Phosphorylation Of Polypeptides Research Articles

Characterization of Ca2+-dependent endogenous phosphorylation of 160,000- and 150,000-Dalton proteins of sarcoplasmic reticulum

The 160 and 150 kDa proteins of sarcoplasmic reticulum (SR) are phosphorylated endogenously. The phosphorylation of both proteins has a marked requirement for Ca2+. Half-maximal and maximal phosphorylation was obtained at about 1 nM- and 1 microM-Ca2+ respectively, and a Hill coefficient of about 0.5 was calculated. The phosphorylation is also dependent on NaF as an inhibitor of the SR phosphoprotein phosphatase. The phosphorylation of these proteins is very rapid, and maximal phosphorylation is achieved in less than 15 s. The phosphorylation of the 160 kDa and 150 kDa polypeptides is completely inhibited by 5 mM-MgCl2 and by 75 microM-LaCl3, by very low concentrations of different detergents, and by preincubation of the SR for 2 min at 60 degrees C. The inhibition by Mg2+ is due to stimulation of ATP hydrolysis, thereby decreasing ATP concentration. Different phosphorylated peptides were obtained by digestion with protease V8 of the 160 kDa and 150 kDa protein bands, suggesting that the 160 kDa and 150 kDa proteins are distinct. The two phosphorylated proteins are present in different fractions and preparations of SR, with or without [3H]PN200-110 binding capacity. These and other results suggest that the phosphorylated SR proteins are distinct from the alpha 1 and alpha 2 subunits of the voltage-gated Ca2+ channel of the T-system membranes. Different inhibitors and activators of protein kinase C and calmodulin-dependent protein kinase have no effect on the endogenous phosphorylation of both polypeptides, suggesting that the phosphorylation is regulated solely by Ca2+. A possible regulatory function for this phosphorylation system is described in the accompanying paper [Gechtman. Orr & Shoshan-Barmatz (1991) Biochem. J. 276.97-102].

Involvement of protein phosphorylation in activation of Ca2+ efflux from sarcoplasmic reticulum

Preincubation of sarcoplasmic reticulum (SR) membranes with a combination of ATP and NaF resulted in inhibition of Ca2+ accumulation and stimulation of Ca(2+)-ATPase and Ca2+ efflux. Under the same conditions, the activity of the SR phosphoprotein phosphatase was inhibited and the phosphorylation of two polypeptides with apparent molecular masses of 160 and 150 kDa was obtained. The effect of ATP is specific, since the ATP analogue adenosine 5'-[beta gamma-imido]triphosphate did not replace for ATP. In the absence of NaF, ATP was ineffective. The phosphorylation of the 160 kDa and/or 150 kDa proteins and the stimulation of Ca2+ efflux are clearly related. The phosphorylation of both proteins and the increase in Ca2+ efflux show a similar dependence on the concentration of ATP. The level of protein phosphorylation and the stimulation of Ca2+ efflux were also controlled by the NaF concentration which inhibits the phosphatase and of net Ca2+ accumulation, as well as for the stimulation of phosphorylation of both polypeptides. Quantitative analysis revealed a linear correlation between these three activities. Dicyclohexylcarbodi-imide, which inhibited Ca2+ efflux, also inhibited the phosphorylation of the two polypeptides. These results suggest the involvement of the phosphorylation/dephosphorylation of 160 kDa and/or 150 kDa polypeptides in the activation of Ca2+ release from SR membranes.

The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers.

Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.

In Vitro and in Vivo Phosphorylation of Polypeptides in Plasma Membrane and Tonoplast-Enriched Fractions from Barley Roots

Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with gamma-[(32)P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg(2+) was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46, and 28 kilodaltons required millimolar Mg(2+) concentrations and was greatly enhanced by Ca(2+). When roots of intact plants were labeled with [(32)P]orthophosphate, polypeptides at approximately 135, 116, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mm NaCl had no effect.

Phosphorylation of small molecular weight polypeptides in the iris-ciliary complex, aqueous humor and vitreous humor

The polypeptides of vitreous, aqueous humor and iris-ciliary complex cells of eyes were phosphorylated with [γ- 32P]ATP without exogenous protein kinase. Phosphorylated polypeptides were analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography. The phosphorylated polypeptides of rabbit vitreous humor showed many high molecular weight prominent bands, but no detectable phosphoproteins were found in the 12 kDa or lower range. Bovine vitreous humor has predominantly acidic polypeptides and some of them are below 20 kDa. Rabbit and bovine iris-ciliary complex and rabbit aqueous humor showed a prominent common 4 kDa phosphopolypeptide which could also be synthesized by cloned populations of cells from the bovine iris and the rabbit iris-ciliary body. It is possible that the 4 kDa phosphopolypeptide of the aqueous humor is synthesized by the iris-ciliary complex cells.

Calcium and protein phosphorylation in the transduction of gravity signal in corn roots.

The involvement of calcium and protein phosphorylation in the transduction of gravity signal was studied using corn roots of a light-insensitive variety (Zea mays L., cv. Patriot). The gravitropic response was calcium-dependent. Horizontal placement of roots preloaded with 32P for three minutes resulted in changes in protein phosphorylation of polypeptides of 32 and 35 kD. Calcium depletion resulted in decreased phosphorylation of these phosphoproteins and replenishment of calcium restored the phosphorylation.

The In Vitro Effects of Glycyrrhizin and the Derivatives of Glycyrrhetinic Acid on the Activity of cAMP-Dependent Protein Kinase and Phosphorylation of Cellular Polypeptide by the Kinase from Ehrlich Ascites Tumor Cells.

The effects of glycyrrhizin (GL) and the derivatives of glycyrrhetinic acid (GA) on the activity of cAMP-dependent protein kinase (A-kinase) and the phosphorylation of cellular polypeptides by the kinase purified from Ehrlich ascites tumor cells had been investigated in vitro. It was found that (i) the derivatives [3 beta-hydroxy-olean-11,13 (18)-diene-30-oic acid Na, olean-9(11), 12 diene-3 beta, 30-diol-3 beta, 30-di-o-hemiphthalate 2Na and olean-12-ene-3 beta, 30-diol 3 beta, 30-di-o-phosphate 2Na] of GA inhibited the activity of A-kinase at the concentrations higher than 25 microM; (ii), at 10 microM, these derivatives and native GL stimulated the activity of the kinase significantly; and (iii) the inhibitory and stimulatory effects of some GA derivatives were clearly correlated with their chemical structures. Moreover, sodium dodecyl sulfate polyacrylamide gel electrophoresis and two dimensional gel electrophoresis followed by autoradiography detected several acidic polypeptides, including polypeptides with approximate molecular weights of 35,000 (pI 4.3), 27,000 (pI 4.5) and 18,000-21,000 (pI 4.5), phosphorylated by A-kinase, to be functioning as mediators in response to these drugs. This observation suggests that the GL-induced inhibition of phosphorylation of these cellular polypeptides by A-kinase may be physiologically implicated in the biochemical mechanisms involved in the anti-inflammatory effect of the drug.

Phosphorylation of murine CD8α is not essential for responses of T cell hybridomas to antigen

CD8 T cell differentiation antigens, expressed on class I-restricted T cells, have a key role in the control of recognition and response of these cells to antigen. It has been suggested that these molecules function as co-receptors together with antigen-specific T cell receptors to regulate T cell responses. We have addressed the question of whether cytoplasmic serine phosphorylation, which occurs on CD8 molecules after activation by antigen or phorbol esters, is relevant to its co-receptor function. By mutagenesis, we show that phorbol ester-induced phosphorylation occurs exclusively on CD8 alpha serine residue 216. However, inhibition of CD8 polypeptide phosphorylation does not appear to have a detrimental effect on several responses of CD8-dependent transfectants to antigen. This is in contrast to results reported with CD4 (N.Glaichenhaus, N.Shastri, D.R. Littmann and J.M.Turner. 1991. Cell, 64:511), suggesting that CD4 and CD8 molecules may play somewhat different roles in the control of T cell activation.

Modulation of phosphorylation and dephosphorylation of keratin and other polypeptides by estradiol-17β in rat vaginal epithelium

Phosphorylation of keratin polypeptides was studied by incubating vaginal tissues (removed from estradiol primed and unprimed 30-day-old rats) with 32Pi. Analysis by SDS-PAGE and autoradiography showed that on treatment with estradiol phosphorylation of 63 and 58 kDa keratin polypeptides increased 3- and 2-fold respectively. Phosphorylation was maximal after 30 min of estradiol priming and decreased thereafter. Phosphorylation of some non-keratin polypeptides (37, 34, 32 and 25 kDa) also showed time dependent variation. The results showed that estradiol can modulate phosphorylation-dephosphorylation of keratins and other polypeptides in rat vaginal epithelial cells.

Characterization of a basic polypeptide-activated protein kinase in the eggs of the silkworm, Bombyx mori

A basic polypeptide-activated protein kinase (designated BA-kinase), which requires Mn 2+ rather than Mg 2+ for its activity, has been highly purified from a 1.5 M KCl extract of fertilized and unfertilized eggs of the silkworm, Bombyx mori. It was found that (i) the molecular weight of the purified protein kinase was approx. 260000; (ii) the kinase phosphorylated vitellin, protamine and casein in a cAMP-, cGMP- and Ca 2+-independent manner; (iii) the kinase phosphorylated only threonine residues when casein was used as a phosphate acceptor; (iv) protein phosphorylation by the kinase was remarkably stimulated by basic polypeptides, such as histone, polyArg and polyLys; and (v) the purified kinase had an affinity with DNA, but not with RNAs. Moreover, the observations that (a) the BA-kinase activity and phosphorylation of polypeptides by the kinase in the 1.5 M KCl egg extract greatly increased after fertilization, and (b) polypeptides, from this extract, of 16.5–21 kDa, which were highly phosphorylated by the kinase but not by A- or G-kinase, appeared within 4 h after fertilization, suggest that a basic polypeptide activated protein kinase (BA-kinase) may play an important role in the progression of embryogenesis through specific phosphorylation of yolk proteins by the kinase, in the presence of nucleic basic proteins.

Susceptibility of human cells to killing by the parvoviruses H-1 and minute virus of mice correlates with viral transcription

Human fibroblasts and epithelial cells differing in their susceptibility to killing by the autonomous parvoviruses H-1 and minute virus of mice were compared for their capacity to express viral mRNAs and proteins. The transition from a parvovirus-resistant to a parvovirus-sensitive phenotype correlated with a proportional increase in the production of the three major viral transcripts and of structural and nonstructural proteins. In contrast, cell sensitization to parvovirus could not be correlated with detectable changes in virus uptake, intracellular localization of gene products, stability of viral mRNAs, or phosphorylation of viral nonstructural polypeptides. Moreover, the H-1 virus-sensitive keratinocyte line studied did not sustain a greater level of viral DNA amplification than its resistant derivative. Therefore, the differential susceptibility of the human cells tested to parvovirus infection appears to be mainly controlled at the level of transcription of the viral genome. Parvoviral gene expression could not be elevated by increasing the input multiplicity of infection in either of the cell systems analyzed. Together, these data suggest that a cellular factor(s) regulating parvoviral transcription may be modulated by oncogenic transformation or by differentiation, as both features have been shown to affect cell susceptibility to parvoviruses.

Altered protein phosphorylation in murine muscular dystrophy

Protein phosphorylation has been studied in the dydy murine muscular dystrophy, both in intact muscle cells and in various membrane fractions derived from them. The results obtained showed that several polypeptides were more heavily phosphorylated in dystrophic myotubes in culture as well as in dystrophic muscle fibers isolated from tibialis anterior. In vitro phosphorylation studies revealed that a large polypeptide of apparent molecular weight of 170 000-150 000 was phosphorylated under basal conditions (3 mM EGTA) in dydy microsomal membranes. The phosphorylation of this polypeptide was not stimulated further by cAMP, calmodulin, cGMP or 12- O-tetradecanoylphorbol 13-acetate (TPA). Under no condition was the corresponding polypeptide phosphorylated at an appreciable rate in normal microsomal membranes. An antibody raised against the voltage-dependent calcium channel reacted, in an immunoblot assay, with a polypeptide, present in both normal and dydy microsomes, which had migration characteristics identical to the phosphorylated 170-150 kDa polypeptide after one- or two-dimensional gel electrophoresis. Additional differences were identified in the phosphorylation of smaller polypeptides of microsomal membranes. When sarcolemmal membranes of normal and dydy muscle were phosphorylated in vitro, no major differences were observed. These results show the existence of an alteration of protein phosphorylation in dystrophic muscle cells in vitro and in vivo, leading to abnormal phosphorylation of the voltage-dependent calcium channel. The possible causes and consequences of this alteration are discussed.

Role of nuclear protein kinase C in the mitogenic response to platelet-derived growth factor

We have assessed the involvement of nuclear envelope protein phosphorylation in the mitogenic response to platelet-derived growth factor (PDGF) in NIH/3T3 fibroblasts. We find that stimulation of quiescent NIH/3T3 cells with PDGF or with the mitogenic protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) or bryostatin 1 (bryo) leads to rapid, dose-dependent phosphorylation of several nuclear envelope polypeptides. The predominant nuclear envelope targets for mitogen-induced phosphorylation are immunologically identified as the nuclear envelope lamins. All three lamin species (A, B and C) are phosphorylated in response to PMA or bryo, while lamins A and C are preferentially phosphorylated in response to PDGF. Phosphopeptide mapping and phosphoamino acid analysis indicate that similar serine sites on the lamins are phosphorylated in response to PDGF, PMA and bryo. Both mitogenicity and lamina phosphorylation induced by these mitogens can be inhibited by the selective PKC inhibitor staurosporine at 2 nM. Treatment of quiescent NIH/3T3 cells with PDGF, PMA or bryo leads to rapid translocation of PKC to the nuclear envelope. These data indicate that rapid nuclear events, including translocation of cytosolic PKC to the nuclear membrane and lamina phosphorylation, may play a role in the transduction of the mitogenic signals of PDGF from the cytoplasm to the nucleus in NIH/3T3 fibroblasts.

Heparin-binding growth factor-1 stimulation of human endothelial cells induces platelet-derived growth factor A-chain gene expression.

Heparin-binding growth factor-1 (HBGF-1), also known as acidic fibroblast growth factor, is a potent mitogen for a variety of cell types including vascular endothelial and smooth muscle cells. Studies using murine 3T3 fibroblasts have shown that HBGF-1 induces numerous cellular responses such as the tyrosine phosphorylation of specific polypeptides and the increased expression of actin mRNA. Here we report that the addition of HBGF-1 to quiescent human umbilical vein endothelial cells increases the level of platelet-derived growth factor (PDGF) A-chain mRNA but not PDGF B-chain mRNA. In contrast, factors that inhibit endothelial cell proliferation such as phorbol myristate acetate and the cytokines interleukin-1, interleukin-6, and tumor necrosis factor-alpha increase both PDGF A-chain and B-chain mRNA levels. HBGF-1 induction of PDGF A-chain mRNA expression occurs in the presence of the protein synthesis inhibitor cycloheximide and thus does not require de novo protein synthesis. HBGF-1 also increases c-fos, c-jun, and c-myc mRNA levels; in the presence of cycloheximide, PDGF A-chain and protooncogene mRNA accumulation kinetics are similar. Nuclear run-on experiments indicate that the transcription rate of the PDGF A-chain gene transiently increases after HBGF-1 addition. Immunoprecipitation analysis using PDGF A-chain-specific antibodies indicates that HBGF-1-stimulated cells synthesize and secrete an increased amount of PDGF relative to unstimulated cells. If HBGF-1 can regulate PDGF expression by vascular endothelial cells in vivo, then HBGF-1 availability would be an important component of smooth muscle cell growth control. For example, HBGF-1 within the vessel wall would promote smooth muscle cell proliferation by (a) direct interaction with smooth muscle cell HBGF-1 receptors, and (b) increasing the amount of endothelial cell-derived PDGF available for binding to smooth muscle cell PDGF receptors.

Modulation of keratin intermediate filament distribution in vivo by induced changes in cyclic AMP‐dependent phosphorylation

Treatment of PtK1 cells with 5 mM acrylamide for 4 hr induces reversible dephosphorylation of keratin in concert with reversible aggregation of intermediate filaments (Eckert and Yeagle, Cell Motil. Cytoskeleton 11:24-30, 1988). We have examined this phenomenon by 1) in vitro phosphorylation of isolated PtK1 keratin filaments and 2) combined treatments of PtK1 cells with both acrylamide and agents which elevate intracellular cAMP levels. PtK1 keratins were incubated in gamma-32P-ATP in the presence or absence of cAMP-dependent kinase (A-kinase) and cAMP. Levels of phosphorylation were analyzed by electrophoresis and autoradiography. Phosphorylation of keratin polypeptides (56 kD, 53 kD, 45 kD, 40 kD) occurred without added kinase, suggesting the presence of an endogenous kinase which remains with intermediate filaments in residues of Triton X-100 extracted cells. Phosphorylation levels were increased by A-kinase but not by cAMP alone, indicating the presence of cAMP-dependent phosphorylation sites in addition to sites phosphorylated by the endogenous kinase. To study the possible role of cAMP-dependent phosphorylation in acrylamide-induced aggregation of keratin filaments, we treated cells with acrylamide in the presence of 8-bromo-cAMP (brcAMP), pertussis toxin (PT), isobutylmethylxanthine (IBMX), or forskolin, which increase intracellular cAMP levels. The distribution and phosphorylation levels of keratin filaments, as well as intracellular cAMP levels, were determined for each of these treatments. In addition to aggregation and dephosphorylation of keratin filaments reported previously, treatment of cells with acrylamide alone also results in reduced levels of intracellular cAMP. 8-bromo-cAMP, IBMX, and forskolin prevent acrylamide-induced aggregation of keratin filaments and result in both normal levels of keratin phosphorylation and normal intracellular cAMP levels. PT was apparently ineffective. These observations suggest that 1) PtK1 keratins are phosphorylated by cAMP-dependent kinase and an endogenous, cAMP-independent kinase and 2) alteration of levels of cAMP-dependent phosphorylation may be involved in aggregation of keratin filaments in response to acrylamide.

Novel Regulatory Role of Phosphorylated Clathrin Light Chain β in Bovine Brain Coated Vesicles

The 50-kilodalton (kDa) assembly polypeptide of bovine brain clathrin coated vesicles (CCVs) is phosphorylated in a cyclic nucleotide- and Ca2+-independent manner and is dephosphorylated by a Mg2+-ATP-dependent CCV phosphatase. This report provides evidence for modulation of the phosphorylation reaction of the 50-kDa assembly polypeptide by phosphorylated clathrin light chain beta (pLC beta). In vitro, phosphorylated LC beta inhibits phosphorylation of the 50-kDa polypeptide in CCVs. Furthermore, incubation of previously phosphorylated 50-kDa polypeptide in CCVs with phosphorylated LC beta results in a rapid dephosphorylation of the 50-kDa assembly polypeptide. Both phenomena are time and concentration dependent. Monoclonal antibodies to LC beta prevent the modulatory effect of phosphorylated LC beta on the 50-kDa assembly polypeptide phosphorylation in CCVs. The results obtained indicate for the first time, to our knowledge, that phosphorylated LC beta has a modulatory role in CCVs. The data also suggest that phosphorylated LC beta promotes activation of a coated vesicle phosphatase.

Interleukin-3 and Bryostatin 1 Mediate Rapid Nuclear Envelope Protein Phosphorylation in Growth Factor-dependent FDC-P1 Hematopoietic Cells: A possible role for nuclear protein kinase C

Abstract Interleukin-3 (IL-3) is a lymphokine which stimulates the proliferation of normal and transformed multilineage hematopoietic cells. Recently we reported that bryostatin 1, a macrocyclic lactone and potent activator of protein kinase C, could stimulate normal multipotential hematopoietic progenitor cells in vitro in the absence of added polypeptide growth factors. We have now used the murine IL-3-dependent cell line FDC-P1, derived from normal murine marrow cells, to examine the early biochemical events associated with stimulation of hematopoietic cells. We find that both IL-3 and bryostatin 1 are mitogenic and stimulate the growth of FDC-P1 cells. Cells grown for extended periods in the presence of bryostatin 1 (1 nM) alone retain IL-3 responsiveness, indicating that bryostatin 1 does not induce an IL-3-independent state. Protein phosphorylation studies in cells treated with either IL-3 or bryostatin 1 indicate that both stimulators can mediate the rapid (within 5 min) serine-specific phosphorylation of several nuclear envelope polypeptides, including lamin B. Both IL-3- and bryostatin 1-mediated nuclear envelope phosphorylation is dose-dependent, occurring at concentrations which are mitogenic to FDC-P1 cells. The extent of nuclear envelope phosphorylation mediated by IL-3 and bryostatin 1 correlates with the mitogenic response. Furthermore, both mitogens mediate the rapid immunologic translocation of protein kinase C to the nuclear envelope where phosphorylation occurs. These data indicate that the early mitogenic signal(s) generated by IL-3 and bryostatin 1 may converge at the level of the nuclear envelope, perhaps through a protein kinase C-like activity which mediates phosphorylation of specific nuclear envelope polypeptides such as lamin B.

Light‐dependent, chilling effects on phosphorylation of thylakoid proteins and consequences for associated photochemical activities in maize

When maize (Zea mays L. cv. LG11) leaves are exposed to low temperatures and high light modifications to both photosystem 2 (PS2) and the light‐harvesting chlorophyll a/b protein complex associated with photosystem 2 (LHC2) occur. This study examines the consequences of these modifications for phosphorylation of LHC2 and PS2 polypeptides and the associated changes in electron transport. Maize leaves were chilled at 5°C for 6 h under photon flux densities of 1 500 and 250 μmol m‐2 s‐1. Thylakoids were then isolated from the leaves and their abilities to phosphorylate LHC2 and PS2 polypeptides and modify electron transport activities were determined. Measurements of chlorophyll fluorescence induction in the thylakoids were also made. Thylakoids isolated from leaves chilled under high light and from leaves kept in the ambient growth environment had similar phosphoprotein profiles. However, polypeptide phosphorylation in thylakoids from the chilled leaves did not produce a decrease in PS2 electron transport. Chilling leaves under low light produced a decrease in the ability of isolated thylakoids to phosphorylate PS2, but not LHC2, polypeptides, which was not associated with any change in the phosphorylation‐induced decrease in PS2 electron transport. Chilling under high, but not low, light appears to produce changes in membrane organisation that do not affect the ability of the thylakoids to phosphorylate PS2 and LHC2 polypeptides, but which do prevent the phosphorylation‐induced decrease in excitation energy transfer from LHC2 to PS2.

Phospholipid-stimulated protein kinase in plants

In membrane fractions from zucchini (Cucurbita pepo L.) hypocotyls, catalytic properties of a platelet-activating factor (PAF)-activated protein kinase were investigated. In the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, phosphorylation of a 55-kDa membrane polypeptide and, to a lesser extent, several others, including a 120-kDa polypeptide, was stimulated by PAF. The phosphorylation of the 55-kDa polypeptide was used for quantification of the PAF-stimulated protein kinase. Stimulation of protein phosphorylation by PAF increased in a concentration range from 10-200 micrograms/ml (= 19-380 microM) PAF up to 10-fold above the control. Addition of Ca2+ ions in the micromolar range in the presence and in the absence of PAF increased the phosphorylation of the 55- and the 120-kDa polypeptide. Other phospholipids and lipids tested including phorbol ester, diglyceride, mono- and triglyceride, and oleic acid were ineffective. The same lipid specificity was previously observed for the activation of ATP-dependent H+ transport in microsomes (Scherer, G.F.E., Martiny-Baron, G., and Stoffel, B. (1988) Planta 175, 241-253). Lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) were able to stimulate the phosphorylation of the same polypeptides as PAF and H+ transport but both to a lesser extent (PAF greater than LPC greater than LPE). In the presence of EGTA, PAF-stimulated phosphorylation of a 55- and a 57-kDa polypeptide was predominantly associated with vacuolar membranes and those of 42, 61, 63, and 120 kDa were predominantly associated with plasma membranes. Stimulation of ATP-dependent H+ transport by PAF was found in tonoplast vesicles whereas plasma membrane vesicles had only little transport activity and, therefore, an effect of PAF on plasma membrane H+ transport could not be measured. Stimulation of ATP hydrolysis by PAF was observed both in tonoplast- and plasma membrane-containing fractions.

Protein phosphorylation and control of excitation energy transfer in photosynthetic purple bacteria and cyanobacteria

The function of phosphorylation of light-harvesting polypeptides is well characterised in chloroplasts of green plants, but the prokaryotic cyanobacteria and purple photosynthetic bacteria have quite different light-harvesting polypeptides whose structure and function cannot be controlled in precisely the same way. Nevertheless, cyanobacteria show light-dependent phosphorylation of membrane polypeptides associated with photosystem II and with the light-harvesting phycobilisome, and purple bacteria show light-dependent phosphorylation of low molecular-weight chromatophore membrane polypeptides. In both cases membrane protein phosphorylation is associated with functional changes observed by chlorophyll fluorescence spectroscopy or chlorophyll fluorescence induction kinetics. Here we report on our recent protein sequence and other data concerning the identities of these phosphoproteins. We also discuss the significance of these findings for regulation by protein phosphorylation of photosynthesis in prokaryotes.