Regulation of the Phosphorylation of Chromatin-associated Proteins in Lemna and Hordeum

This paper represents attempts to observe alterations in the pattern of chromatin protein phosphorylation in Lemna and barley (Hordeum vulgare).     

The Phosphorylation of Ribosomal Protein in Lemna minor

Sterile cultures of Lemna minor have been labeled with ³²P₁, and the ribosomal proteins have been examined for radioactivity. In relatively short term labeling a radioactive protein was found which ran as a single component in both urea/acetic acid and sodium lauryl sulfate gel electrophoresis. Acid hydrolysis of the labeled protein permitted the isolation of serine phosphate. After labeling to equilibrium with ³²P₁, calculation indicated only 0.6 to 0.75 atom of this protein phosphorus per ribosome.     

Phosphorylation of Proteins in Normal and Regenerating Goldfish Optic Nerve

Within 6 h after radiolabeled phosphate was injected into the eye of goldfish, labeled acid-soluble and acid-precipitable material began to appear in the optic nerve and subsequently also in the lobe of the optic tectum, to which the optic axons project. From the rate of appearance of the acid-precipitable material, a maximal velocity of axonal transport of 13-21 mm/day could be calculated, consistent with fast axonal transport group II. Examination of individual proteins by two-dimensional gel electrophoresis revealed that approximately 20 proteins were phosphorylated in normal and regenerating nerves. These ranged in molecular weight from approximately 18,000 to 180,000 and in pI from 4.4 to 6.9. Among them were several fast transported proteins, including protein 4, which is the equivalent of the growth-associated protein GAP-43. In addition, there was phosphorylation of some recognizable constituents of slow axonal transport, including alpha-tubulin, a neurofilament constituent (NF), and another intermediate filament protein characteristic of goldfish optic axons (ON2). At least some axonal proteins, therefore, may become phosphorylated as a result of the axonal transport of a phosphate carrier. Some of the proteins labeled by intraocular injection of 32P showed changes in phosphorylation during regeneration of the optic axons. By 3-4 weeks after an optic tract lesion, five proteins, including protein 4, showed a significant increase in labeling in the intact segment of nerve between the eye and the lesion, whereas at least four others (including ON2) showed a significant decrease. When local incorporation of radiolabeled phosphate into the nerve was examined by incubating nerve segments in 32P-containing medium, there was little or no labeling of the proteins that showed changes in phosphorylation during regeneration. Segments of either normal or regenerating nerves showed strong labeling of several other proteins, particularly a group ranging in molecular weight from 46,000 to 58,000 and in pI from 4.9 to 6.4. These proteins were presumably primarily of nonneuronal origin. Nevertheless, if degeneration of the axons had been caused by removal of the eye 1 week earlier, most of the labeling of these proteins was abolished. This suggests that phosphorylation of these proteins depends on the integrity of the optic axons.     

Phosphorylation of Neurofilament Proteins in Isolated Goldfish Mauthner Axoplasm

Abstract: The six neurofilament proteins (NFPs) in the goldfish Mauthner axon (M-axon) have molecular sizes of 235, 145, 123, 105, 80, and 60 kDa. To determine if NFPs in the M-axon are phosphorylated, isolated Mauthner axoplasm (M-axoplasm) and a neurofilament-enriched extract (NFE) prepared from M-axoplasm were incubated with ³²P, which resulted in the radiolabeling of NFPs as determined by their detection on autoradiograms. Kinase inhibitors directed against cyclic AMP-dependent kinases (PKAs) or cofactor-independent kinases significantly reduced the in vitro phosphorylation of NFPs in NFE, whereas inhibitors directed against protein kinase C did not significantly reduce the in vitro phosphorylation of NFPs in NFE. Experiments using two kinase inhibitors directed against different kinases significantly reduced the in vitro phosphorylation of NFPs in NFE to a greater extent than the reduction produced using any single kinase inhibitor. These data suggest that NFPs in the M-axon are phosphorylated and that the in vitro (and perhaps the in vivo) phosphorylation of NFPs is mediated by PKA and/or cofactor-independent kinases that copurify with NFPs.     

Brain Coated Vesicle Destabilization and Phosphorylation of Coat Proteins

Abstract: Two basic polypeptides, bee venom melittin and poly-L-lysine, induced concentration-dependent destabilization of bovine brain coated vesicles. Ultrastructurally the changes observed were aggregation of clathrin coats and segregation of the vesicle membrane, concomitant with the appearance of elongated cisternae of various sizes. Changes in coated vesicle morphology induced by melittin and poly-L-lysine were concurrent with stimulation of phosphate incorporation in proteins of the coat lattice: M, 33,000 and 100,000. Melittin-stimulated phosphorylation was Ca²⁺ sensitive and inhibited by EGTA. The initiation of vesicle membrane segregation by melittin, followed by fusion and formation of elongated membrane cisternae, paralleled an increase of endogenous phospholipase A₂ activity. The data suggest that a correlation exists between the state of assembly of the coat proteins on coated vesicles and protein phosphorylation.     

Calcium- and Calmodulin-Promoted Phosphorylation of Membrane Proteins during Senescence in Apples

Sodium dodecylsulfate-polyacrylamide gel electrophoresis ofmicrosomal membrane proteins from post-climacteric apples atan early and an advanced stage of senescence showed only slightqualitative changes in the protein pattern. Though there wasa 30% reduction in the total microsomal protein content in applesat an advanced stage of senescence, a polypeptide with 18,000molecular weight increased in quantity during senescence. Invitro phosphorylation of several proteins was promoted by calciumin membranes from apples at an early stage of senescence. Phosphorylationof proteins with molecular weights of 95,000, 91,000, 53,000and 50,000 was promoted by calcium and calmodulin. Phosphorylationof these proteins increased with increasing calcium concentration.Proteins with molecular weights of 53,000 and 50,000 showedmarked promotion of phosphorylation over the calcium-promotedlevel when the amount of calmodulin in the assay mixture wasincreased. Calcium- and calmodulin-promoted phosphorylationof membrane proteins showed considerable decrease when the appleswere at an advanced stage of senescence. Moreover, increasingthe concentrations of calcium and calmodulin in the assay mixturedid not have any promoting effect on the phosphorylation ofthese proteins. Phosphoprotein phosphatase activity as measuredby the loss of label from phosphorylated proteins followingchase with cold ATP, did not differ to a great extent in membranepreparations from normal and senesced apples. Hydrolysis ofATP by senesced apple membrane preparation, however, was foundto be relatively higher. The significance of these observationsin relation to senescence is discussed. ¹ Scientific Paper No. 7084, College of Agriculture and HomeEconomics, Washington State University, Pullman, Project 0321. ² Supported in part by grants from the Washington State TreeFruit Research Commission, and National Science Foundation GrantPCM-8208408.     

Phosphorylation of Chromosomal Proteins in Resting and Wounded Potato Tuber Tissues

Wounding of quiescent white potato tuber tissue enhances chromatin-boundprotein phosphokinase activity, which exhibits two distinctphases during wound-healing. A moderate activation of the enzymesup to 20 hr after injury is followed by a dramatic increasein activity with a peak at 50 hr. This time-course resemblesthat of chromatinbound DNA-dependent RNA polymerase with a peakin activity at about 48 hr after wounding. The kinases phosphorylateendogenous proteins as well as added histones, phosvitin andcasein. The incorporated phosphate is stable under standardassay conditions, indicating the absence of protein phosphatases.Sensitivity of the incorporated phosphate toward trypsin andalkali, but not DNase, RNase, hydroxylamine or succinic acidpoints to seryl- and threonyl-bonds and proteins as acceptormolecules. Kinases from resting tissues are only weakly stimulatedeven by 100 mM MgCl₂, those from wounded tissues exhibit pronouncedMg^$$-optima at 5–10 mM with endogenous proteins, phosvitinand casein and 50 mM MgCl₂ with histones. Wounding also increasesthe sensitivity of the kinases toward p-hydroxymercuribenzoate. Chromatin preparations from both resting and wounded tissuescontain about 40 protein bands after polyacrylamide disc gelelectrophoresis. In vitro phosphorylation of these proteinsin chromatin from quiescent tissues is comparably low and uniform.Wounding induces changes in the protein and phosphorylationpattern with a general enhancement of phosphorylative capacityand preferential phosphorylation of low molecular weight proteins. (Received August 10, 1981; Accepted November 18, 1981)     

2,4-Dichlorophenoxyacetic Acid-enhanced Phosphorylation of Soybean Nuclear Proteins

In vitro nuclear protein phosphorylation is enhanced in nuclei isolated from 2,4-dichlorophenoxyacetic acid (2,4-d)-treated mature soybean (Glycine max) hypocotyl relative to nuclei from untreated tissue. Increased nuclear protein phosphorylation correlates with increased levels of nuclear protein kinase activity. These changes generally parallel previously reported 2,4-d-enhanced RNA polymerase activity of these nuclei and the in vivo levels of RNA synthesis. Phosphate incorporation represents bona fide protein phosphorylation, with 87% of the label being identified as phosphoserine and 7% as phosphothreonine. Label from [γ-³²P]adenosine 5′-triphosphate is incorporated primarily into various nonhistone fractions with the greatest accumulation in loosely associated fractions (either released during incubation with ATP or removed by 0.15 m Nacl). Although electrophoretic analysis on sodium dodecyl sulfate gels shows no differences in the protein profiles of the loosely associated or sodium dodecyl sulfate-soluble nonhistone proteins, there are changes in the pattern of phosphorylation of other proteins, after 2,4-d treatment. Acid-soluble basic nuclear proteins are phosphorylated to a much lower extent than are the other nuclear protein fractions. While histone F₁ is subject to slight phosphorylation when nuclei are labeled in vitro, phosphorylation of the other histones is undetectable. One acid-soluble protein shows a substantial increase in quantity and in phosphorylation after 2,4-d treatment. This protein is similar in electrophoretic mobility to pea histone F₁ but its identity is unknown. Urea-acetic acid gels of the acid-soluble nuclear proteins show that auxin treatment results in increased quantities and in increased phosphorylation of various low mobility nonhistone basic nuclear proteins.     

Enzymatic Phosphorylation of Soybean Proteins by Protein Kinase

Soybean proteins were subjected to phosphorylation with cyclic adenosine monophosphate- dependent protein kinase (A-kinase). As a result, acidic subunits of the 11S fraction were found to be phosphorylated by A-kinase. To estimate the effect of the phosphorylation, 11S acidic subunits were isolated and subjected to A-kinase phosphorylation. The optimal enzyme amount and Mg^{2 +} concentration for the phosphorylation of 11S acidic subunits were determined to be 1.5U/ml and 1.6 mm, respectively. The rate of phosphorylation was 2mol/mol acidic subunits (MW 38,000) under the above conditions. The protein structures of 11S acidic subunits, as determined from UV and CD spectra, were slightly affected by the enzymatic phosphorylation.     

Phosphorylation Characteristics of Brain Clathrin-Coated Vesicle Endogenous Proteins

Clathrin-coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle-associated substrates: a 50,000-Mr polypeptide (pp50) and clathrin-associated protein2 (CAP2; the faster-migrating clathrin light chain). Various exogenous substrates, e.g., casein, phosvitin, histone II, and histone III, also are phosphorylated. The pp50 protein kinase activity of clathrin-coated vesicles is not modulated by Ca2+, calmodulin, phosphatidylserine, or cyclic AMP. On the other hand, phosphorylation of the other endogenous substrates requires certain activators, including histone, polylysine, polyarginine, or polyethylenimine. Phosphate incorporation into pp50 was sensitive to divalent cations that inhibit sulfhydryl-dependent enzymes in the following order of potency: Zn2+ greater than Hg2+ greater than Cd2+, Cu2+, and Pb2+. Phosphate incorporation into CAP2 with polylysine present was insensitive to divalent cations. The alkylating agents dithiodinitrobenzene, phenacyl bromide, and N-ethylmaleimide inhibited phosphate incorporation into pp50 up to 90% without affecting incorporation into the other substrates. Vanadium pentoxide inhibited phosphorylation of CAP2 but had a minimal effect on pp50. CAP2 kinase activity was separated from the coated vesicle membrane and from dis-assembled clathrin triskelions, coeluting with the assembly polypeptide complex on a Sepharose 4B column. It retained phosphorylation properties similar to those of intact vesicles. These data imply that clathrin-coated vesicle kinases are elements of the coat proteins and may be involved in the assembly/disassembly of clathrin triskelions or interactions of coated vesicles with other cellular components.     

Phosphorylation of Superior Cervical Ganglion Proteins During Regeneration

The incorporation of radioactive phosphate into proteins of both normal and regenerating ganglia of the sympathetic nervous system of the rat is reported. The incorporation reactions were carried out in vitro by incubating homogenates of excised ganglia with [gamma-32P]ATP under various conditions. It was found that incorporation of phosphate into proteins of regenerating ganglia in the molecular mass range 10,000-100,000 daltons increased up to 40% over incorporation into proteins from control ganglia during the first 3 days following injury and returned to control levels after 14 days. Analysis of the proteins by two-dimensional electrophoresis revealed that only few, i.e., less than 20, became radioactively labelled in homogenates of superior cervical ganglia in the presence of Ca2+, and even fewer in the presence of cyclic AMP. Furthermore, all these proteins fell within a narrow pI range of 4-6. The growth-associated protein, variously designated GAP-43, B-50, F-1, and pp46, has an enhanced level of expression and phosphorylation in regenerating ganglia compared with controls at day 3. Injury also caused consistently higher levels of incorporation into two other proteins with molecular masses at positions 55,000 and 85,000 and pI values of 5.1 and 4.5, respectively; the former protein most probably is beta-tubulin. The fact that both proteins are found in the 15,000 g pellet after the tissue has been solubilized in 0.5% nonionic detergent indicates that they may indeed by components of filament assemblies. Thus, the results suggest that protein phosphorylation is a mechanism involved in cytoskeletal function in regenerating nerve.     

Receptor-Mediated Phosphorylation of Astroglial Intermediate Filament Proteins in Cultured Astroglia

Primary cultures of purified astroglia have been shown to exhibit a variety of membrane receptors that regulate intracellular cyclic AMP levels. The experiments described in this paper were completed to examine the effect of such receptor agonists on protein phosphorylation in intact astroglia. An analysis of 32P-labelled proteins derived from whole cell extracts and separated via two-dimensional gel electrophoresis indicated that increasing cyclic AMP levels in astroglia stimulated the phosphorylation of two distinct proteins that had apparent molecular weights/isoelectric points (pI) of 51K/6.0 and 57K/5.7. Similar experiments with cultured meningeal cells indicated that only the 57K/5.7 protein was phosphorylated in response to elevated levels of cyclic AMP. The 51K/6.0 protein was never observed in gels derived from meningeal cells. Immunoblot experiments indicated that the 51K/6.0 protein stained with antiserum to glial fibrillary acidic protein (GFAP) and the 57K/5.7 protein stained with antibodies to vimentin. Concentration-effect studies indicate that these proteins are maximally phosphorylated at concentrations of receptor agonists that only slightly elevate cyclic AMP levels. All receptor agonists that have been shown to increase cyclic AMP levels appear similarly efficacious with respect to increasing the phosphorylation of the two proteins. These experiments suggest that the membrane receptors present on astroglia function, in part, to regulate phosphorylation of the intermediate filament proteins GFAP and vimentin.     

Phosphorylation of Simian Virus 40 Proteins in a Cell-Free System

We have shown previously that all the structural proteins of simian virus 40 (SV40) are phosphoproteins. Virus phosphorylated in vivo could be further phosphorylated with exogenous cellular protein kinases in a cell-free system containing gamma-(32)P-ATP as phosphate donor. In intact infectious virus only polypeptides 1 and 2 (mol wt 49,000 and 40,800, respectively) were further phosphorylated in vitro. However, when infectious SV40 was partially disrupted, treated with nucleases, and then phosphorylated in vitro, all five structural polypeptides accepted additional phosphate groups. Similarly, all polypeptides of intact empty capsids, derived from infected cells, were further phosphorylated in vitro. Phosphorylation of empty capsids and infectious SV40 in vitro was enhanced from 4- to 11-fold after prior treatment of virus with alkali. The phosphate group was linked only to serine residues of the viral polypeptides phosphorylated both in vitro and in vivo.     

Phosphorylation of Bcl-2 and Bax Proteins during Hypoxia in Newborn Piglets

Studies indicate that phosphorylated Bcl-2 cannot form a heterodimer with Bax and thus may lose its antiapoptotic potential. The present study tests the hypothesis that graded hypoxia in cerebral tissue induces the phosphorylation of Bcl-2, thus altering the heterodimerization of Bcl-2 with Bax and subsequently leading to apoptosis. Anesthetized, ventilated newborn piglets were assigned to a normoxic and a graded hypoxic group. Cerebral cortical neuronal nuclei were isolated and immunoprecipitated; immune complexes were separated and reacted with Bcl-2 and Bax specific antibodies. The results show an increased level of serine/tyrosine phosphorylated Bcl-2 in nuclear membranes of hypoxic animals. The level of phosphorylated Bcl-2 protein increased linearly with decrease in tissue PCr. The level of phosphorylated Bax in the neuronal nuclear membranes was independent of cerebral tissue PCr. The data shows that during hypoxia, there is increased phosphorylation of Bcl-2, which may prevent its heterodimerization with Bax and lead to increased proapoptotic activity due to excess Bax in the hypoxic brain. Further increased phosphorylation of Bcl-2 may alter the Bcl-2/Bax-dependent antioxidant, lipid peroxidation and pore forming activity, as well as the regulation of intranuclear Ca²⁺ and caspase activation pathways. We speculate that increased phosphorylation of Bcl-2 in neuronal nuclear membranes is a potential mechanism of programmed cell death activation in the hypoxic brain.     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Characterization of Cyclic AMP-Dependent Phosphorylation of Neuronal Membrane Proteins

We examined the patterns of cyclic AMP-dependent protein phosphorylation in membranes prepared from rat cortical synaptosomes following gel electrophoresis and autoradiography. We determined the optimum pH (6.2), time (20 s), Mg2+ concentration (10 mM) and cyclic AMP concentration (5 microM) for the reaction. We also found that the detergents Triton X-100 and gramicidin S enhanced cyclic AMP-dependent protein phosphorylation. Inhibitors of the Na+, K+ ATPase (ouabain, NaF, vanadate) enhanced protein phosphorylation. This effect occurred in the presence but not in the absence of detergent. The addition of purified bovine brain cyclic AMP-dependent protein kinase catalytic subunit enhanced membrane protein phosphorylation. The addition of homogeneous neural (bovine brain) and non-neural (bovine skeletal muscle) cyclic AMP-dependent protein kinase type II regulatory subunit partially inhibited protein phosphorylation. Both neural and non-neural regulatory subunits behaved similarly. In addition to cyclic AMP-dependent phosphorylation, the alpha-subunit of pyruvate dehydrogenase (Mr = 41,000) is phosphorylated in a cyclic AMP-independent fashion. We also examined the phosphorylation pattern of membranes prepared from rat heart and found that the number of acceptor substrates was much less than that from the nervous system.     

Cell Cycle-Dependent Phosphorylation of Theileria annulata Schizont Surface Proteins

The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1), are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr), serine (p-Ser) and threonine-proline (p-Thr-Pro) epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state.