulocytes

Abstract

Purified variants of asl casein and p casein have been used to examine the substrate-specificity of two adenosine-3': 5'-monophosphate-independent protein kinase activities purified from rabbit reticulocytes. These enzymes, identified as casein kinase I and I1 were stimulated by monovalent cations. Casein kinase I had a pH optimum between 6.8 and 8.0, used ATP as the phosphate donor and preferentially phosphorylated serine. Casein kinase I1 had optimal activity at pH 8.5 to 10, utilized both ATP and GTP in the phosphotransferase reaction and modified predominantly threonine residues. The chymotryptic peptides of the phosphorylated casein variants were analyzed by thin-layer electrophoresis and ascending chromatography and the phosphorylated peptides were identified by autoradiography. In the p caseins, the phosphorylated peptides observed with casein kinase I were different from those obtained with casein kinase 11. With the asl caseins, the same phosphorylated peptide was observed with both protein kinases. The phosphorylation sites in the aSl and p caseins have been assigned on the basis of the amino acid analyses of the phosphorylated chymotryptic peptides and the phosphoserine and phosphothreonine determinations in conjunction with the known primary sequences. Casein kinase I phosphorylated Ser-22 in p-A2 casein and Ser-41 in aSl-A casein, suggesting that this protein kinase recognized the primary sequence Glu-X-Ser. Thr-41 in p-A2 casein and Thr-49 in aSl-A casein were the primary sites phosphorylated by casein kinase 11. The recognition determinants for casein kinase I1 appeared to be Thr-Glu-Asp. When endogenous phosphate was removed from the caseins, the rate of phosphorylation of / 3 casein was diminished by greater than six-fold indicating that negative charge near the phosphorylation site facilitated phosphorylation.Two CAMP-independent protein kinases which modify casein have been highly purified from rabbit reticulocytes by ion-exchange chromatography as previously described by Hathaway and Traugh [l]. These enzymes have been designated casein kinase I and casein kinase I1 in order of elution from DEAEcellulose. Casein kinase I is a single polypeptide chain of molecular weight 37 000 and preferentially uses ATP. Casein kinase I1 is a tetramer ( M , 130000) and utilizes both ATP and GTP. Enzymes similar to casein kinase I1 have also been highly purified from rat liver [2] and Novikoff ascites tumor cells [3].Little is known about the mechanism of action or the biological role of the CAMP-independent protein kinases from reticulocytes, although several translational initiation factors have been identified as endoAbbreviation. Cyclic AMP or CAMP, adenosine 3': 5'-monoEnzymes. Chymotrypsin (EC 3.4.21.1); acid phosphatase (EC phosphate. 3.1.3.2).genous substrates [4 -91. Initiation factors eIF-4B and eIF-5 have been shown to be phosphorylated by casein kinase I while initiation factors eIF-2, eIF-3, eIF-4B and eIF-5 are modified by casein kinase I1 [4]. In these studies the purified casein variants have...

Two genes in Saccharomyces cerevisiae encode a membrane-bound form of casein kinase-1.

“…CK1 is a monomer in solution with an unusually high isoelectric point and a strict requirement for ATP as cosubstrate. Because dephosphorylation of casein renders it a less efficient substrate for CK1 (Tuazon et al, 1979), phosphorylated amino acid residues may comprise part of the substrate recognition site for the enzyme (Flotow and Roach, 1991;Meggio et al, 1991). Casein kinase-2 is distinguished by its heterotetrameric structure (aa'f(3), its extreme heparin sensitivity (IC50 < 0.15 ,ug/ml), its ability to use either ATP or GTP as cosubstrate, and its welldefined selectivity for acidic substrates (Pinna, 1990; Marshak and Carroll, 1991).…”

Determinants on simian virus 40 large T antigen are important for recognition and phosphorylation by casein kinase I

“…Early studies examining phosphorylation of casein variants by casein kinase I, illustrated the effectiveness of glutamate residues at the -2 position preceding the phosphorylatable serine for recognition by casein kinase I [5]. However, the sites phosphorylated on SV40 large T antigen and glycogen synthase by casein kinase I lack an acidic residue at the -2 position.…”

“…Casein kinase I is a monomer and is present in the nucleus, cytoplasm and membrane of all eukaryotic cells examined. The enzyme from reticulocytes has a molecular mass ranging over 34-37 kDa, depending on the Early studies examining phosphorylation of casein variants by casein kinase I, illustrated the effectiveness of glutamate residues at the -2 position preceding the phosphorylatable serine for recognition by casein kinase I [5]. However, the sites phosphorylated on SV40 large T antigen and glycogen synthase by casein kinase I lack an acidic residue at the -2 position.…”

Phosphorylation of the Type‐II Regulatory Subunit of Cyclic‐AMP‐Dependent Protein Kinase by Glycogen Synthase Kinase 3 and Glycogen Synthase Kinase 5

“…The amino acid sequences surrounding the phosphorylation sites on these three proteins and j A z casein are remarkably similar, the sequence Ser(P)/Thr(P)-Glu-Asp-Glu being common to thrce of them (Table 4). The importance of acidic amino acids C-terminal to the phosphorylated residue in determining the substrate specificity of this enzyme is well documented [41,42].…”