Protamine phosphorylation

Phosphoproteins.—A chemical synthesis of partially and fully phosphorylated protamines has been described during the past year,98 and structural requirements for the enzymatic phosphorylation of phosvitin have been delineated.99 Phosphorylated forms of phosphofructokinase100 and fatty acid synthetase101 have been discovered recently both may be concerned with the regulation of their respective enzymes. 

This enzyme [EC 2.7.1.70], also referred to as protamine kinase, catalyzes the reaction of ATP with protamine to produce ADP and an O-phosphoseryl residue in protamine. The enzyme will also phosphorylate histones and it requires cAMP. 

The activity of protein kinase is readily assayed by measuring the rate of transfer of P from [y- P]ATP to a protein substrate, such as histone, casein, or protamine [77,80,82]. The phosphorylated substrate is collected on a filter, eluted, and counted. 

Cyclic AMP-independent protein kinases are found in numerous animal tissues. Some are specific for basic proteins such as the histones and protamines. Others are specific for acidic proteins such as phosvitin and casein, while others may show both types of activity. Enzymes that catalyze the phosphorylation of phosvitin and casein have been purified... 

Protamine is phosphorylated on serine residues adjacent to arginine residues (102) ... 

Dephosphorylation. In vivo, phosphoprotein phosphatases (phos-phoprotein phosphohydrolase EC 3.1.3.16) participate in the regulation of phosphate turnover and in rapid reversal of the phosphorylating reactions, yet there is very little information on this important group of enzymes. Alkaline (104) and acid (105) phosphatases of milk can hydrolyze the phosphate groups of phosphoserine residues in the caseins. E. coli alkaline phosphatase dephosphorylates many phosphoproteins (106,107). A phosphoprotein phosphatase of liver dephosphorylates phosphorylated histones and protamines but has little or no activity on casein or phosvitin (108). The 60-fold purified enzyme had an apparent Km for dephosphorylation of histone I (FI) of 2 X 10"5M and a pH optimum of 7-8. Histone phosphatase activity was detected in all eukaryotic cells examined, but it was not found in the extracts of several prokaryotes. 

Using a similar approach to the isolation of acetyl-CoA carboxylase phosphatase. Lent and Kim (70) isolated a cyclic AMP-independent protein kinase for acetyl-CoA carboxylase (Table IV). The properties of this cyclic AMP-independent protein kinase are different from those reported by Shiao et al. (105). The enzyme had a Km for acetyl-CoA carboxylase of 85 nAf and requires CoA for activity. The significance of this CoA requirement is further discussed in Section V,C. Phos-phorylase b and HMG-CoA reductase do not serve as substrates for this kinase however, protamine, casein, and histones are phosphorylated. The subunit molecular weight, as measured under denaturing conditions, was 160,000, which is similar to that reported by Shiao et al. (105). However, the CoA-requiring enzyme exists in multimers under nondenaturing conditions, with molecular weights between 700,000 and 2 x 10 . 

Recent studies have shown that cyclic AMP-dependent protein kinases are present in many cells, and that these may phosphorylate protamine, histone, casein, phosphorylase kinase, and presumably other proteins as well. Kuo and Greengard (21) have proposed that cyclic AMP mediates all of its actions by activating specific protein kinases in various tissues. The tissue-specific effects of cyclic AMP on a given tissue would then be accounted for in terms of the specific protein kinase and its substrates in that tissue. The apparent Michaelis constants for activation by cyclic AMP are from 2 X 10 Af to 3 X 10 M, and it has been suggested that the... 

At the terminal stage of spermatogenesis of salmon fish, nucleohistones are replaced by nucleoprotamine. Not much is known about the mechanisms by which one kind of basic polypeptide is replaced by another. What seems certain is that protamines are made in the cytoplasm and transferred to the nucleus before the displacement can take place. Prior to the exchange, both types of basic proteins, preformed histones and newly synthesized protamines, are phosphorylated. 

The role that phosphorylation plays in the exchange is not clear. Phosphorylation does not seem to modify the affinity of histones for DNA but slightly decreases the affinity of protamines for DNA [64]. 

An energy-dependent histone phosphorylation was discovered in calf and rat thymus nuclei [68, 69], A histone phosphokinase has been purified from liver, and some of its properties have been studied. The kinase phosphorylates only the serine residues of histones or protamines. It is inactive on other proteins and therefore different from the typical phosphopro-tein kinase. In vitro the enzyme phosphorylates all histone fractions, but FI appears to be a preferred substrate. 

Evidence that intracellular cyclic 3, S -AMP may account for the growth promoting action of many hormones is increasing. This action as well as the activation of new enzyme synthesis, and the increased synthesis of normally present enzymes takes place at the transcription level A possible mechanism of such an action of 3, S -AMP has been demonstrated. It is the activation of the phosphorylation of histones and protamines. A consequence of this phosphorylation may be the unmasking of DNA and the stimulation of RNA synthesis. 

Phosphorylation of histones is another mechanism for the neutralization of basic proteins. The phosphorylation reaction occurs after the completion of the histone synthesis. It is reversible. Phosphorylation of the lysine- and arginine-rich histones precedes an increase in RNA synthesis. This was found in transforming lymphocytes, in the regenerating liver and pancreas, etc. Histones and protamines are phosphorylated at the final stages of spermatogenesis (see review of Allfrey et al, 1964, 1972). Specific kinases which participated in the transport of phosphorus groups from ATP to histones are stimulated by cyclic AMP (Allfrey et al., 1972). 

The newly synthesized protamine is phosphorylated by adenosine triphosphate in cytoplasm catalyzed by protamine kinase (Langan and Smith, 1967 Ingles and Dixon, 1967 Marushige etal. 1969 Jergil and Dixon, 1970). The phosphorylation occurs at the hydroxyl groups of serine residues (Trevithick et al., 1967 Ingles and Dixon, 1967). Protein kinase has been found in various mammalian tissues... 

Ingles, C. J., Dixon, G. H. Phosphorylation of protamine during spermatogenesis in trout testis. Proc. nat. Acad. Sci. (Wash.) 58, 1011—1018 (1967). 

Langan, T. a.. Smith, L. K. Phosphorylation of histones and protamines by a specific protein kinase from liver. Fed. Proc. 26, 603 (1967). 

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