V. Moret

Phosphorylation of casein fractions by rat liver ‘phosvitin kinase’

Previous work from our laboratory [l] had shown that the ‘phosvitin kinase’ activity of crude rat liver cytosol is resolved by Sepharose 6B gel-filtration into two enzyme fractions. Both fractions, further purified by P-cellulose chromatography, still phosphorylate endogenous substrates, such as mitochondrial membrane proteins [2] as well as exogenous proteins like phosvitin and casein, but display only neglegible activity, if any, towards histones and protamines. However the two enzyme fractions, which are CAMP-independent, exhibit different catalytic activities since the less retarded and predominant Sepharose fraction phosphorylates both threonine (thr) and serine (ser) residues of whole casein, ‘TSform’, while the other fraction is active only on serine residues, ‘S-form’ [ 1 ] . It should be noted that whole casein, used in the investigations on protein kinases, is a heterogeneous mixture of several different ‘caseins’. The aim of this work is to study the phosphorylation of the single casein fractions and determine in which fractions are located the serine and threonine residues involved in the kinase reaction. The results of such an investigation, combined with the knowledge of the caseins’ primary structure, might provide new information about the specificity of ‘phosvitin casein-kinases’ and the structural features that determine the phosphorylation of the proteins by these enzyme fractions.

Comparative study of mitochondrial and cytosol protein kinase activities

Both cytosol and mitochondria of rat liver display protein kinase activity, cyclic AMP-independent, which is resolved by Sepharose 6B filtration and P-cellulose chromatography into multiple forms phosphorylating, besides endogenous mitochondrial membrane-bound proteins, also exogenous phosphoproteins such as casein and phosvitin. However, the forms by far predominant in the cytosol phosphorylate both phosphorylserine and phosphorylthreonine residues of casein, while most of the activity associated to mitochondrial structures is due to the forms phosphorylating only phosphorylserine residues.

Preferential dephosphorylation of protein bound phosphorylthreonine and phosphorylserine residues by cytosol and mitochondrial “casein phosphatase”

The partial purification of a rat liver cytosol protein phosphatase is described, resulting in a preparation active on casein but not on phosvitin, cytosol phosphopeptides, ATP, ADP and p-nitrophenylphosphate, which, on the contrary, are still dephosphorylated by the protein phosphatase purified from rat liver mitochondria. Moreover the activity of the former enzyme on casein appears to involve only a limited amount of phosphoric sites which are also preferentially phosphorylated by soluble protein kinase. The isolation and evaluation of 32P-serine and 32P-threonine from protein-kinase-dependently labelled phosvitin and casein, before and after incubation with the two enzymes, led to the conclusion that mitochondrial protein phosphatase hydrolyzes more actively the phosphorylserine residues, while the cytosol “casein phosphatase” promotes a preferential breakdown of the 32P-threonine residues.

Functional correlation between the Ser/Thr-phosphorylation of band-3 and band-3-mediated transmembrane anion transport in human erythrocytes

In human erythrocytes, okadaic acid, a potent inhibitor of certain protein phosphatases, promotes a marked increase of Ser/Thr-phosphorylation of membrane proteins, including band-3 protein. Moreover, okadaic acid also increases the band-3-mediated oxalate transport across the membranes, thus suggesting that this process is regulated by Ser/Thr-phosphorylation of transporter band-3 protein.

Phosphorylation of membrane proteins by cytosolic casein kinases in human erythrocytes. Effect of monovalent ions, 2,3-bisphosphoglycerate and spermine.

SummaryMembrane proteins of human erythrocytes can be phosphorylated not only by membrane casein kinase (MS) but also by cytosolic casein kinases CS and CTS, resembling casein kinase I and II, respectively.Casein kinase CS, like membrane casein kinase MS, preferentially phosphorylates membrane proteins such as band 2 (spectrin, β-subunit) and band 3, which are the major phosphate-acceptor proteins in the endogenous phosphorylation of isolated ghosts in the presence of [γ-32P]ATP.By contrast, cytosolic casein kinase CTS phosphorylates, in addition to band 2, some membrane proteins, whose endogenous phosphorylation in isolated ghosts under the same conditions is negligible, if any.The CS- and CTS-catalyzed phosphorylations exhibit different response to increasing NaCl (or KCI) concentrations up to physiological levels (140 mM KCI, 20 mM NaCI); i.e. CS-and MS-catalyzed phosphorylations are strongly inhibited by 75–150 mM KCI (or NaCl), while CTS-catalyzed phosphorylation is practically unaffected.In the absence of added NaCl, CS- and MS-catalyzed phosphorylations are markedly inhibited by 1.5-3 mM 2,3-bisphosphoglycerate, whereas CTS-catalyzed phosphorylation appears to be practically unaffected.Finally, CS- and MS-catalyzed phosphorylations are slightly inhibited also by 1 mM spermine, while CTS-catalyzed phosphorylation is enhanced by this polycation concentration.

Endogenous proteinkinase-dependent phosphorylation of rat liver mitochondrial membranes

Abstract Isolated rat liver mitochondrial membranes are found to contain tightly bound protein substrate(s) which can be phosphorylated in the presence of ATP by protein kinase(s) previously extracted with 0.7 M NaCl from the membranes themselves and by phosvitin kinase purified from liver cytosol. Such proteinkinase-dependent phosphorylation, which seems to be cyclic AMP-independent, involves the seryl and threonyl residues of the protein substrate(s).

Multiple forms of phosvitin kinase from rat liver cytosol

Abstract Rat liver cytosol phosvitin kinase can exist in more than one active form, distinguishable by phosphorylated cellulose chromatography and Sephadex filtration. It is probable that they consist of different types of aggregates dissociable in solutions of high ionic strength.

Rat liver cytosol phosphoprotein: Purification and enzymatic phosphorylation and dephosphorylation

Abstract A phosphoprotein fraction containing both phosphorylserine and phosphoryl threonine has been isolated from rat liver cytosol and purified up to 100-fold by DEAE-cellulose column chromatography followed by gel filtration through Sephadex G-200 and G-100. The purified phosphoprotein, whose alkali labile phosphate content approximates 9 μg per mg protein, is resolved into at least five phosphorylated bands upon electrophoresis on cellulose-acetate strips, pH 7.2. The phosphorylation of cytosol phosphoprotein by [ 32 P]ATP is catalyzed by both cytosol and microsomal phosvitin kinase, free of any protamine kinase activity. A partial dephosphorylation of the 32 P-labelled phosphoprotein can be accomplished through the reversal of the protein kinase reaction, using both ADP and GDP as phosphate acceptors. An almost complete release of previously incorporated 32 P from the protein is catalyzed by a mitochondrial protein phosphatase. In contrast, no dephosphorylation could be observed with crude preparations of cytosol protein phosphatase.

On the possible role of phosphoproteins as iron carriers.

The chemical analysis of purified cytosol phosphoproteins disclosed further similarities with phosvitin, i.e. a very high content of phosphate residues and of protein-bound Fe (see table 1). The amount of iron bound to phosvitin depends on the preparation procedure [3]: when prepared according to Mecham and Olcott [4] phosvitin exhibits an iron content fairly different from that of cytosol phosphoproteins. However both phosphoproteins when exposed to an excess of FeC13 reach the same saturation level corresponding to a Fe/P molar ratio of about 0.5. The effect of several conditions on the stability of [s9 Fe]-phosvitin complex is shown in table 2. It can be seen that only unlabelled Fe 3+ promotes a massive release of s 9 Fe, as expected through a mechanism of isotopic dilution. Among the remaining diand trivalent cations tested, a slight release of s9 Fe was observed only in the presence of high Co 2÷ concentrations, the remaining cations being quite ineffective, even those, like Mn 2+, Mg 2+ and protamine, which bind to phosvitin through electrostatic forces resulting in insoluble complexes. Very high ionic strength (2 M NaC1) was

Metabolic depletion effect on serine/threonine- and tyrosine-phosphorylations of membrane proteins in human erythrocytes

The response of serine/threonine-phosphorylation of the major transmembrane protein (band 3) in human erythrocytes to the metabolic state of the cells is different from that exhibited by the tyrosine-phosphorylation of the same protein. Precisely, both serine- and tyrosine-phosphorylation are decreased during metabolic depletion of the erythrocytes. However, the depletion-induced tyrosine-phosphorylation decrease of band 3 is not reversed by the subsequent metabolic repletion of the depleted cells, being accompanied by an irreversible inactivation of both membrane-bound and cytosolic tyrosine-protein kinase(s). By contrast, the depletion-induced phosphoserine-dephosphorylation is reversed by the following repletion, being accompanied by a reversible translocation of casein kinase(s) between cytosolic and membrane compartments. A possible functional correlation between the serine-phosphorylation state of band 3 protein and the band 3-mediated anion transport across the membrane is discussed.