Örjan Zetterqvist

The minimum substrate of cyclic AMP-stimulated protein kinase, as studied by synthetic peptides representing the phosphorylatable site of pyruvate kinase (type L) of rat liver

Synthetic peptides, representing part of the phosphorylatable site of rat liver pyruvate kinase, were phosphorylated by (32P)ATP and the catalytic subunit of cyclic AMP-stimulated protein kinase. The shortest peptide which could be significantly phosphorylated was Arg-Arg-Ala-Ser-Val, with an apparent Km of 0.08 mM. The apparent Km for Arg-Arg-Ala-Ser-Val-Ala was 0.02 mM and that for Leu-Arg-Arg-Ala-Ser-Val-Ala was less than 0.01 mM. Peptides in which threonine was substituted for serine, or leucine for the one or the other arginine of the pentapeptide were not detectably phosphorylated. Substitution of phenylalanine for valine increased, and substitution of lysine or glycine for valine considerably decreased the rate of phosphorylation.

Regulation in vitro of rat liver pyruvate kinase by phosphorylation-dephosphorylation reactions, catalyzed by cyclic-AMP dependent protein kinases and a histone phosphatase

1. Cyclic-AMP dependent protein kinases, resolved by chromatography on DEAE-cellulose and hydroxylapatite, catalysed the phosphorylation of rat liver pyruvate kinase and calf thymus histones by [gamma32P]ATP. [32P]phosphopeptides, from acid hydrolysates of pyruvate kinase phosphorylated by the different protein kinase fractions, displayed identical electrophoretic patterns. Phosphorylation inhibited pyruvate kinase activity. 2. Full activity was restored when phosphorylated pyruvate kinase was dephosphorylated by a histone phosphatase from the soluble fraction of rat liver. These results are consistent with the hypothesis that pyruvate kinase is regulated by phosphorylation-dephosphorylation reactions.

Phosphorylation and dephosphorylation reactions of bovine brain (Na+K+)-stimulated ATP phosphohydrolase studied by a rapid-mixing technique

Abstract 1. 1. In order to allow a study of K + -dependent dephosphorylation of (Na + K + )-stimulated ATP phosphohydrolase (EC 3.6.1.3, (Na + K + )-stimulated ATPase), a rapid-mixing apparatus with two consecutive mixing chambers and four syringes was developed. The shortest incubation time was about 1.5 ms, excluding the quenching time which was about 1 ms. 2. 2. The pseudo first-order rate constant of the Na + -dependent phosphorylation was estimated to be about 11 000 min −1 at 21 °C in the presence of 3 mM MgCl 2 , 120 mM NaCl and 100 μM [γ- 32 P]ATP. This rate seemed to have reached a maximum at this concentration of [γ- 32 P]ATP. The same seemed to hold for the amount of 32 P-labelled enzyme. Since this concentration of [γ- 32 P]ATP only gives half maximal rate of the (Na + K + )-stimulated hydrolysis of ATP the present data suggest a complex role of ATP, which is consistent with reports by others. 3. 3. When studying the disappearance of protein-bound acyl-[ 32 P]phosphate upon the addition of 10 mM KCl, an apparent first-order rate constant of at least 14 000 min −1 was obtained at 21 °C. Within the time resolution of the rapid-mixing apparatus this rate constant seemed to be the same irrespective of which concentration of ATP was used in the range of 5–100 μM. 4. 4. Since the steady-state concentration of the phosphorylatable enzyme form was unknown, the steady-state velocity of the phosphorylation could not be estimated. The concentration of 32 P-labelled enzyme at steady-state was about 0.2 nmole·mg −1 of protein. If it is assumed that all of this phosphoenzyme is dephosphorylated in one step, characterized by a first-order rate constant of at least 14 000 min −1 , the calculated rate of dephosphorylation at steady-state is at least 2800 nmoles·mg −1 ·min −1 . However, the steady-state rate of (Na + K + )-stimulated hydrolysis of ATP at 21 °C was only about 280 nmoles·mg −1 ·min −1 in the presence of 3 mM MgCl 2 , 120 mM NaCl and 10 mM KCl at 100 μM ATP. The consistency of these results with the current hypothesis on two different forms of the phosphorylated intermediate is discussed.

Non-dependence on native structure of pig liver pyruvate kinase when used as a substrate for cyclic 3′,5′-AMP-stimulated protein kinase

Abstract Alkali-inactivated pig liver pyruvate kinase, type L, and a cyanogen bromide fragment from the same enzyme were shown to be phosphorylated by ( 32 P)ATP and cyclic 3′,5′-AMP-stimulated protein kinase. In both cases the rate of phosphorylation was higher than with the native enzyme. Pyruvate kinases types A and M were not phosphorylated under the same conditions. From the 32 P-labelled cyanogen bromide fragment ( 32 P)phosphorylserine was isolated. The electrophoretic pattern of ( 32 P)phosphopeptides obtained on partial acid hydrolysis of the fragment indicated that the phosphorylated site of the fragment was identical with that of the native pyruvate kinase.

The structural requirements of substrates of cyclic AMP-dependent protein kinase

The primary structure required by substrates of cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been detailed by the aid of synthetic peptides, representing the phosphorylatable site of pyruvate kinase (EC 2.7.1.40) of rat and pig livers [ 1,2]. The minimum substrate emerging from these studies may be represented by the general sequence: Arg-Arg-X-Ser-X [3]. The substitution of either arginine residue with other ammo acids considerably increases the app. Km for cyclic AMP-dependent protein kinase [2]. The distance between the phosphorylatable serine residue and the block of 2 arginine residues is critical, as elucidated in [4] using synthetic dodecapeptides of the general sequence: (Gly),-Arg-Arg-(Gly)y-Ala-Ser-Leu-Gly and the peptide: It has been deduced from this and other examples that the required structure of substrates of cyclic AMPdependent protein kinase is met also by the general structure Lys-Arg-X-X-&r-X [3]. However, in experiments with synthetic peptides of the general structure (Gly),-Lys-Arg-(Gly)y-Ala-Ser-I_euGly, the spacing of the Lys-Arg-block from the phosphorylatable serine residue by 2 neutral residues gave extremely high Km-values [4]. Additional structural elements are apparently required to form the optimal environment of the phosphorylatable serine residue in the P-subunit of phosphorylase kinase.

Amino acid sequence at the phosphorylated site of rat liver pyruvate kinase

One dominating peptic phosphopeptide, Asx-Thr-Lys-Gly-Pro-Glx-Ile-Glx-Thr-Gly-Val-Leu-Arg-Arg-Ala-(32P)SerP-Val-Ala-Glx-Leu, was obtained from rat liver pyruvate kinase (type L) phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase from the same tissue. The sequence around the phosphorylated serine residue is similar to that of a corresponding but smaller peptic phosphopeptide previously isolated from pig liver (type L) pyruvate kinase, Leu-Arg-Arg-Ala-(32P)SerP-Leu.

Phosphorylation of bovine brain Na+, K+-stimulated ATP phosphohydrolase by adenosine [32P]triphosphate studied by a rapid-mixing technique

Abstract 1. 1. A bovine brain Na + , K + -stimulated ATPase preparation was incubated with [γ- 32 P]ATP by use of a rapid-mixing technique, the reaction time being 8–40 msec. The phosphorylated material was analysed for 32 P-labelled acyl-phosphate, phosphoserine, phosphothreonine and phospholipids. 2. 2. With 5 μM [γ- 32 P]ATP, 3 mM MgCl 2 and 120 mM NaCl at pH 7.4 and 21° the acyl-[ 32 P]phosphate appeared at a rate of 0.55 μmole/mg protein per min. At the same conditions the enzyme preparation hydrolysed 0.06 μmole of ATP/mg protein per min. This was the rate of ATP hydrolysis irrespective of whether or not 20 mM KCl was present. After 40 msec the acyl-[ 32 P] phosphate comprised more than 99 % of the [ 32 P]phosphate of the high-molecular-weight material. 3. 3. It is concluded that the [ 32 P]phosphorylenzyme, having a similar pH stability to acyl-[ 32 P]phosphate, is formed rapidly enough to be an intermediate of the Na + , K + -dependent ATPase reaction.

Isolation of [32P]phosphohistidine from different rat-liver cell fractions after incubation with [32P]adenosine triphosphate

Summary 1. The particulate microsomal fraction and. soluble high-molecular weight material from the cell sap, nuclear and mitochondrial fractions of rat liver were incubated for 15 sec at 0° with highly 32 P-labelled adenosine triphosphate. After alkaline hydrolysis, [ 32 P]phosphohistidine could be isolated from all cell fractions by Dowex-1 chromatography. It was obtained in highest yield from the cell sap. The amount of [ 32 P]phosphohistidine obtained from the nuclear, mitochondrial and microsomal fractions was about 5, 50 and 100 times less, respectively. 2. Succinyl-CoA synthetase (EC 6.2.1.4) activity could not be detected in the cell sap. This enzyme, which has been reported by others to be phosphorylated on a histidine residue by adenosine triphosphate, was detected only in the mitochondrial and nuclear fractions. 3. In the hydrolysate from the cell sap, an unknown 32 P-labelled component was detected which was acid-labile but did not yield [ 32 P]phosphohistidine on further alkaline treatment. It has not yet been identified.

Detection and identification of substrates for protein kinases: Use of proteins and synthetic peptides

Publisher Summary This chapter focuses on the detection and identification of substrates for protein kinases active on serine and threonine residues, with emphasis on the substrates and kinases present in mammalian tissues. The crude tissue extract may consist in isolated cell supernatant or the extracts of particulate cell fractions. When the extracts are incubated with [γ-32P]ATP and Mg 2+ , considerable incorporation of [32P]phosphate into proteins occurs, owing to the presence of protein kinases and endogenous substrates. The method of the interruption of phosphorylation greatly influences the amount and type of [32P]phosphoproteins obtained. When further studies on native phosphoprotein are to be made, the reaction may be interrupted by the removal of free Mg 2+ with ethylenediaminetetraacetic acid (EDTA). Phosphoprotein phosphatases may be inhibited by sodium fluoride or orthophosphate. The 32P-labeled phosphoproteins are separated from most of the low molecular weight, 32P-labeled compounds by rapid gel filtration. The considerable amount of these compounds may be adsorbed to the protein of crude extracts after the gel filtration. To obtain reliable estimates of the true protein phosphorylation, the adsorbed material must be removed by denaturation procedures.

Protein-bound phosphorylserine in different tissues and organisms.

Abstract The amount of protein-bound phosphorylserine in different calf tissues, in human and turkey erythrocytes, as well as in several microorganisms, was estimated after acid hydrolysis of the isolated protein fraction. The phosphorylserine was isolated by chromatography on Dowex 50 and Dowex 1, using a small amount of highly 32 P-labelled phosphorylserine as a marker. 0.12–0.25 μmole of phosphorylserine (uncorrected for an approximate loss of 75% during acid hydrolysis) per g of wet material was isolated from five different calf tissues and bakers yeast. The corresponding amount from six bacterial strains, as well as human erythrocytes, was less than 0.02 μmole, while 0.09 μmole was obtained from turkey erythrocytes. The results support the view of Langan 7 that phosphoproteins with serine-bound phosphate occur preferentially in eucaryotic cells.