Caseins phosphorylated amino acid

Figure 4.10 Amino acid sequence of bovine asJ-casein A, showing nine of the 10-13 phosphorylation sites (from Swaisgood, 1992).

Phosphorylation occurs in the Golgi membranes of the mammary cell, catalysed by two serine-specific casein kinases. Only certain serines are phosphorylated the principal recognition site is Ser/Thr.X.Y, where Y is a glutamyl and occasionally an aspartyl residue once a serine residue has been phosphorylated, SerP can serve as a recognition site. X may be any amino acid but a basic or a very bulky residue may reduce the extent of phosphorylation. However, not all serine residues in a suitable sequence are phosphorylated, suggesting that there may be a further topological requirement, e.g. a surface location in the protein conformation. 

The primary structure of asl-casein B-8P, the major component of this variant, is shown in Figure 3.1. The additional phosphorylated serine residue in the minor component, a9i-casein B-9P, occurs at position 41 (Manson et al. 1976,1977). The B-variant consists of 199 amino acid... 

Cobb and Novotny (7) obtained improved separations using C)8 microcolumns as a method for separating quantities on the order of 4 picomoles of tryptic peptides of phosphorylated and dephosphorylated /3-casein. Figure 4 shows two peaks with different retention times, corresponding to the phosphorylated and dephosphorylated forms of the same peptide. The rest of the peptide map is similar. Using this microcolumn, phosphorylation of a single amino acid on a protein can be detected. The method is reproducible with standard deviations smaller than 2%. Characterization of bovine /3-lg tryptic peptides by RP-HPLC on a Nucleosil Cl 8 column was also reported (123). 

Caseins were once defined as the phosphoproteins that precipitate from milk at pH 4.6 but are now defined by their amino acid sequences. In the elucidation of the primary structures of the caseins a French group has taken a prominent part (Ribadeau Dumas et al., 1975). The four polypeptide chains of bovine casein are the aSi-, as2-, P-, and K-caseins. Of these, the two as-caseins and K-casein are each phosphorylated to different extents and K-casein also exists in a number of glycosylated forms. 

At least five genetic variants of asi-casein have been identified, but in Western breeds of cattle the B variant is predominant. The protein comprises a single polypeptide chain of known amino acid sequence (Fig. 2) which is found in milk as a major and a minor fraction. The major fraction, previously designated aSi-casein, contains 8 mol P/ mol (aSi-CN-8P), whereas the minor component (previously aSo-cas-ein) has an additional phosphorylated serine residue at position 41... 

Four genetic variants have been identified, with variant A the most common in Western commercial breeds. The complete amino acid sequence of as2-CN A-l IP is shown in Fig. 3. Although the sites of phosphorylation are not all identified, it is thought that aS2-casein exists, at least predominantly, in four phosphorylated forms containing 10-13 mol P/mol. 

Mercier, J.-C. 1981. Phosphorylation of caseins, present evidence for an amino acid triplet code postranslationally recognised by specific kinases. Biochimie 63, 1-17. 

Regulation of glycogen synthase by multisite phosphorylation. The location of phosphorylation sites ( ) and the protein kinases that phosphorylate at these sites (boxes) are shown. Phosphorylations occur only at N- and C-terminal regions of the enzymes, as indicated by CB-N and CB-C, respectively. The single-letter abbreviations for amino acids are used (see Chapter 2). cAMP-PK = cyclic AMP-dependent protein kinase CAM-MPK = Ca +/calmodulin-dependent multiprotein kinase PhK = phosphorylase kinase GSK = glycogen synthase kinase CK = casein kinase NIO-PK = A novel protein kinase. [Reproduced with permission from P. Cohen, Protein phosphorylation and hormone action. Proc. R. Soc. Lonrf. (Biol.) 234, 115(1988).]... 

In a dilute protein solution, the nano length scale or the molecular structure of protein molecules determines the thermodynamic equilibrium between protein-protein and protein-water interactions. The consequent surface and hydrodynamic properties of proteins are resulted from the proportion of hydrophobic, hydrophilic, and charged amino acid residues. For example, caseins could adopt a random coil structure due to their flexible structure as a result of phosphorylated serine residues caseins indeed lack the ordered structures of a-helix, 3-sheet, and 3-turn found in globular proteins. This gives rise to better multifunctionality of caseins over globular proteins. 

On the basis of data presently available, one would conclude that phosphorylation of serine in proteins probably occurs when the serine residues are adjacent to or in close proximity (sequence-wise) to basic or acidic amino acid residues. In the tertiary structure it would be expected that these residues would be near the surface of the protein. Recently, it has been shown for a wide variety of O-phosphoryl proteins that protein substrates of intracellular protein kinases all have a common feature when the caseins are excluded in general, all phosphorylated sites are separated from either lysine or arginine (in two cases, histidine) by no more than two amino acid residues (103a). 

Casein kinases exist as two distinct sets of enzymes [88]. The casein kinase in mammary tissue that normally provides in vivo phosphorylation of nascent proteins is distinct from the multisubstrate and ubiquitous casein kinase (CK-2) that is responsible for in vitro casein phosphorylation. The substrate specificity of the two enzymes is also different. For the casein kinase from mammary gland, the recognition sequence corresponds to the tripeptide Ser/Thr-X-Glu/Ser-P or Ser/ Thr-X-Glu/Ser in nonphosphorylated proteins, where X is any amino acid [89]. For CK-2, the recognition sites have been identified as Ser-Glu-Ala-Glu-Glu-Glu and Ser-Ala-Ala-Glu-Glu-Glu [88]. 

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