Light-induced Phosphorylation Research Articles

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.

Correlation of Blue Light-Induced Phosphorylation to Phototropism in Zea mays L.

The physiology of light-induced phototropic curvature has been studied extensively in coleoptiles of grasses, particularly in Avena and Zea mays L. In Z. mays L., we have found that, in addition to curvature, blue light also induces rapid phosphorylation of a 114-kD protein in the tips of coleoptiles, and, in a previous report, we reported several characteristics of the phosphorylated substrate protein and kinase (J.M. Palmer, T.W. Short, S. Gallagher, W.R. Briggs [1993] Plant Physiol 102: 1211-1218). Here, we compare the phosphorylation response to several known aspects of phototropism physiology. Blue light-induced phosphorylation occurs only in the upper portion of the coleoptile and is absent from the node and mesocotyl. The specific activity of phosphorylation is highest in the extreme apical portion of the tip, which is also the site of maximal sensitivity to phototropic stimuli (A. W. Galston [1959] In Physiology of Movements, Encyclopedia of Plant Physiology, Springer, Berlin). Fluence-response determinations indicate that light dosage levels that stimulate curvature also stimulate phosphorylation. However, the threshold for inducing detectable phosphorylation in maize cannot be matched to the threshold for curvature induction. The recovery of sensitivity to phototropic stimuli after exposure to high fluences of light occurs with kinetics that are very similar to those for recovery of the phosphorylation response after a previous high-fluence light exposure. In addition, wavelengths of light in the blue and near-ultraviolet regions of the spectrum that maximally stimulate phototropic curvature also maximally stimulate in vitro phosphorylation in maize. The pattern of stimulation matches the absorption spectra of flavoproteins, which have been proposed as candidates for blue light photoreceptors.

Redox dependence of the blue-light-induced phosphorylation of a 100-kDa protein on isolated plasma membranes from tips of coleoptiles

A blue-light-induced rapid phosphorylation of a 100-kDa protein localized in plasma membranes of phototropically sensitive tips of maize (Zea mays L.) coleoptiles was studied. Since, under in-vivo conditions or in a crude homogenate of tips, cytosolic ATP is the phosphate donor for the light-induced phosphorylation of this protein, a subsequent in-vitro phosphorylation by [32P]ATP is prevented. However, in-vitro irradiation of microsomal membranes isolated from non-irradiated tips followed by a 1-min incubation with [32P]ATP resulted in a strong phosphorylation (labelling) of the 100-kDa plasma-membrane protein. This process was saturated by a 7-s light pulse (200 μmol photons·m−2·s−1). In the absence of [32P]ATP the capacity for in-vitro phosphorylation of the 100-kDa protein after a 30-s light pulse declined slowly within 60 min but could be reconstituted by a new light pulse in the presence of reducing compounds. Moreover, when plasma membranes which had been stored frozen were used, reducing compounds such as NADH, NADPH, ascorbate, glutathione or dithiotreithol enhanced the light-triggered in-vitro phosphorylation. These compounds were unable to elicit or enhance the phosphorylation in the dark. It is suggested that the transfer of (blue-light) excited electrons from the chromophore moiety of the receptor to the target (either the 100-kDa protein or the protein kinase itself) is facilitated when reducing compounds instantly eliminate the positive charge generated at the chromophore. The transferred electrons could finally alter the redox state and-or the conformation of either the 100-kDa protein, rendering it susceptible to the action of a protein kinase, or the protein kinase which would then be capable of phospho-rylating the 100-kDa protein.

Recollections

About 1939, Sam Ruben and Martin Kamen introduced me to the emergent application of artificial radio-isotopes in the study of photosynthesis. While my own experiments on CO2 fixation by isolated chloroplasts turned out to be negative, their laboratory provided me with an informative and exciting experience. Also, there were many stimulating contacts with Cornelis van Niel, Robert Emerson, Don DeVault and many other outstanding scientists. Efforts on my part to obtain a better understanding of intermediary metabolism, eventually led me to Fritz Lipmann's laboratory. There I was encouraged to study the metabolic activities of cell-free preparations of photosynthetic purple bacteria. Investigations of oxidative phosphorylation by isolated bacterial chromatophores in the dark raised questions about the possible effects of light on the phosphorylation activities of such preparations. Surprisingly, high rates of phosphorylation were observed in the light in the absence of molecular oxygen ('light-induced phosphorylation'). In this process, adenosine diphosphate (ADP) and inorganic phosphate (Pi) could be converted quantitatively into adenosine triphosphate (ATP). It was postulated that this process was 'cyclic' in nature, as only catalytic concentrations of added electron donors were required. Later, at Minnesota, it could be shown that similar chromatophore preparations, in the presence of suitable electron donors, could reduce nicotinamide-adenine dinucleotide (NAD(+)) to NADH in the light. It was then demonstrated that the chromatophores of Rhodospirilum rubrum, as well as the smaller membrane components derived from them, must contain the active metabolic components for these photosynthetic reactions.These observations, and studies on the kinetics of the formation and decay of light-induced free radicals, appeared to demonstrate the usefulness of bacterial chromatophores and of their membrane fragments in the study of partial reactions of bacterial photosynthesis. Since that time, numerous investigators elsewhere have carried out remarkable research on the purification and eventual crystallization of distinct bacterial membrane components, capable of carrying out well characterized photochemical and electron transport reactions.

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.

Blue Light-Induced Phosphorylation of a Plasma Membrane Protein in Pea: a Step in the Signal Transduction Chain for Phototropism

A 117 kDa polypeptide associated with the plasma membrane isolated from the growing zones of etiolated pea epicotyls is phosphorylated upon a brief exposure to blue light. The literature pertaining to this photosensitive system to date is briefly summarised prior to a short experimental section. In the experimental work the polypeptide has been studied in its non-denatured and denatured state in order to investigate its possible role in blue light-induced signal transduction. The polypeptide has a molecular weight near 117 kDa. Under non-denaturing conditions, following solubilisation in non-ionic detergent, and after electrophoresis, a complex migrating at approximately '335 kDa' still retains the ability to perceive the blue light signal and undergo phosphorylation. This complex is resolved into a phosphorylated protein of approximate molecular mass of 117 kDa by second dimensional analysis on denaturing polyacrylamide gels. It is not at present possible to determine whether this protein is the only one involved or whether two or more different proteins are required for the light-inducible phosphorylation. This reaction has been established elsewhere by genetic and physiological criteria to be an early step in the signal transduction pathway for phototropism. Two other reactions that are inducible in vitro in isolated plasma membranes from etiolated seedings - activation of GTPase activity and reduction of a b-type cytochrome-are almost certainly independent pathways unrelated to the phosphorylation reaction or to each other. Neither has as yet been associated definitively with a known physiological response.

Structural and functional properties of rhodopsin from rod outer segment disk and plasma membrane

The structural and functional properties of bovine rhodopsin from rod outer segment disk and plasma membranes were compared by high performance liquid chromatography (HPLC), mass spectrometric analyses, and in vitro rhodopsin phosphorylation assays. Disk and plasma membranes separated by a ricin gold-dextran affinity perturbation method were treated with trypsin or cynogen bromide, and the N-terminal and C-terminal rhodopsin peptides were isolated by immunoaffinity chromatography using antirhodopsin monoclonal antibodies coupled to Sepharose. Reverse phase HPLC chromatograms of the C-terminal and N-terminal peptides from disk and plasma membrane rhodopsin were found to be similar. Mass spectrometric, PicoTag, and hexose analyses of the tryptic 1–16 N-terminal peptides further indicated that the post-translational glycosylation of plasma membrane rhodopsin is identical to that of disk membrane rhodopsin. HPLC analysis of soluble peptides obtained from cyanogen bromide and tryptic digestion of immunoaffinity purified rhodopsin also indicated that no significant differences exist between disk and plasma membrane rhodopsin. Light-induced phosphorylation of rhodopsin in disk and plasma membranes were also compared using in vitro phosphorylation assays. Plasma membrane rhodopsin was found to undergo light-dependent, rhodopsin kinase catalyzed phosphorylation to the same extent as disk membrane rhodopsin. These results indicate that the bulk rhodopsin in rod outer segment plasma membranes appears to be identical to rhodopsin in disk membranes in regard to primary structure, post-translational glycosylation and light-dependent phosphorylation. On this basis, it is unlikely that the sorting of rhodopsin between disk and plasma membranes occurs by a mechanism based on differences in structural properties of rhodopsin.

ATP-independent deactivation of squid rhodopsin.

Deactivation of light-activated squid rhodopsin was studied in vitro using GTP gamma S binding by G-protein as a direct measure of rhodopsin activity. Deactivation was inhibited by dilution of the retinal suspension or by removal of soluble components. Deactivation could be restored by addition of soluble material to washed membranes. These results indicate that the deactivation is not due entirely to a conformational transition within rhodopsin itself, but depends on the interaction with other molecules. The possibility that phosphorylation is involved in the deactivation was studied. Deactivation occurred in the presence and absence of added ATP. Deactivation also occurred in the presence of kinase inhibitors and after addition of apyrase, which reduced residual ATP levels to below 1 microM. These results indicate that light-induced phosphorylation is not required for deactivation of squid rhodopsin. In this regard deactivation of squid rhodopsin is different from that of vertebrate rhodopsin, which requires phosphorylation.

Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana.

Blue light is known to cause rapid phosphorylation of a membrane protein in etiolated seedlings of several plant species, a protein that, at least in etiolated pea seedlings and maize coleoptiles, has been shown to be associated with the plasma membrane. The light-driven phosphorylation has been proposed on the basis of correlative evidence to be an early step in the signal transduction chain for phototropism. In the Arabidopsis thaliana mutant JK224, the sensitivity to blue light for induction of first positive phototropism is known to be 20- to 30-fold lower than in wild type, whereas second positive curvature appears to be normal. While light-induced phosphorylation can be demonstrated in crude membrane preparations from shoots of the mutant, the level of phosphorylation is dramatically lower than in wild type, as is the sensitivity to blue light. Another A. thaliana mutant, JK218, that completely lacks any phototropic responses to up to 2 h of irradiation, shows a normal level of light-induced phosphorylation at saturation. Since its gravitropic sensitivity is normal, it is presumably blocked in some step between photoreception and the confluence of the signal transduction pathways for phototropism and gravitropism. We conclude from mutant JK224 that light-induced phosphorylation plays an early role in the signal transduction chain for phototropism in higher plants.

Phosrestins I and II: Arrestin homologs which undergo differential light-induced phosphorylation in the [formula omitted] photoreceptor in vivo

The 49-kDa phosphoprotein gene and Dmarrestin gene encode two distinct arrestin homologs in the Drosophila photoreceptor. We find that two DNA fragments representing the Dmarrestin gene hybrid-selected a mRNA the in vitro translation of which produced a protein corresponding to the 39-kDa phosphoprotein previously reported by us. We propose to name these phosphorylated homologs of arrestin phosrestin I (49-kDa protein) and phosrestin II (39-kDa protein or the Dmarrestin gene product). We find that phosrestins I and II follow different time courses of phosphorylation in vivo ; in the time period (∼seconds) during which 43% of phosrestin I became phosphorylated, the phosphorylated state of phosrestin II remained unchanged from that of the nonilluminated flies. These results indicate that phosrestins I and II probably occupy different functional roles in the Drosophila photoreceptor.

Relationships between heterogeneity of the PSII core complex from grana particles and phosphorylation

Isoelectrofocusing of photosystem II enriched membranes from spinach reveals the presence of at least four different populations of PSII core complex. The four bands are neither equally populated nor equally active in electron transport from diphenylcarbazide to 2,6-dichlorophenolindophenol. Under conditions of a low and high phosphorylation level a change in the relative populations of the PSII core isoforms is observed and the amount of radiolabelled phosphate incorporated into the four types of complexes is correlated to the value of their isoelectric point suggesting that the origin of the heterogeneity evidenced in vitro is at least partially due to different levels of light-induced phosphorylation. A 9 KD phosphoprotein, previously described in PSII, is found in our core complex preparation at a concentration which decreases as the total phosphorylation level on D 1 D 2 polypeptides increases.

A 49-Kilodalton Phosphoprotein in the Drosophila Photoreceptor Is an Arrestin Homolog

The gene encoding the 49-kilodalton protein that undergoes light-induced phosphorylation in the Drosophila photoreceptor has been isolated and characterized. The encoded protein has 401 amino acid residues and a molecular mass of 44,972 daltons, and it shares approximately 42 percent amino acid sequence identity with arrestin (S-antigen), which has been proposed to quench the light-induced cascade of guanosine 3',5'-monophosphate hydrolysis in vertebrate photoreceptors. Unlike the 49-kilodalton protein, however, arrestin, which appears to bind to phosphorylated rhodopsin, has not itself been reported to undergo phosphorylation. In vitro, Ca2+ was the only agent found that would stimulate the phosphorylation of the 49-kilodalton protein. The phosphorylation of this arrestin-like protein in vivo may therefore be triggered by a Ca2+ signal that is likely to be regulated by light-activated phosphoinositide-specific phospholipase C.

Cyclic nucleotides and GTP analogues stimulate light-induced phosphorylation of octopus rhodopsin

Light-induced phosphorylation of octopus rhodopsin in microvillar membrane was shown to be stimulated by cyclic nucleotides in contrast to vertebrate rhodopsin kinase. Non-hydrolyzable GTP analogues, GTPγS and GppNHp, greatly enhanced the light-induced phosphorylation of octopus rhodopsin, but the non-hydrolyzable GDP analogue, GDPβS, was not effective. These results suggest that rhodopsin A-kinase is involved in regulating the interaction between rhodopsin and G-protein in octopus photoreceptors. Rhodopsin; Rhodopsin kinase; Kinase, A-; Protein, G-; Photoreceptor; (Octopus, Invertebrate)

Dynamics of Photosystem II and Its Light Harvesting System in Response to Light Changes in the Halotolerant Alga Dunaliella salina

A photosystem two (PSII) core complex consisting of five major polypeptides (47, 40, 32, 30, and 10 kilodaltons) and a light harvesting chlorophyll a/b complex (LHC-2) have been isolated from the halotolerant alga Dunaliella salina. The chlorophyll and polypeptide composition of both complexes were compared in illuminated and dark-adapted cultures. Dark adaptation is accompanied by a decrease in the chlorophyll a to chlorophyll b (Chl a/Chl b) ratio of intact thylakoids without any change in total chlorophyll. These changes occur with a half-time of 3 hours and are reversed upon reillumination. Analyses of PSII enriched membrane fragments suggest that the decrease in the Chl a/Chl b is due partly to an increase in the Chl b content of LHC-2 and partly to changes in the relative levels of the two complexes. Apparently during dark adaptation there is: (a) a net synthesis of chlorophyll b, (b) removal of PSII core complexes resulting in a 2-fold drop in the PSII cores to LHC-2 chlorophyll ratio. These changes should dramatically increase the light harvesting capacity of the remaining PSII reaction centers. Presumably this adjustment of antenna size and composition is a physiological mechanism necessary for responding to shade conditions. Also detected, using (32)P, are light-induced phosphorylation of the LHC-2 (consistent with the ability to undergo State transitions) and of the 40 and 30 kilodalton subunits of the PSII core complex. These observations indicate that additional mechanisms may also exist to help optimize the interception of quanta during rapid changes in illumination conditions.

Light stimulates phosphorylation of two large membrane proteins in frog photoreceptors.

By photoactivating rhodopsin, light indirectly initiates a series of biochemical reactions within photoreceptors as part of the visual process. I herein report that one of the light-stimulated reactions in bullfrog photoreceptors is the phosphorylation of two previously unreported proteins (220 and 240 kDa). Their phosphorylation by endogenous kinase(s) is readily observed in freshly isolated, fragmented rods. On subcellular fractionation, the labeled proteins copurify with the membranes of the outer segments, from which they cannot be extracted with low ionic strength. They appear to be integral membrane proteins of the disk or plasma membranes. Their light-induced phosphorylation is also observed in intact receptors when excised frog retinas are incubated under in vivo conditions with 32PO4. Thus, appropriate kinase(s) is (are) present within outer segments and presumably is (are) the one(s) responsible for phosphorylation in fragmented cells. In the presence of adenosine 5'-(gamma-[35S]thiotriphosphate) [( 35S] ATP-gamma-S), light can also stimulate thiophosphorylation, leading to preferential labeling of the 220-kDa protein. On the basis of four criteria (electroporetic mobility, membrane location, binding of concanavalin A, and mobility shifts with SH oxidation), the 220-kDa protein appears to correspond to the membrane protein previously identified at the rims of rod disks [Papermaster, D.S., Schneider, B.G., Zorn, M.A. & Kraehenbuhl, J.P. (1978) J. Cell Biol. 78, 415-425]. Identity of the other substrate protein is unknown. When fragmented cells are illuminated with a flash of 1-ms duration, the half-time for phosphorylation is about 1 min with ATP at 0.1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of monoclonal antibody binding and proteolysis by light-induced phosphorylation of rhodopsin.

Light-induced phosphorylation of rhodopsin in bovine rod outer segment disk membranes inhibits the binding of three carboxyl-terminal-specific anti-rhodopsin antibodies and the cleavage of the carboxyl-terminal region of rhodopsin by trypsin and Staphylococcus aureus V-8 protease. Two monoclonal antibodies, rho 3A6 and rho 1C5, which previously have been shown to preferentially bind to the 8'-12' and the 9'-18' carboxyl-terminal segments of rhodopsin, respectively, are both highly sensitive to phosphorylation. When an average of one phosphate is incorporated per rhodopsin, the binding reactivity of rhodopsin for these antibodies decreases to 30% that of nonphosphorylated rhodopsin as measured in radioimmune competition assays. Reactivity of the rho 1D4 antibody whose primary binding site is localized in the 1'-8' C-terminal segment of rhodopsin is unaffected at this level of phosphorylation but decreases to 30% when three phosphates on average are incorporated per rhodopsin. Direct binding studies using 125I-labeled antibodies indicate that phosphorylation of rhodopsin decreases the maximum extent of rho 3A6 and rho 1C5 binding to rhodopsin. For rho 1D4, the maximum extent of binding is unaffected by phosphorylation, but the dissociation constant is increased by 10-fold. Phosphorylation of rhodopsin also inhibits cleavage of the 1'-9' and 1'-7' carboxyl-terminal peptides by trypsin and S. aureus V-8 protease, respectively. When an average of one phosphate per rhodopsin is incorporated, cleavage decreases to 40% that of nonphosphorylated rhodopsin as measured by high-performance liquid chromatography. Phosphorylation of rhodopsin had no effect on S. aureus cleavage of rhodopsin into the F1 (Mr 25 000) and F2 (Mr 12 000) fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Light-induced phosphorylation of retina-specific polypeptides of Drosophila in vivo.

A moderate light stimulus induced isoelectric point (pI) changes in three classes of retina-specific polypeptides (80, 49, and 39 kilodaltons) of Drosophila in vivo. When inorganic phosphate labeled with phosphorus-32 was fed to flies, the radioactive label was incorporated into these polypeptides during the pI changes, indicating light-induced phosphorylation of the polypeptides. A 1-millisecond flash induced a detectable amount of phosphorylation in the 80- and 49-kilodalton polypeptides within 3 seconds. These results, and our previous results with norpA mutants, suggest that phosphorylation of these two polypeptides may be involved in some early stages of photoreceptor excitation or its modulation.

Reversible phosphorylation of opsin induced by irradiation of blowfly retinae

Light-induced phosphorylation and dephosphorylation of the visual pigment protein, opsin, was investigated in isolated retinae of the blowfly making use of the fact that photon capture by rhodopsin leads to the formation of a thermostable metarhodopsin. Retinae were exposed, in the presence of exogenous32P-orthophosphate, to an intense blue light which initiated the phosphorylation of opsin (half-time about 5 min at 25 °C). Subsequent exposure of the retina to red light converted all the metarhodopsin present into rhodopsin and triggered a relatively rapid dephosphorylation of rhodopsin (half-time less than 20 s). It is proposed that the phosphorylated forms of rhodopsin and metarhodopsin represent inactive states of the pigment, i.e. phosphorylated metarhodopsin does not initiate reactions leading to the excitation of the photoreceptor cell and phosphorylated rhodopsin cannot be converted into physiologically active metarhodopsin without first being dephosphorylated.

Modification by immobilization of the microenvironment of chromatophores of Rhodopseudomonas capsulata. The influence on light-induced ADP phosphorylation coupled to cyclic electron transport.

Rhodopseudomonas capsulata chromatophores were immobilized with a co-crosslinking method. Immobilization was used as a tool for a defined modification of the chromatophore environment to study ATP production over a long period of time. The light-induced phosphorylation of ADP as a function of time was studied with chromatophores under different conditions: (a) native chromatophores with and without the hexokinase ATP-trapping system; (b) immobilized chromatophores without hexokinase, with the enzyme added in the bulk solution and with the enzyme co-immobilized in the matrix. The overall amount of ATP produced as a function of ADP concentration was studied for native and immobilized chromatophores. The global phosphorylation performed was also studied as a function of the amount of biological material used. The results can be explained by an effect of the ATP/ADP ratio. The results given by the immobilization show that the important point is not the ATP/ADP ratio in the bulk solution but the ratio value in the microenvironment of the chromatophore itself.

Light-induced phosphorylation of rhodopsin in cattle photoreceptor membranes: substrate activation and inactivation.

Light-induced phosphorylation of rhodopsin in cattle photoreceptor membranes: substrate activation and inactivation.