Blue Light-Induced Phosphorylation of a Plasma Membrane-Associated Protein in Zea mays L

Blue light induces a variety of photomorphogenic responses in higher plants, among them phototropic curvature, the bending of seedlings toward a unidirectional light source. In dark-grown coleoptiles of maize (Zea mays L.) seedlings, blue light induces rapid phosphorylation of a 114-kD protein at fluence levels that are sufficient to stimulate phototropic curvature. Phosphorylation in response to blue light can be detected in vivo in coleoptile tips preincubated in 32Pi or in vitro in isolated membranes supplemented with [[gamma]-32P]ATP. Phosphorylation reaches a maximum level in vitro within 2 min following an inductive light pulse, but substantial labeling occurs within the first 15 s. Isolated membranes remain activated for several minutes following an in vitro blue light stimulus, even in the absence of exogenous ATP. Phosphoamino acid analysis of the 114-kD protein detected phosphoserine and a trace of phosphothreonine. The kinase involved in phosphorylating the protein in vitro is not dependent on calcium. The 114-kD protein itself has an apparent binding site for ATP, detected by incubating with the nonhydrolyzable analog, 5[prime]-p-fluorosulfonyl-benzoyladenosine. This result suggests that the 114-kD protein, which becomes phosphorylated in response to blue light, may also be capable of kinase activity.     

Light-Induced Changes in Phosphorylation Status of Low Molecular Weight Wheat Nuclear Proteins

A large number of polypeptides were phosphorylated when in vitro protein phosphorylation was carried out in nuclei isolated from dark-grown seedlings. For studying the effect of light, dark-grown seedlings were exposed to light and the isolated nuclear proteins phosphorylated in vitro. Although 4 h of white light was sufficient to alter the phosphorylation status of at least two polypeptides of 19 and 17 kD but the effect of light was more pronounced after irradiation for 8 h or more, leading to virtual disappearance of a 19 kD and emergence of a 17 kD phosphopolypeptide. Studies using norflurazon, a bleaching herbicide, indicate that, in addition to 19 and 17 kD phosphopolypeptides, another 21 kD phosphopolypeptide may be involved in the de-etiolation process. However, the nature of the photoreceptor involved in these light-induced changes in nuclear protein phosphorylation remains to be established.     

Detection in Vivo of Very Rapid Red Light-Induced Calcium-Sensitive Protein Phosphorylation in Etiolated Wheat (Triticum aestivum) Leaf Protoplasts

Etiolated wheat (Triticum aestivum cv Mercia) leaf protoplasts respond to brief red-light irradiation by increasing in volume over a 10-min incubation period (M.E. Bossen, H.A. Dassen, R.E. Kendrick, W.J. Vredenberg [1988] Planta 174: 94-100). When the calcium-sensitive dye Fluo-3 was incorporated into these protoplasts, red-light irradiation initiated calcium transients lasting about 2 min (P.S. Shacklock, N.D. Read, A.J. Trewavas [1992] Nature 358: 153-155). Release of calcium in the protoplasts by photolysis of incorporated 1-{2-amino-5-[1-hydroxy-1-(2-nitro-4, 5-methylenedioxyphenyl)-methyl]-phenoxy}-2-(2[prime]-amino-5[prime]-methylp henoxy)-ethane-N,N, N[prime],N[prime] -tetraccetic acid, tetrasodium salt (caged calcium) or caged inositol trisphosphate frequently induced transient increases in intracellular calcium levels, although the kinetics of these changes showed variation between experiments. Upon exposure to red light, a pronounced increase in the phosphorylation of a 70-kD and to a lesser extent a 60-kD peptide was observed, commencing within 15 s and continuing for up to 2 min. Simultaneous far-red and red irradiation attenuated the response. Upon release of incorporated caged calcium by cage photolysis, the labeling of these two peptides was greatly increased. When incorporated caged inositol trisphosphate was photolyzed, only the labeling of the 70-kD peptide was enhanced. Phosphorylation of the 70-kD peptide was also increased when extracellular calcium was elevated, but it decreased with increasing extracellular EGTA. These data thus provide direct evidence for the operation of an in vivo transduction sequence involving red light-dependent, calcium-sensitive protein phosphorylation.     

Rapid Changes in Plasma Membrane Protein Phosphorylation during Initiation of Cell Wall Digestion

Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [γ-³²P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M_r 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M_r 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M_r 22,000. These effects appeared not to be due to nonspecific protease activity and neither in vivo nor in vitro exposure to driselase caused a significant loss of Coomassie blue-staining bands on the gels of the isolated plasma membranes. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic ³²P also showed a response to the Driselase treatment. An enzymically active driselase preparation was required for the observed responses.     

A Pea Plasma Membrane Protein Exhibiting Blue Light-Induced Phosphorylation Retains Photosensitivity following Triton Solubilization

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase.     

Protein Changes Associated with Auxin-Induced Stimulation and Kinetin-Induced Inhibition of Lateral Root Initiation in Lettuce (Lactuca sativa) Roots

Protein changes associated with hormonal regulation of lateralroot initiation in lettuce (Lactuca sativa L.) roots were examined.Naphthalene acetic acid (NAA) stimulates the induction of lateralroots (Maclsaac, Sawhney, and Pohorecky, 1989) and this wasaccompanied by an increase in soluble proteins as well as thesynthesis of several polypeptides, including specific polypeptidesof 32 and 31 kD. The synthesis of these polypeptides coincidedwith the onset of cell division in the pericycle of NAA-treatedroots. Application of cycloheximide at different times showedthat NAA-induced protein synthesis is essential for the initiationand development of lateral root primordia. Kinetin inhibitedthe formation of lateral roots as well as the level of solubleproteins in NAA-treated roots. In addition, kinetin-treatedroots contained 22 and 21 kD polypeptides not found in othertreatments. This study suggests that the mechanisms of NAA-stimulationand kinetin-inhibition of lateral root initiation are probablydifferent. Key words: Lactuca sativa, lateral roots, proteins     

Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility

Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function.     

Diagravitropism in Corn Roots

The diagravitropic behavior of Merit corn (Zea mays L.) roots grown in darkness provides an opportunity for comparison of two qualitatively different gravitropic systems. As with positive gravitropism, diagravitropism is shown to require the presence of the root cap, have a similar time course for the onset of curvature, and a similar presentation time. In contrast with positive gravitropism, diagravitropism appears to have a more limited requirement for calcium, for it is insensitive to the elution of calcium by EGTA and insensitive to the subsequent addition of a calcium/EGTA complex. These results are interpreted as indicating that whereas the same sensing system is shared by the two types of gravitropism, separate transductive systems are involved, one for diagravitropism, which is relatively independent of calcium, and one for positive gravitropism, which is markedly dependent on calcium.     

Effect of Fluoride on Ribosomes from Corn Roots. Changes with Growth Retardation

Growth inhibition caused by fluoride was further studied by analyzing free and bound ribosomes, and the ribosomal components in corn roots. Ribosomes were isolated by differential centrifugation. Ribosomal components were analyzed by column chromatography and electrophoresis. Fluoride reduces the amounts of both free and bound ribosomes. Fluoride also modifies the RNA to protein ratio of bound ribosomes more than that of free ribosomes. Fluoride does not affect base ratios of the ribosomal RNA, but reduces its content and changes the structure of the ribosomal protein moiety.     

Stage-Specific Changes in Calcium-Regulated Protein Phosphorylation in Developing Tomato Fruits

The role of calcium and calmodulin in the in vitro phosphorylationof soluble and membrane proteins was studied in relation togrowth and development of tomato fruits. Calcium at micromolarconcentrations promoted the phosphorylation of both solubleand membrane proteins. The calmodulin antagonists, chlorpromazineand trifluoperazine, inhibited the phosphorylation of severalproteins. Qualitative changes were observed in the pattern ofprotein phosphorylation at different developmental stages. Therewas a general decrease in protein phosphorylation towards ripening.These results indicated that calcium may be involved in theregulation of phosphorylation of different proteins at differentstages of fruit development. ¹ Scientific Paper No. 7149, College of Agriculture and HomeEconomics, Washington State University, Pullman, Project 0321. ² Supported in part by a grant from the National Science Foundation.DCB-8502215. (Received May 14, 1985; Accepted September 12, 1985)     

In the Beginning,There Was Protein Phosphorylation

The importance of reversible protein phosphorylation to cellular regulation cannot be overstated. In eukaryotic cells, protein kinase/phosphatase signaling pathways regulate a staggering number of cellular processes, including cell proliferation, cell death (apoptosis, necroptosis, necrosis), metabolism (at both the cellular and organismal levels), behavior and neurological function, development, and pathogen resistance. Although protein phosphorylation as a mode of eukaryotic cell regulation is familiar to most biochemists, many are less familiar with protein kinase/phosphatase signaling networks that function in prokaryotes. In this thematic minireview series, we present four minireviews that cover the important field of prokaryotic protein phosphorylation.     

Light-Induced CREB Phosphorylation and Gene Expression in Rat Retinal Cells

Abstract: The signal pathway for light-induced expression of c- fos and the neuropeptide somatostatin (SS) in rat retinal cells was investigated. Flashing light induced c- fos and SS mRNA in the inner nuclear layer and the ganglion cell layer. As both c- fos and SS genes have a cyclic AMP response element (CRE) in their promoters, CRE-binding protein (CREB) phosphorylation in retinal cells was examined with a phospho-CREB-specific antibody. Both flashing light and administration of the L-type Ca²⁺ channel activator Bay K 8644 induced phosphorylation of CREB in the nuclei of the amacrine cells and the ganglion cells where c- fos /SS mRNAs were expressed. These cells could be double-stained with anti-calmodulin kinase II (anti-CaM kinase II) monoclonal antibody and phospho-CREB-specific polyclonal antiserum after Bay K 8644 administration, indicating the colocalization of phosphorylated CREB at Ser¹³³ and CaM kinase II in the neural retina.     

Rapid Phytochrome-Controlled Protein Phosphorylation and Dephosphorylation in Avena sativa L.

The time course for in vivo changes in the protein phosphorylationpattern was measured after red and red/far-red light. Avenacoleoptile tips were incubated in ³²P-labeled phosphate andirradiated. The supernatant fractions of homogenates were subjectedto SDS-poly-acrylamide gel electrophoresis and then autoradiographed.Within seconds, the radioactive label of two proteins decreasedand the radioactive label of one protein increased. These datasuggest that the phosphorylation states for these proteins maybe under phytochrome control. (Received July 20, 1987; Accepted July 20, 1988)     

Light-Induced Absorption Changes in Etiolated Coleoptiles

Blue (or green) light induced reversible and irreversible absorption changes in etiolated wheat (Triticum aestivum) coleoptiles. Immediately after a 10 s pulse of blue light there was an absorbance increase at 440 nm and a decrease at 420 nm. The absorbance at 440 nm returned to the original level in a biphasic way, with first-order rate constants of 0.05 s^?1 and 0.006 s^?1 at 25°C. The change at 440 nm was partly, but not completely, inhibited by iodide. In the 500–600 nm region complex changes occurred, indicating the participation of at least two different cytochromes, one of which was oxidized during a 10 s light pulse and the other oxidized more slowly during the following dark period.     

Negative Body Image Associated with Changes in the Visual Body Appearance Increases Pain Perception

Changing the visual body appearance by use of as virtual reality system, funny mirror, or binocular glasses has been reported to be helpful in rehabilitation of pain. However, there are interindividual differences in the analgesic effect of changing the visual body image. We hypothesized that a negative body image associated with changing the visual body appearance causes interindividual differences in the analgesic effect although the relationship between the visual body appearance and analgesic effect has not been clarified. We investigated whether a negative body image associated with changes in the visual body appearance increased pain. Twenty-five healthy individuals participated in this study. To evoke a negative body image, we applied the method of rubber hand illusion. We created an “injured rubber hand” to evoke unpleasantness associated with pain, a “hairy rubber hand” to evoke unpleasantness associated with embarrassment, and a “twisted rubber hand” to evoke unpleasantness associated with deviation from the concept of normality. We also created a “normal rubber hand” as a control. The pain threshold was measured while the participant observed the rubber hand using a device that measured pain caused by thermal stimuli. Body ownership experiences were elicited by observation of the injured rubber hand and hairy rubber hand as well as the normal rubber hand. Participants felt more unpleasantness by observing the injured rubber hand and hairy rubber hand than the normal rubber hand and twisted rubber hand (p<0.001). The pain threshold was lower under the injured rubber hand condition than with the other conditions (p<0.001). We conclude that a negative body appearance associated with pain can increase pain sensitivity.     

Nitrogenase Activity Associated with Roots and Stems of Field-Grown Corn (Zea mays L.) Plants

Corn (Zea mays L.) plants were assayed for nitrogenase activity (C₂H₂ reduction) during early ear development. Hybrid corn and inbred lines were grown separately at two experimental fields in New Jersey. Acetylene-dependent ethylene production was observed a few hours after harvest, from the field, on intact plants, root-soil cores, lower stem segments, and excised roots, all assayed under air and not preincubated previously. Incubation of excised roots at 1% O₂ resulted in lower rates of C₂H₂ reduction. The time course of C₂H₂ reduction by excised roots, assayed in air, was similar for all genotypes studied (two hybrids, eight inbreds, and a cross of corn × teosinte) and indicated that a long preincubation at reduced O₂ is not absolutely required for early detection of nitrogenase activity. Isolation of N₂-fixing bacteria from within the roots and stems, together with the diurnal fluctuation of nitrogenase activity in response to day/night cycles, were indicative of a close association with plant function. Collectively, the results provided strong evidence for the occurrence of nitrogenase activity associated with corn plants growing in a temperate climate and dependent upon indigenous N₂-fixing bacteria.     

Changes in Membrane Lipids of Roots Associated with Changes in Permeability: I. EFFECTS OF UNDISSOCIATED ORGANIC ACIDS

Previous work has shown that undissociated forms of organic acids, such as formic, acetic, and propionic acids, increase the permeability of barley roots to ions. The work here was undertaken to test whether these undissociated acids affect the lipids from the root membranes in such a way as to account for the permeability increase. Relative amounts of the principal fatty acids from barley root membranes were measured as a function of organic acid concentration, pH, and time of treatment of barley roots under conditions similar to those of the previous studies.