Single Phosphoprotein Research Articles

Human T-Cell Leukemia Virus Type 2 Rex Carboxy Terminus Is an Inhibitory/Stability Domain That Regulates Rex Functional Activity and Viral Replication

Human T-cell leukemia virus (HTLV) regulatory protein, Rex, functions to increase the expression of the viral structural and enzymatic gene products. The phosphorylation of two serine residues (S151 and S153) at the C terminus is important for the function of HTLV-2 Rex (Rex-2). The Rex-2 phosphomimetic double mutant (S151D, S153D) is locked in a functionally active conformation. Since rex and tax genes overlap, Rex S151D and S153D mutants were found to alter the Tax oncoprotein coding sequence and transactivation activities. Therefore, additional Rex-2 mutants including P152D, A157D, S151Term, and S158Term were generated and characterized ("Term" indicates termination codon). All Rex-2 mutants and wild-type (wt) Rex-2 localized predominantly to the nucleus/nucleolus, but in contrast to the detection of phosphorylated and unphosphorylated forms of wt Rex-2 (p26 and p24), mutant proteins were detected as a single phosphoprotein species. We found that Rex P152D, A157D, and S158Term mutants are more functionally active than wt Rex-2 and that the Rex-2 C terminus and its specific phosphorylation state are required for stability and optimal expression. In the context of the provirus, the more active Rex mutants (A157D or S158Term) promoted increased viral protein production, increased viral infectious spread, and enhanced HTLV-2-mediated cellular proliferation. Moreover, these Rex mutant viruses replicated and persisted in inoculated rabbits despite higher antiviral antibody responses. Thus, we identified in Rex-2 a novel C-terminal inhibitory domain that regulates functional activity and is positively regulated through phosphorylation. The ability of this domain to modulate viral replication likely plays a key role in the infectious spread of the virus and in virus-induced cellular proliferation.

Enrichment of Phosphopeptides Using Biphasic Immobilized Metal Affinity-Reversed Phase Microcolumns

Immobilized metal affinity chromatography (IMAC) based on Fe (3+) or Ga (3+) chelation is the most widely employed technique for the enrichment of phosphopeptides from biological samples prior to mass spectrometric analysis. An IMAC resin geared mainly toward phosphoprotein enrichment, Pro-Q Diamond, has been assessed for its utility in phosphopeptide isolation. Using both single phosphoprotein tryptic digests of beta-casein and ovalbumin and synthetic mixtures composed of tryptic digests of phosphorylated and nonphosphorylated protein standards, the selectivity and sensitivity of Pro-Q Diamond resin in an immobilized metal affinity-reversed phase microcolumn format were compared to an alternate titanium dioxide approach. The biphasic microcolumn method was found to be superior to metal oxide-based phosphopeptide capture in biological samples of increasing complexity. The lower limit of mass spectrometric detection for the immobilized metal affinity-reversed phase microcolumn approach was determined to be 10 pmol of beta-casein monophosphorylated peptide in 20 microL of a solution of human serum protein digest (from 200 microg total serum protein after digestion and desalting).

A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry.

Recent advances in phosphopeptide enrichment prior to mass spectrometric analysis show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissociation (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MS(n), for phosphopeptide ion dissociation in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simultaneously stored, the method generates a "composite" spectrum containing fragments derived from multiple precursors. In comparison to analysis by conventional MS/MS (CAD), Pseudo MS(n) shows improved phosphopeptide ion dissociation for 7 out of 10 synthetic phosphopeptides, as judged by an automated search algorithm (TurboSEQUEST). A similar overall improvement was observed upon application of Pseudo MS(n) to peptides generated by enzymatic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixture, several phosphopeptides mis-assigned by TurboSEQUEST under the conventional CAD approach were successfully identified after analysis by Pseudo MS(n).

Preparation and application of polyclonal and monoclonal sequence-specific anti-phosphoamino acid antibodies.

This unit discusses the issues that must be considered in the design, production, and characterization of polyclonal and monoclonal sequence-specific anti-phosphoamino acid antibodies. Protocols are provided for generating and purifying such antibodies, and methods are also provided for producing useful polyclonal antibodies in a non-purified form. Support protocols describe coupling of peptides or phosphotyrosine to a solid support for use in affinity chromatography. An example of the generation, purification, and characterization of two sequence-specific anti-phosphopeptide antibodies specific for different sequences of a single phosphoprotein is described. The cross-reactivity of such antibodies, which is a common problem with anti-peptide antibodies, is also discussed.

Cholesterol ester hydrolysis and hormone-sensitive lipase in lactating rat mammary tissue

Neutral cholesterol esterase activity is expressed in extracts of mammary epithelial cells. The identity of the enzyme catalyzing this hydrolysis was investigated. Anti-hormone-sensitive lipase immunoglobulin elicited the total inhibition of this activity and also immunoprecipitated a single phosphoprotein of M r 84 kDa from mammary cell extracts previously phosphorylated in vitro with [γ- 32P]ATP and cyclic AMP-dependent protein kinase. It is concluded that mammary cell cholesterol esterase activity results from the presence of hormone-sensitive lipase.

Identification and isolation of the phosphorylated intermediate of the calcium pump in rat intestinal basolateral membranes.

Transport of Ca2+ by the ATP-dependent Ca2+ pump has been demonstrated previously in rat intestinal basolateral-membrane vesicles. To identify the Ca2+-pump protein, duodenal basolateral membranes were phosphorylated with [gamma-32P]ATP in the presence of Ca2+ and La3+, under conditions conducive for maximal formation of the phosphorylated intermediate of the Ca2+ pump. Four major phosphoprotein bands were seen on autoradiograms of acidic SDS/polyacrylamide gels; the properties of a phosphoprotein (pp) at 130 kDa (pp130) were consistent with those expected for the plasma-membrane Ca2+ pump. This phosphoprotein was markedly enhanced by La3+, exhibited the characteristics of an acyl-phosphate bond, was preferentially phosphorylated from ATP and inhibited by micromolar concentrations of vanadate. Another phosphoprotein of 115 kDa possibly represented the endoplasmic reticulum Ca2+ pump or a fragment of pp130. Other phosphoproteins of 75 and 95 kDa were predominantly expressions of alkaline phosphatase. Formation of pp130 was highest in duodenal basolateral-membrane preparations when compared with those of jejunum and ileum or other subcellular fractions. A similar correlation between Ca2+-pump activity and pp130 formation was not found in membranes from villus-tip and crypt cells or in vitamin D-deficient animals. pp130 was isolated as a single phosphoprotein by calmodulin-affinity chromatography. We conclude that pp130 represents the phosphorylated intermediate of the rat intestinal basolateral-membrane Ca2+ pump, which can be separated from other phosphoproteins using its properties as a calmodulin-binding protein.

A specific insulin receptor and tyrosine kinase activity in the membranes of Neurospora crassa

Cells of the wall-less ("slime") strain of Neurospora crassa possess specific high affinity insulin binding sites on their cell surface. 125I-labeled bound insulin was not displaced from these cells by insulin-like growth factor II (IGF-II), and was only weakly displaced by IGF-I and proinsulin. Cross-linking of 125I-labeled insulin with N. crassa cells using disuccinimidyl suberate resulted in the labeling of a single band of ca. 67 kDa m.w. on a polyacrylamide gel. Two proteins of ca. 66 and 59 kDa m.w. were purified from detergent solubilized plasma membrane preparations by passage over an insulin-agarose affinity matrix. Antibodies against an autophosphorylation site on the human and Drosophila insulin receptors (anti P2) immunoprecipitated a single phosphoprotein of ca. 50 kDa m.w. from detergent solubilized plasma membranes, which possessed protein tyrosine kinase activity when histone H2 was used as substrate.

Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein.

Histidinoalanine, a naturally occurring cross-link derived from phosphoserine and histidine residues in mineral-binding phosphoproteins.

Native mineral-containing phosphoprotein particles were isolated from the Heterodont bivalve Macrocallista nimbosa. The native particles are discrete structures about 40 nm in diameter which migrate as a single band during electrophoresis in agarose gels. Removal of the mineral component with ethylenediaminetetraacetic acid dissociates the native protein into nonidentical subunits. The lower molecular weight subunits, representing 8% of the total protein, were obtained by differential centrifugation. The native protein is characterized by a high content of aspartic acid, phosphoserine, phosphothreonine, histidine, and the bifunctional cross-linking residue histidinoalanine. The low molecular weight subunits have the same amino acid composition except for a reduction in histidinoalanine and a corresponding increase in phosphoserine and histidine residues, demonstrating that the alanine portion of the cross-link is derived from phosphoserine residues. Ion-exchange chromatography and molecular sieve chromatography show that the low molecular weight subunits have a similar charge density but differ in molecular weight, and the relative mobilities of the subunits on agarose gels indicate that they are polymers of a single phosphoprotein molecule. The minimum molecular weight of the monomer is about 140 000 on the basis of the amino acid composition. The high molecular weight subunits are rich in histidinoalanine and too large to be resolved by either molecular sieve chromatography or gel electrophoresis. On the basis of the ultrastructural, electrophoretic, chromatographic, and compositional evidence, native phosphoprotein particles are composed of subunits ionically cross-linked via divalent cations. These subunits are variable molecular weight aggregates of a single phosphoprotein molecule covalently cross-linked via histidinoalanine residues. Evidence for a nonenzymatic cross-linking mechanism is discussed.

Multiple phosphorylation of pp30, a rat brain polyribosomal protein, sensitive to polyamines and corticotropin.

A rat brain polyribosomal protein with an apparent Mr of 30 000, designated pp30, was further characterized. The protein was identified by its phosphorylation by an endogenous protein kinase sensitive to both corticotropin and spermine. Two-dimensional separation of a polyribosomal fraction was applied, combining non-equilibrium pH-gradient-gel electrophoresis in the first and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the second dimension. In this system, pp30 was separated into at least five defined phosphoprotein spots. Pulse-labelling with [gamma-32P]ATP followed by a chase for various time periods with excess unlabelled ATP resulted in a shift of the distribution of radioactivity and protein staining along the spots towards the anode. This suggests that the various spots of pp30 may represent multiple phosphorylation states. Limited proteolysis of the five spots with three different proteinases resulted in the same one-dimensional peptide maps with a given proteinase, indicating that all five spots represent different forms of a single phosphoprotein. Inhibition of the overall phosphorylation of pp30 by corticotropin or spermine was accompanied by a shift in the recovery of labelled phosphate towards spots nearer the cathode. Immunoblotting with monoclonal antibodies directed against ribosomal protein S6 stained only one band, a protein that had an apparent Mr of 34 000 and was clearly distinct from pp30.

A study of platelet protein phosphorylation in duchenne muscular dystrophy: Further evidence against the generalised membrane defect theory

As a test of the generalised defect theory for Duchenne muscular dystrophy (DMD), basal and calcium-dependent platelet protein phosphorylation was examined in order to determine if the increased concentration of calcium in DMD skeletal muscle is reflected in DMD platelets. Protein phosphorylation was quantitated by gradient slab gel electrophoresis and autoradiography. The number of phosphoproteins in each phosphoprotein peak was determined by comparison with two-dimensional gel electrophoresis. Many phosphoprotein peaks were present in unstimulated platelet preparations both in whole platelet homogenates and in intact platelets. Two of these phosphoprotein peaks were calciumdependent, one was a single phosphoprotein, the other consisted of 4 phosphoproteins. No disease-related differences were observed in either basal or calcium-stimulated phosphoproteins. These results do not support previous reports of platelet abnormalities in DMD, and provide further evidence that the biochemical defect in Duchenne muscular dystrophy is neither generalised nor a membrane defect. The biochemical defect in DMD should be regarded as a skeletal muscle abnormality until proved otherwise.

31P nuclear magnetic resonance spectroscopic evidence for ternary complex formation of fetal dentin phosphoprotein with calcium and inorganic orthophosphate ions.

The single phosphoprotein of fetal calf dentin, having a molecular weight of approximately 94,000 and a phosphorus content of 8% (w/w), was examined by 31P NMR spectroscopy. The single resonance at 3.7 ppm at pH 10 and its chemical shift during acid titration established the phosphomonoester nature of the organic phosphorus moiety. During titration of the phosphoprotein with CaCl2 in the presence of inorganic orthophosphate ions, line broadening for the orthophosphate resonance was both phosphoprotein- and calcium-dependent, indicating ternary complex formation. The data indicate that the phosphoprotein of fetal calf dentin binds both calcium and inorganic orthophosphate ions and therefore has the requisite physical chemical properties necessary for it to facilitate the heterogeneous nucleation of a Ca-PO4 solid phase from solution during tissue mineralization.

The short term hormonal control of cytoplasmic protein phosphorylation in hepatocytes from fed rats

1. 1. Exposure of 32P-prelabelled isolated hepatocytes to vasopressin affected the phosphorylation of nine of the 26 phosphoproteins resolved by sodium dodecyl sulphate gel electrophoresis. 2. 2. Glucagon (2 nM) or cyclic AMP elicited significant changes in the phosphorylation of only four phosphoproteins. 3. 3. A very high concentration of glucagon (1000 nM) affected additional phosphoproteins. 4. 4. Insulin alone significantly increased the phosphorylation of a single protein. 5. 5. Vasopressin, A23187, glucagon and cyclic AMP all induced the dephosphorylation of a single phosphoprotein of mol. wt 20,000. 6. 6. The significance of these results with respect to the short-term control of hepatic metabolism is discussed.

Protein modification enzymes associated with the protein-synthesizing complex from rabbit reticulocytes. Protein kinase, phosphoprotein phosphatase, and acetyltransferase.

A number of protein modification activities are present in the protein-synthesizing complex isolated from rabbit reticulocytes. These enzymes are solubilized by sedimentation of the ribosomes through buffered sucrose containing 0.5 M KCl, and have been partially purified from the high salt wash fraction by chromatography on DEAE-cellulose and phosphocellulose. The ribosomal-associated enzymatic activities include cyclic AMP-regulated and cyclic nucloetide-independent protein kinase, phosphoprotein phosphatase, and acetyltransferase activities. These enzymatic activities have been shown to modify specific ribosomal and ribosomal-associated proteins. The cycli c AMP-regulated protein kinase phosphorylate the 40 S ribosomal subunit from rabbit reticulocytes. One of the cyclic nucleotide-independent protein kinase catalyzes the phosphorylation of two different factors involved in the initiation of hemoglobin synthesis. A single phosphoprotein phosphatase activity is shown to remove phosphate from 40 S ribosomal subunits. The major acetyltransferase activity associated with ribosomes acetylates a 60 S ribosomal protein.

Substrate specificity of glycogen synthase phosphatase from bovine heart: Action on phosphorylase [formula omitted] and histone

Glycogen synthase phosphatase has been purified from bovine heart. This preparation catalyzes conversion of synthase D into I and phosphorylase a into b and is able to dephosphorylate synthase D, phosphorylase a , active phosphorylase kinase, and phosphorylated histone and casein. The activity of phosphatase was assayed with synthase D, phosphorylase a , and histone as substrates after chromatography on Sephadex G-100, after sucrose gradient centrifugation, and after isoelectric focusing in a sucrose gradient. In all cases no separation of enzyme activity was observed with the above substrates. The phosphatase activity on all substrates was lost at the same rate by heat denaturation. These results indicate that this enzyme preparation contains a single phosphoprotein phosphatase which is responsible for the activity observed on the above substrates.

Composition of the Casein-Containing Particles in Milk

Summary 1.Roughly 75 to 90% of the casein in fresh untreated skimmilk appears to exist as a single phosphoprotein, which is probably a definite mixture or combination of α-, β-, and γ-caseins. The organic phosphorus:nitrogen ratio for this phosphoprotein is in close agreement with that for a mixture of these caseins in the proportions 16:4:1. This phosphoprotein is presumed to be contained in all the colloidal particles comprising the broad intermediate size range. An alternative assumption, that these individual caseins exist in separate particles having comparable or identical size ranges, is possible, however. 2.In the range of sizes referred to, the phosphoprotein may be considered to be combined with calcium and tricalcium phosphate as a complex. The proportion of inorganic phosphorus is variable as between milks and decreases with decreasing particle size. For milks of low calcium content, however, and for the smallest particles removed, a definite minimum proportion of inorganic phosphorus is indicated, such that the mol proportion of calcium to organic phosphorus to inorganic phosphorus is approximately 5:2:2. 3.The very largest casein-containing particles in skimmilk, which are not included in the size range above referred to, differ in composition. They appear to contain less organic phosphorus, but they also contain nonproteinaceous substances, and more refined analyses are required to characterize either the phosphoprotein or the inorganic components. 4.A residual fraction of the casein-containing particles, representing from 5 to 10% of the total casein, was not removed in the centrifuging times used.