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

Summary Membrane 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, -subunit) and band 3, which are the major phosphate-acceptor proteins in the endogenous phosphorylation of isolated ghosts in the presence of [-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.     

Phosphorylation of actin-binding proteins by casein kinases 1 and 2

Filamin and vinculin from chicken gizzards were significantly phosphorylated in vitro by casein kinases 1 and 2, but not by alpha-actinin. Antisera raised against these actin-binding proteins immunoprecipitated the phosphorylated proteins corresponding to filamin and vinculin, but no phosphoprotein corresponding to alpha-actinin was detected. These results suggest that filamin and vinculin are phosphorylated in vivo but alpha-actinin is not.     

Phosphorylation and dephosphorylation of soluble proteins in human eosinophils

The effect of phorbol 12-myristate 13-acetate (PMA), calcium ionophore (A23187), opsonized zymosan (OZ), and N-formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe) on protein phosphorylation was examined in purified eosinophils (eos) isolated from human peripheral blood. Eos were prelabeled with [32P]orthophosphate, stimulated with several activating agents for varying periods of time. The soluble proteins were then analyzed by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. In resting eos, there was phosphorylation of endogenous soluble proteins with molecular weights of 12, 16, 21, 40, and 66 kilodaltons (kDa). PMA, a potent activator of oxidative metabolism, induced phosphorylation of 19-, 40-, and 67-kDa proteins. A23187, a strong degranulating stimulus, caused phosphorylation of 40-, 53-, and 67-kDa proteins. OZ, a relatively weak stimulus for eos function, caused phosphorylation of 30-34-, 59-, 67-, and 93-kDa proteins. In addition, all the above stimuli caused a time-dependent dephosphorylation of 21-kDa protein. In contrast, f-Met-Leu-Phe caused neither phosphorylation of new proteins nor dephosphorylation of preexisting eos proteins. These findings demonstrate that selected stimuli affect phosphorylation of soluble eos protein. These results also suggest that phosphorylation of specific proteins in eos is an intermediary step in external stimulus-induced cell activation, which may involve many different cell functions.     

Phosphorylation of the insulin receptor by casein kinase I

Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.     

Identity and substrate specificity of human erythrocyte membrane-bound and cytosolic casein kinases

The relationship and substrate specificity of the human erythrocyte membrane kinase and casein kinase A were investigated. Based on Staphylococcus aureus V8 protease digestion patterns, the 2 kinases appeared to be structurally homologous. These enzymes also exhibited the same substrate specificity and phosphorylated the same synthetic peptides and domains of ankyrin. Both kinases did not utilize GTP effectively as a substrate and were not inhibited by low concentrations of heparin, suggesting that they were type I casein kinases. An analysis of synthetic peptide phosphorylation failed to reveal a specific pattern of recognition of the amino acid sequence surrounding the phosphorylation site.     

Purification and properties of 2,3-bisphosphoglycerate phosphatase-mutase from erythrocytes of day-old chicks.

After tryptic digestion of intact Staphylococcus aureus the residual portion of protein A that was still bound to the cell wall was cleaved off with lysostaphin. From the two digests Fc-binding fragments were isolated and the following characteristics observed. (a) There are four Fc-binding, highly homologous regions, each consisting of 58--62 amino acid residues. (b) These regions are consecutively arranged from the N-terminal part of the protein. (c) The residual C-terminal part, approximately 150-residues long, differs to a great extent with respect to primary and secondary structures from the four active regions. Furthermore, it is suggested that the protein is bound to the bacterial cell wall structure through this C-terminal part.     

Interrelationship between energy metabolism from various substrates and the 2,3-bisphosphoglycerate bypass in human erythrocytes

Metabolism of the substrates D-ribose, xylitol, D-Xylulose, D-fructose, D-glucose and mixtures of these compounds were studied in human erythrocytes. The metabolic rates obtained with the various substrates affected the intracellular levels of ATP and 2,3-bisphosphoglycerate. Small amounts of substrate utilization resulted in a decrease of the ATP and more pronounced of the 2,3-bisphosphoglycerate concentration while carbon utilization rates beyound 14 microgram atom C/ml packed cells/120 min yielded constant levels of ATP and 2,3-bisphosphoglycerate. From these results it can be concluded that a carbon utilization rate of 14 microgram atom C/ml cells/120 min is able to cover the ATP requirement of the red cells under steady state conditions. Based on the carbon utilization rates obtained with the various substrates and the rates of 2,3-bisphosphoglycerate decomposition an attempt is made to calculate the contribution of the 2,3-bisphosphoglycerate bypass to substrate metabolism. In case of xylitol as substrate the decrease in the 2,3-bisphosphoglycerate content provides the regeneration of NAD thus facilitating uptake and metabolism of xylitol.     

Phosphorylation of human DNMT1: implication of cyclin-dependent kinases

DNA methylation plays a central role in the epigenetic regulation of gene expression during development and progression of cancer diseases. The inheritance of specific DNA methylation patterns are acquired in the early embryo and are specifically maintained after cellular replication via the DNA methyltransferase 1 (DNMT1). Recent studies have suggested that the enzymatic activity of DNMT1 is possibly modulated by phosphorylation of serine/threonine residues located in the N-terminal domain of the enzyme. In the present work, we report that cyclin-dependent kinases (CDKs) 1, 2 and 5 can phosphorylate Ser154 of human DNMT1 in vitro. Further evidence of phosphorylation of endogenous DNMT1 at position 154 by CDKs is also found in 293 cells treated with roscovitine, a specific inhibitor of CDK1, 2 and 5. To determine the importance of Ser154 phosphorylation, a mutant of DNMT1 encoding a single-point mutation at position 154 (S154A) was generated. This mutation induced a severe loss of enzymatic activity when compared to wild type DNMT1. Moreover, after treatment with 5-Aza-2′-Deoxycytidine (5-aza-dC), a faster decline in DNMT1 protein level was observed for HEK-293 cells expressing DNMT1(S154A) as compared to cells expressing wild type DNMT1. Our data suggest that phosphorylation of DNMT1 at Ser154 by CDKs is important for enzymatic activity and protein stability of DNMT1. Considering that tumour-associated cell cycle defects are often mediated by alterations in CDK activity, our results suggest that dysregulation of cell cycle via CDKs could induce abnormal phosphorylation of DNMT1 and lead to DNA hypermethylation often observed in cancer cells.     

An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose

A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.     

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent andcAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase

Ca,phospholipid-dependent (PKC) andcAMP-dependent (PKA) protein kinases phosphorylate the -subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol³²P/mol of -subunit, respectively. PKA (in contrast to PKC) phosphorylates the -subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that³²P is incorporated into the N-terminal 41-kDa fragment of the -subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the -subunit molecule. These findings suggest that PKC phosphorylates the -subunit of the Na,K-ATPase within the region restricted by C₃ and T₁ cleavage sites.     

Thiamin transport by human erythrocytes and ghosts

Summary Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20°C in the presence of [³H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na⁺ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 m and at 37°C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes,K _m =0.12, 0.11 and 0.10 m andJ _max=0.01, 0.02 and 0.03 pmol·l^–1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts,K _m =0.16 and 0.51 m andJ _max=0.01 and 0.04 pmol·l^–1 intracellular water after 15 and 30 min incubation times, respectively. At 20°C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence ofd-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein-(probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN^– showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.     

Purification of protein methylase II from human erythrocytes

Protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC. 2.1.1.24) which methyl esterifies free carboxyl groups of protein substrate using S-adenosyl-L-methionine as the methyl donor, has been purified from human erythrocytes approximately 13000-fold with a yield of 12%. The purified enzyme was over 95% pure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A bulk of hemoglobin present in the erythrocyte lysate, which severely limited the use of affinity chromatography for purification, was effectively removed by ammonium sulfate precipitation and by the subsequent salt washing of the precipitates followed by molecular sieve chromatography on Sephadex G-75. This preparation can be further purified by affinity chromatography, in which S-adenosyl-L-homocysteine is covalently linked to Sepharose-4B, followed by Sephadex G-75 chromatography to yield an enzyme with an activity of 27000 pmol methyl group transferred/mg/min at 37 degrees C.     

Phosphorylation and nucleotidylation of proteins in Rhodocyclus gelatinosus

Abstract Cells of Rhodocyclus gelatinosus were radioactively labeled by addition of [³²P]orthophosphate, [¹⁴C]inosine or [¹⁴C]orotic acid during anaerobic growth on citrate in the light. Protein analysis by two-dimensional gel electrophoresis and autoradiography of the gels revealed the presence of several radioactively labeled protein species in this organism. The molecular mass and the isoelectric point of all these proteins were determined. Treatment of the ³²P-labeled protein fractions with acid and alkaline phosphatase clearly showed that at least 8 protein species were modified by phosphorylation. The experiments conducted with the ¹⁴C-labeled precursors of purines and pyrimidines indicated the presence of 4 protein species which were modified by a compound containing a purine and phosphate, and a single protein simultaneously being labeled with pyrimidine and phosphate.     

Phosphorylation of vinculin in human platelets spreading on a solid surface.

Vinculin is a cytoskeletal protein believed to be involved in linking microfilaments to the cell membrane. It is a substrate for the Ca(2+)- and phospholipid-dependent protein kinase C. We show here that when human platelets attach and spread on a solid surface, the alpha isoforms of vinculin become phosphorylated at serine and/or threonine residues. Phosphorylation is dependent on adhesion to a surface, since suspended, unattached platelets can produce filopodia but no phosphorylation of vinculin. Phosphorylation is also dependent on actin polymerization, as it does not occur when platelets had been pretreated with cytochalasin B. Most likely, protein kinase C is responsible for the phosphorylation of vinculin, since phosphorylation also occurs when platelets are treated with a phorbol ester, which activates protein kinase C, and is blocked by treatment with a staurosporine derivative which inhibits this enzyme. These results suggest that phosphorylation plays a role in anchoring vinculin at sites of microfilament-membrane interaction.     

Tyrosine Phosphorylation of Extracellular Signal-Regulated Protein Kinase 4 in Response to Growth Factors

Abstract: Extracellular signal-regulated protein kinases (ERKs) are members of the mitogen-activated protein kinase family that are rapidly phosphorylated and activated in response to various extracellular stimuli, including growth factors. Of these, the ERK1 and ERK2 forms are by far the most abundant and the most studied. Much less is known about other ERK forms, including one previously designated ERK4 on the basis of its cross-reactivity with ERK1 and ERK2. We report here that ERK4 in rat PC12 pheochromocytoma cells can be immunoprecipitated by anti-ERK antiserum R2 and have used this reagent to characterize this species further. We find that ERK4 rapidly becomes tyrosine-phosphorylated in response to nerve growth factor (NGF) and epidermal growth factor (EGF) and, to a lesser degree, in response to insulin and a permeant cyclic AMP analogue. As in the case of ERK1 and ERK2, tyrosine phosphorylation of ERK4 occurs by a ras-dependent pathway in response to NGF and EGF and shows prolonged kinetics for NGF but not EGF treatment. Recognition by multiple antisera directed against various domains of ERK1 supports classification of ERK4 within the ERK family; however, two-dimensional gel analysis clearly distinguishes ERK4 from isoforms of ERK1. These findings thus reveal an additional member of the ERK family that is responsive to growth factors and that could play a distinct role in intracellular signaling.     

Phosphorylation at Tyr-694 of Nogo-A by Src-family kinases

Nogo-A is a neurite outgrowth inhibitor protein associated with myelin in the central nervous system. Unexpectedly, targeted disruption of Nogo-A in mice results in little or no improvement of axonal regeneration, suggesting that Nogo-A has other functions and/or receives complex regulations to exert its inhibitory functions. Here, we have found that Nogo-A becomes phosphorylated at Tyr-694 in the N-terminal region. The phosphorylation is mediated co-operatively by Src-family tyrosine kinases, which play many important roles in the nervous system. Levels of tyrosine phosphorylation of Nogo-A seem to be irrelevant to developmental stages of oligodendrocytes, and might be regulated by specific extracellular stimuli. Identification of tyrosine phosphorylation of Nogo-A will introduce an additional level of complexity into Nogo-A functions.     

Phosphorylation and DNA binding of nuclear rat liver proteins soluble at low ionic strength.

Proteins were extracted from isolated rat liver nuclei with 0.15 M NaCl and 0.35 M NaCl at pH 8.0. The number of phosphoproteins in these extracts was determined by labeling with 32P and autoradiography after two-dimensional gel electrophoresis. Two proteins, B22p and B24p, contained small amounts of 32P and sedimented with the 30S nuclear informofer particle. With the exception of two phosphoproteins, CB and CN', all of the phosphoproteins found in the 0.35 M NaCl extract. Approximately 20% of the 0.15 M NaCl soluble proteins bound to rat liver DNA in 0.05 M KCl-0.05 M Tris-HCl (pH 8). Of these proteins, 1-2% bound to DNA in 0.15 M KCl and were eluted with 2 M KCl. This DNA bound fraction which contained both phosphorylated and nonphosphorylated proteins was similar in both the 0.15 and 0.35 M NaCl extracts. However, two major proteins (C13 and C14) and three minor proteins (C15, C25, Cg') were present only in the 0.15 M NaCl extract. The results of the present study show that there are marked similarities in the two-dimensional gel electrophoretic, phosphorylation, and DNA binding properties of rat liver nuclear proteins soluble in either 0.15 or 0.35 M NaCl.