Induction of stress response and differential expression of 70 kDa stress proteins by sodium fluoride in hela and rat brain tumor 9l cells

We herein demonstrate that sodium fluoride (NaF) acts as a stress response inducer on HeLa and 9L rat brain tumor cells. NaF is only slightly cytotoxic, and inhibitory to Ser/Thr-phosphatases but not to Tyr-phosphatases in both cell lines. After treatment with 5 mM NaF for 2 h, the phosphorylation levels of vimentin and an alkali-resistant 65-kDa phosphoprotein were enhanced, a common phenomenon detected in cells under a variety of stress conditions. Under an identical treatment protocol, in which the cells were treated with 5 mM NaF for 2 h and then allowed to recover under normal growing conditions for up to 12 h, NaF differentially induced the cytoplasmic/nuclear heat-shock protein70s (including both the inducible and the constitutively expressed members of this protein family) in HeLa cells and the endoplasmic reticulum residing heat-shock protein70 (the glucose-regulated protein with an apparent molecular weight of 78 kDa) in 9L cells. Electrophoretic mobility shift assays (EMSA) using probes containing well-characterized regulatory elements revealed the activation of the heat-shock factor in HeLa but not in 9L cells; this is in good agreement with the stress protein induction pattern. Additional differential induction of binding activities toward EMSA probes individually containing NF-κB, AP-2, and CRE-like elements were detected in NaF-treated cells. The possible involvement of these binding sites as well as the corresponding factors in the stress response are discussed. J. Cell. Biochem. 69:221–231, 1998. © 1998 Wiley-Liss, Inc.     

Modulation of protein phosphorylation and stress protein expression by okadaic acid on heat shock cells

We have demonstrated that pretreatment but not post-treatment with okadaic acid (OA) can aggravate cytotoxicity as well as alter the kinetics of stress protein expression and protein phosphorylation in heat shocked cells. Compared to heat shock, cells recovering from 1 hr pretreatment of OA at 200 nM and cotreated with heat shock at 45°C for the last 15 min of incubation (OA→HS treatment) exhibited enhanced induction of heat shock proteins (HSPs) 70 and 110. In addition to enhanced expression, the attenuation of HSC70 and HSP90 after the induction peaks was also delayed in OA→HS-treated cells. The above treatment also resulted in the rapid induction of the 78 kDa glucose-regulated protein (GRP78), which expression remained constant in cells recovering from treatment with 200 nM OA for 1 hr, heat shocked at 45°C for 15 min, or in combined treatment in reversed order (HS→OA treatment). Enhanced phosphorylation of vimentin and proteins with molecular weights of 65, 40, and 33 kDa and decreased phosphorylation of a protein with a molecular weight of 29 kDa were also observed in cells recovering from OA→HS treatment. Again, protein phosphorylation in cells recovering from HS→OA treatment did not differ from those in cells treated only with heat shock. Since the alteration in the kinetics of stress protein expression and protein phosphorylation was tightly correlated, we concluded that there is a critical link between induction of the stress proteins and phosphorylation of specific proteins. Furthermore, the rapid induction of GRP78 under the experimental condition offered a novel avenue for studying the regulation of its expression. © 1996 Wiley-Liss, Inc.     

Tumor necrosis factor-α induces changes in the phosphorylation,cellular localization,and oligomerization of human hsp27, a stress protein that confers cellular resistance to this cytokine

The stress protein hsp27 is constitutively expressed in several human cells and shows a rapid phosphorylation following treatment with tumor necrosis factor-α (TNF-α). hsp27 usually displays native molecular mass ranging from 100 to 700 kDa. Here, we have analyzed the TNF-α-mediated changes in the phosphorylation, cellular localization, and structural organization of hsp27 in HeLa cells. We report that the TNF-α-mediated hsp27 phosphorylation is a long-lasting phenomenon that correlates with the cytostatic effect of this cytokine. Following TNF-α treatment, the rapid phosphorylation of hsp27 occurred concomitantly with complex changes in the intracellular distribution and structural organization of this protein. This resulted in the quantitative redistribution of hsp27 toward the soluble phase of the cytoplasm. In addition, during the first 2 h of TNF-α treatment, a transient increase in the native molecular mass of most hsp27 molecules (≤ 700 kDa) occurred. Then, by 4 h of TNF-α treatment, the native size of this stress protein drastically regressed (< 200 kDa). During this phenomenon, the phosphorylated isoforms of hsp27 remained concentrated in the small or medium-sized oligomers (< 300 kDa) of this protein. We also analyzed the properties of human hsp27 in transfected murine L929 cell lines that constitutively express this protein. In these cells, TNF-α induced modifications in the phosphorylation, intracellular distribution, and oligomerization of human hsp27 similar to those observed in HeLa cells. Moreover, the expression of hsp27 in L929 cells was found to correlate with a reduced cytotoxicity of this cytokine. Hence, the complex changes in the phosphorylation, intracellular locale and structural organization of human hsp27 may be related to the protective activity of this protein against the deleterious effects induced by TNF-α.     

Polyamines stimulate endogenous protein phosphorylation in thyroid cytosol

The polyamine, spermine (1-5 mM), when added to rat thyroid cytosol, increases the phosphorylation of a 107 kDa protein 4-fold as analyzed by sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis (SDS-PAGE) and autoradiography; spermidine was less effective and putrescine was without effect. Sodium chloride, when tested at equivalent ionic strengths (4-40 mM), did not reproduce the effects of spermine. In addition to stimulating the phosphorylation of a 107 kDa protein, spermine had an apparent biphasic effect on the phosphorylation of 88 and 65 kDa proteins; maximum stimulation of approximately 60-70% was observed at 0.5-2 mM. Both basal and spermine-stimulated protein phosphorylation patterns were identical whether [gamma-32P] ATP or [gamma-32P] GTP was used as phosphate donors. Heparin (1 microgram/ml) reduced spermine-stimulated phosphorylation of the 107 kDa protein by 64%. Phosphorylation of a 107 kDa protein was not restricted to rat thyroid as spermine was found to augment the phosphorylation of 107 kDa protein(s) in mouse and beef thyroid cytosol preparations.     

A cell surface phosphoprotein of 48 kDa specific for myoblast fusion

The data we present here permit us to affirm that a 48 kDa phosphoprotein is the target of extracellular Ca2+ during fusion. It is detected only in fusion-competent L6 myoblasts and not in the fusion-defective spontaneous stable variants we isolated. The phosphorylation of this protein species can be totally inhibited by culturing myoblasts in a medium containing low Ca2+ concentrations (0.250 mM). However, under such conditions myoblasts do not fuse, but withdraw from the cell cycle and accumulate the muscle isoform of creatine kinase (M-CK). The results we have obtained support the following conclusions: (1) in fusion-competent cells, overall Ca2+-dependent phosphorylation of cell surface proteins appears to be necessary, but is not sufficient by itself for myoblast fusion; (2) the phosphorylation of a 48 kDa protein species is required for cell fusion; and (3) the phosphorylation of this 48 kDa protein is independent of other main events of cellular differentiation.     

S100 A9 is expressed and secreted by the oviduct epithelium,interacts with gametes and affects parameters of human sperm capacitation in vitro

Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [³⁵S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [³⁵S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.     

Thiophosphorylation and phosphorylation of saponin-permeabilized cultured chromaffin cells

There is increasing evidence that phosphorylation of cellular proteins plays a role in the control of events surrounding secretion in neurons and chromaffin cells. In previous studies, we have used thiophosphorylation of cell proteins as a means of fixing cellular phosphorylation reactions in the phosphorylated state. Thiophosphorylation of permeabilized chromaffin cells with adenosine-5′-O-(3-thiotriphosphate) results in irreversible inhibition of secretion. Thiophosphate is incorporated primarily by two cellular proteins of 58 and 47 kDa. Calcium enhanced thiophosphorylation of the 47 kDa protein but not the 54 kDa protein. This pattern of thiophosphorylation differed markedly from that for phosphorylation under similar treatment conditions. The phosphoprotein composition of the cells depended upon the medium calcium and ATP concentration. In the absence of exogenous ATP, fewer phosphoproteins were seen in calcium stimulated cells than in unstimulated cells. Proteins labelled with ³²P or ³⁵S migrated to the same position on polyacrylamide gels containing sodium dodecyl sulfate. In the presence of exogenous ATP, ³²P incorporation was similar for both control and calcium-stimulated cells and was found primarily in a 64 kDa protein. Incorporation of [³²P]phosphate by calcium-stimulated cells was reduced to the same extent by pretreatment of the cells with either adenosine-5′-O-(3-thiotriphosphate) or ATP.The different electrophoretic banding patterns for thiophosphorylation and phosphorylation are likely due to the irreversibility of the thiophosphorylation reaction and reversibility of the phosphorylation reaction. The inability to turn over thiophosphate groups, in association with changes in secretion, may permit identification of those phosphoproteins that are putatively involved in secretion.     

Protein phosphorylation in pea root plastids.

Protein phosphorylation has been investigated in non-photosynthetic plastids of pea roots. Intact and lysed preparations of plastids were incubated with [gamma-(32)P]ATP and three stromal proteins of sizes 41, 58 and 62 kDa were phosphorylated on a serine residue. No other proteins were significantly labelled under the conditions used. The 62 kDa protein is probably phosphoglucomutase and represents a phosphoenzyme catalytic intermediate. The protein kinase(s) and phosphatase(s) acting on the other proteins were not sensitive to exogenous calcium but were sensitive to magnesium. The protein phosphatase which acts on the 41 kDa protein is possibly of type 2C, whereas that acting on the 58 kDa phosphoprotein did not fall into any class defined by mammalian systems. Metabolism of exogenous glucose 6-phosphate by the oxidative pentose phosphate pathway in intact plastids abolished the phosphorylation of the 58 kDa protein. Dihydroxyacetone phosphate, phosphoenolpyruvate and 3-phosphoglycerate also inhibited phosphorylation of the 58 kDa protein and had a time-dependent effect on the phosphorylation of the 41 kDa protein. The significance of these results in relation to a possible role for protein phosphorylation in these plastids is considered.     

Comparison of Ca2+ -dependent phosphorylation in viable dispersed brain cells with calmodulin-dependent protein kinase activity in cell-free preparations of rat brain.

Using two depolarizing agents, veratrine and high concentrations of extracellular KCl, we studied depolarization-stimulated phosphorylations in 32P-labelled dispersed brain tissue in order to identify phosphoprotein substrates for Ca2+ - and calmodulin-dependent protein kinase activity at the cellular level, for comparison with findings in cell-free preparations. In intact brain cells, the only prominent depolarization-stimulated phosphorylation was a 77 kDa protein separated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This phosphorylation was dependent on external Ca2+, since chelation of Ca2+ in media with 6 mM-EGTA or the presence of verapamil (a Ca2+ -channel blocker) in the incubation media inhibited depolarization-stimulated phosphorylation of the 77 kDa protein. Phosphorylation of the 77 kDa protein also appeared to be dependent on calmodulin, because depolarization-stimulated phosphorylation was significantly decreased (P less than 0.05) when 100 microM-trifluoperazine was present in the incubation media. Polymyxin B, an inhibitor of Ca2+- and phospholipid-dependent phosphorylation, and 12-O-tetradecanoylphorbol 13-acetate, the phorbol ester enhancing Ca2+- and phospholipid-dependent phosphorylation, had no effect on the phosphorylation of the 77 kDa protein. The 77 kDa phosphoprotein was identified as a protein previously named synapsin I [Ueda, Maeno & Greengard (1973) J. Biol. Chem 248, 8295-8305] on the basis of similar migration of native and proteolytic fragments of the 77 kDa protein with those of authentic synapsin I on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Whereas several studies with cell-free preparations showed that 57 kDa and 54 kDa endogenous phosphoproteins were the most prominent species phosphorylated in a Ca2+ and calmodulin-dependent manner, these results indicate that synapsin is the most prominent Ca2+-and calmodulin-dependent phosphorylation in intact cells. The phosphorylations of 54 kDa and 57 kDa proteins may not be as important in vivo, but instead occur as a result of the disruption of cellular integrity inherent in preparation of cell-free subfractions of brain tissue.     

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.     

Inhibition of phosphotyrosine phosphatases reveals candidate substrates of the PDGF receptor kinase

In normal fibroblasts stimulated by platelet derived growth factor (PDGF), PDGF receptors are transiently phosphorylated on tyrosine and represent the major phosphotyrosine containing protein. The phosphate of the phosphotyrosine groups turns over rapidly, and extensive evidence indicates a dynamic balance between phosphorylation and dephosphorylation reactions. Thus, the effect of an inhibitor of phosphatases, orthovanadate, on the pattern of the tyrosine phosphorylations induced by PDGF in Swiss 3T3 fibroblasts was investigated. Western blot analysis with antibodies against phosphotyrosine indicated that whereas in unstimulated cells no phosphotyrosine containing proteins were detected, treatment of cells with orthovanadate alone elicited the slow phosphorylation of several proteins including a 170 kDa component that was recognized to be the phosphorylated PDGF receptor. Addition of PDGF to cells shortly pretreated with vanadate highly increased the intensity of the 170 kDa band corresponding to the phosphorylated receptor and caused its stabilization during time. In addition, the phosphorylation on tyrosine of other proteins (molecular mass 116, 80, 73, 60, 50 and 39 kDa) was also induced. Both the receptor and the other tyrosine phosphorylated proteins appeared to be associated with the detergent insoluble matrix.     

Phosphorylation of ribosomal proteins S3, L1 and L24 during spherulation in Physarum polycephalum

The phosphate content of ribosomal proteins S3, L1 and L24 has been determined in the course of spherulation of Physarum polycephalum. The major phosphoprotein, S3, was completely dephosphorylated after 4 h of differentiation. The phosphate content of L1 and L24 was not altered during the differentiation. The cellular level of ATP remained constant for at least 5 h. A 3-fold reduction of cyclic AMP concentration occurred in the first hour, followed by a slow increase to a final value of twice the level observed in growing cells. The results showed that the phosphorylation of ribosomal proteins is regulated by at least two different mechanisms and that the dephosphorylation of S3 is not induced by a lack of cellular ATP. Although cyclic AMP might trigger the dephosphorylation of S3, the phosphate content of this protein remained at a very low value even when the cellular concentration of cyclic AMP rose significantly. Since the polysome level remains constant during the first 24 h of spherulation, the phosphorylation of S3 is not necessary for active protein synthesis and the phosphorylation of L1 and L24 is not involved in ribosome inactivation, which occurs after 24 h.     

Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.     

Dynamics of serine/threonine protein kinase activity during the growth of the wild-typeStreptomyces avermitilis strain and its chloramphenicol-resistant mutant

The dynamics of serine/threonine protein kinase activity during the growth of the wild-typeStreptomyces avermitilis strain 964 and its chloramphenicol-resistant (Cml^r) pleiotropic mutant with an enhanced production of avermectins was studied by measuring the transfer of radiolabeled phosphate from [y-³²P]ATP to the serine and threonine residues of proteins in cell-free extracts. In both of the strains studied, radiolabeled phosphate was found to incorporate into polypeptides with molecular masses of 32, 35, 41, 68, 75, 79, 83, and 137 kDa; however, the degree and the dynamics of phosphorylation of particular peptides were different in these strains. The differences revealed could not be accounted for by the interference of ATPases or phosphoprotein phosphatases. The data obtained may be interpreted as evidence that Cml^r mutation activates the protein kinase signalling system ofS.avermitilis cells in the early stationary growth phase and thus enhances the production of avermectins and leads to some other physiological changes in the mutant strain.     

The CD95 (Fas/APO-1) receptor is phosphorylatedin vitro andin vivo and constitutively associates with several cellular proteins

Different CD95 (Fas/APO-1) isoforms and phosphory lated CD95 species were identified in human T and B cell lines. We had shown previously that the CD95 intracellular domain (IC), expressed as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts, was phosphorylatedin vivo. GST-CD95IC was phosphorylatedin vitro by a kinase present in extracts from the human lymphocytic cell lines Jurkat and MP-1 and from murine L929 cells. Phosphoamino acid analysis indicated that phosphorylation occurred at multiple threonine residues and also at tyrosine (Tyr232 and Tyr291) and serine. Amino acids 191 to 275 of CD95 were sufficient for phosphorylation at threonine, tyrosine and serine and also mediated interaction with a 35 kDa cellular protein. Immuno-precipitation of CD95 and chemical cross-linking revealed CD95-associated proteins of approximately 35, 45 and 75 kDa. GST-CD95IC affinity chromatography detected binding of the 35 and 75 kDa protein species. The 75 kDa species may correspond to the CD95-associated proteins RIP or FAF1 and the 35 kDa protein may represent a TRADD analogue. These data indicate that several cellular proteins interact with CD95, possibly in a multi-protein complex, and that a kinase activity is associated with CD95 not onlyin vitro but alsoin vivo. Therefore, receptor phosphorylation may play a role in CD95 signal transduction. This work was in part supported by a grant from the Health Research Council of New Zealand (to JW).     

Light-modulated ADP-ribosylation, protein phosphorylation and protein binding in isolated fly photoreceptor membranes

Rhodopsin (P, lambda max 480 nm) of blowfly photoreceptors R1-6 is converted by light into a thermally stable metarhodopsin (M, lambda max 565 nm). In isolated blowfly rhabdoms photoconversion of P to M affects bacterial toxin-catalyzed ADP-ribosylation of a 41-kDa protein, activates phosphorylation of opsin and induces the binding of a 48-kDa phosphoprotein to the rhabdomeric membrane. ADP-ribosylation of the 41-kDa protein is catalyzed by cholera toxin and is inhibited by P----M conversion. The 41-kDa protein might represent the alpha-subunit of the G-protein, proposed to be part of the phototransduction mechanism [Blumenfeld, A. et al. (1985) Proc. Natl Acad. Sci. USA 82, 7116-7120]. P----M conversion leads to phosphorylation of opsin at multiple binding sites: up to 4 mol phosphate are bound/mol M formed. Dephosphorylation of the phosphate binding sites is induced by photoconversion of M to P. High levels of calcium (2 mM) inhibit phosphorylation of M and increase dephosphorylation of P. Protein patterns obtained by sodium dodecyl sulfate gel electrophoresis of irradiated retina membranes show an increased incorporation of label from [gamma-32P]ATP also into protein bands of 48 kDa, 68 kDa and 200 kDa. Binding studies reveal that in the case of the 48-kDa protein this effect is primarily due to a light-induced binding of the protein to the photoreceptor membrane. The binding of the 48-kDa phosphoprotein is reversible: after M----P conversion the protein becomes extractable by isotonic buffers. These data suggest that in rhabdomeric photoreceptors of invertebrates light-activation of rhodopsin is coupled to an enzyme cascade in a similar way as in the ciliary photoreceptors of vertebrates, although there may be differences, e.g. in the type of G-protein which mediates between the activated state of metarhodopsin and a signal-amplifying enzyme reaction.     

DNA-activated protein kinase in Raji Burkitt's lymphoma cells. Phosphorylation of c-Myc oncoprotein.

Autophosphorylation of a DNA-activated protein kinase (DNA-PK) in Raji Burkitt's lymphoma cells generated a band that corresponded to a phosphoprotein of about 300 kDa on SDS/PAGE. This band corresponds to a 300-350-kDa DNA-PK found previously in HeLa cells. In addition to the 300-kDa phosphoprotein, the band of a highly phosphorylated 58-kDa protein was detected by SDS/PAGE of partially purified DNA-PK preparations after the phosphorylation reaction in the presence of double-stranded DNA. This phosphoprotein was specifically immunoprecipitated by phosphoprotein nor detectable activities of other kinases, phosphorylated recombinant c-Myc proteins in the presence of DNA. The c-Myc phosphorylation by DNA-PK was markedly stimulated by relaxed, double-stranded DNA, but neither by single-stranded DNA nor by RNA. Phosphopeptide mapping and phosphoamino acid analysis indicated that DNA-PK phosphorylates c-Myc in vitro at several serine residues.     

Radiation inactivation analysis of thylakoid protein kinase systems in light and in darkness

Chloroplast thylakoid contains several membrane-bound protein kinases that phosphorylate thylakoid polypeptides for the regulation of photosynthesis. Thylakoid protein phosphorylation is activated when the plastoquinone pool is reduced either by light-dependent electron flow through photosystem 2 (PS2) or by adding exogenous reductants such as durohydroquinone in the dark. The major phosphorylated proteins on thylakoid are components of light-harvesting complex 2 (LHC2) and a PS2 associated 9 kDa phosphoprotein. Radiation inactivation technique was employed to determine the functional masses of various kinases for protein phosphorylation in thylakoids. Under the photosynthetically active radiation (PAR), the apparent functional masses of thylakoid protein kinase systems (TPKXs) for catalyzing phosphorylation of LHC2 27 and 25 kDa polypeptides were 540±50 and 454±35 kDa as well as it was 448±23 kDa for PS2 9 kDa protein phosphorylation. Furthermore, the functional sizes of dark-regulated TPKXs for 25 and 9 kDa proteins were 318±25 and 160±8 kDa. The 9 kDa protein phosphorylation was independent of LHC2 polypeptides phosphorylation with regard to its TPKX functional mass. Target size analysis of protein phosphorylation mentioned above indicates that thylakoid contains a group of distinct protein kinase systems. A working model is accordingly proposed to interpret the interaction between these protein kinase systems.     

Modulation of the rhythmic patterns of expression of phosphoprotein phosphatases in human leukaemia cells

Temporal variations in the expression of phosphoprotein phosphatase 1 (PP1), phosphoprotein phosphatase 2A (PP2A) and protein tyrosine phosphatase 1B (PTP1B) were monitored in the human acute, promyelocytic leukaemia cell line, HL60. Granulocytic differentiation was induced using all-trans retinoic acid (ATRA) and monocytic differentiation by phorbol-12-myristate-13-acetate (PMA). Expression of the enzyme proteins in cell extracts was determined by SDS-PAGE and Western immunoblotting using specific antibodies. For PP1, a single immunospecific band of molecular mass 38 kDa was detected corresponding to the catalytic subunit; induction of differentiation with either ATRA or PMA showed differences in the patterns of expression and, in the case of the latter, the mean value. Two immunospecific bands, of mass 34 and 37 kDa, possibly corresponding to dephosphorylated and phosphorylated forms, respectively, were detected for PP2A, as well as a minor band of mass 46 kDa; dynamic variations in the expression of all 3 forms were observed and there were differences between the control and treated cells. The catalytic domain of PTP1B was detected as a 46 kDa band. A 42 kDa form of the protein was also seen, which may represent a change in phosphorylation state, or be the result of proteolytic cleavage; usually the 46 kDa band was the major form, but on occasion there was a change to predominance of the 42 kDa band.     

Haemonchus contortus: a simple procedure for purifying surface proteins from third- and fourth-stage larvae

Surface proteins were solubilized from exsheathed third (XL3)- and fourth (L4)-stage larvae of Haemonchus contortus by a one-step extraction procedure involving brief heat treatment of the worms in the presence of buffer and 100 mM sodium chloride. Surface proteins also could be preferentially extracted from XL3s, but not from L4s, by heating the worms briefly in 1% sodium dodecyl sulfate. The major proteins extracted by these procedures were similar in molecular weight to those detected by surface-labeling live worms with 125Iodine. Both extraction procedures solubilized a single, major protein with an apparent molecular weight of 68-97 kDa from XL3s. In contrast, extraction of L4s with 100 mM sodium chloride yielded four major proteins with relative molecular weights of 27, 29, 78, and 200 kDa. Antibodies raised in rabbits to surface proteins prepared by the sodium chloride procedure reacted with the surfaces of live worms in indirect immunofluorescence assays. The anti-XL3 surface protein serum was stage specific in immunofluorescence experiments using live worms and in immunoprecipitation experiments using 125Iodine-labeled XL3 and L4 surface proteins. The overall amino acid composition of the surface proteins is hydrophilic. Twenty-six percent of the amino acid residues of the XL3 surface proteins, which consist predominantly of the 68-97 kDa species, are glutamate or glutamine.