Absence Of Phosphorylation Research Articles

Spatial and temporal phosphorylation of a transcriptional activator regulates pole-specific gene expression in Caulobacter.

Polar localization of proteins in the Caulobacter predivisional cell results in the formation of two distinct progeny cells, a motile swarmer cell and a sessile stalked cell. The transcription of several flagellar promoters is localized to the swarmer pole of the predivisional cell. We present evidence that the product of the flbD gene is the transcriptional activator of these promoters. We show that FlbD is distributed in all cell types and in both poles of the predivisional cell. We also demonstrate that FlbD can be phosphorylated, and that a FlbD kinase activity is under cell cycle control. Cells expressing a FlbD mutant that should activate transcription in the absence of phosphorylation, exhibited an alteration in the temporal pattern of flagellin transcription. Furthermore, predivisional cells expressing the mutant FlbD failed to polarly localize flagellin synthesis. We propose that the phosphorylation of FlbD is restricted to the swarmer compartment of the predivisional cell, and serves as the control point for regulating the spatial transcription of flagellar promoters.

Activating and inhibitory mutations in the regulatory domain of CheB, the methylesterase in bacterial chemotaxis.

In the chemotaxis system of Escherichia coli, CheB promotes sensory adaptation by interacting with the chemotaxis receptor-transducer proteins to catalyze removal of their gamma-glutamyl methyl ester groups. CheB is comprised of two functional domains; the C-terminal domain contains the methylesterase active site, and the N-terminal domain regulates the activity of this active site. The chemotaxis system controls CheB methylesterase activity via a mechanism involving phosphorylation of the CheB regulatory domain by the chemotaxis protein kinase CheA. To further explore the communication between the regulatory and methylesterase domains of CheB, I generated mutations in the CheB regulatory domain that affect methylesterase activity in vitro. Three of these mutations (D11K, E58K, and E91K) caused increased methylesterase activity in the absence of phosphorylation, and several other mutations (R42H, R73H, and K107R) caused decreased methylesterase activity in the purified proteins. Several of these mutations (D10N, D11K, R42H, E58K, and K107R) also affected the phosphorylation biochemistry of CheB by reducing the rate of CheA-mediated phosphorylation of CheB and/or by decreasing the autodephosphorylation rate of CheB. In addition, all of these mutations diminished the ability of excess CheA to inhibit CheB methylesterase activity. The locations of these mutations in the deduced three-dimensional structure of the CheB N-terminal domain indicate that the region of the protein surrounding the putative phosphorylation site plays important roles in its interaction with the CheB C-terminal domain as well as in its interactions with CheA.

Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates.

The p34cdc2 protein kinase is a conserved regulator of the eukaryotic cell cycle. Here we show that residues Thr14 and Tyr15 of mouse p34cdc2 become phosphorylated as mouse fibroblasts proceed through the cell cycle. We have mutated these residues and measured protein kinase activity of the p34cdc2 variants in a Xenopus egg extract. Phosphorylation of residues 14 and 15, which lie within the presumptive ATP-binding region of p34cdc2, normally restrains the protein kinase until it is specifically dephosphorylated and activated at the G2/M transition. Regulation by dephosphorylation of Tyr15 is conserved from fission yeast to mammals, while an extra level of regulation of mammalian p34cdc2 involves Thr14 dephosphorylation. In the absence of phosphorylation on these two residues, the kinase still requires cyclin B protein for its activation. Inhibition of DNA synthesis inhibits activation of wild-type p34cdc2 in the Xenopus system, but a mutant which cannot be phosphorylated at residues 14 and 15 escapes this inhibition, suggesting that these phosphorylation events form part of the pathway linking completion of DNA replication to initiation of mitosis.

CAMP-Dependent protein kinase inhibits the chloride conductance in apical membrane vesicles of hunman placenta

The role of adenosine 3',5'-monophosphate (cAMP) dependent protein kinase (PK-A) on the Cl- conductance has been studied in the apical membrane vesicles purified from the chorionic villi of human placenta. In order to phosphorylate the cytosolic side of the membranes, vesicles have been hypotonically lysed, loaded with 100 nM catalytic subunit of PK-A purified from human placenta and 1 mM of the phosphatase resistant adenosine 5'-thiotriphosphate (ATP-gamma-S) and resealed. Cl- conductance has been measured by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) at 23 degrees C with membrane potential clamped at 0 mV. The actual volume of the resealed vesicles was measured in each experiment by trapping an impermeable radioactive molecule ([14C]-sucrose) and included in each Cl- flux calculation. In 19 independent experiments, the mean Cl- conductance in placental membranes in the absence of phosphorylation was 3.67 +/- 3.18 whereas with the addition of PK-A and ATP-gamma-S it was 1.97 +/- 1.75 nmol.sec-1. (mg protein)-1 (mean +/- SD). PK-A dependent phosphorylation reduced the Cl- conductance in 14/19 experiments. The same protocol applied to the apical membranes of bovine trachea, where PK-A is known to activate the Cl- channels, confirmed that the PK-A dependent phosphorylation increased in Cl- conductance in 11/13 experiments, from 1.01 +/- 0.61 to 1.85 +/- 0.99 nmol.sec-1.(mg protein)-1 (mean +/- SD). These studies indicate that the PK-A dependent phosphorylation inhibits one or more Cl- channel(s) of the apical membranes of human placenta.

Negative and positive regulation by transcription factor cAMP response element-binding protein is modulated by phosphorylation.

We have shown that the transcriptional activity of the protooncogene jun (c-jun) promoter is repressed by a transcription factor, the cAMP response element-binding protein (CREB). This repression can be alleviated when CREB is phosphorylated by the catalytic subunit of protein kinase A. Repression cannot be alleviated by a mutant CREB deficient in the protein kinase A phosphorylation site (M1 CREB Ser-133----Ala), suggesting that phosphorylation of CREB at this site is essential for the relief of repression. Repression by CREB requires its binding to the c-jun promoter. In NIH 3T3 cells stably expressing CREB, c-jun is no longer induced by serum, but this repression can be relieved by treatment of the cells with forskolin, an agonist of the adenylate cyclase pathway. Thus, CREB has a dual function, that of a repressor in the absence of phosphorylation and an activator when phosphorylated by protein kinase A.

Inhibition of Conformational Change in Smooth Muscle Myosin by a Monoclonal Antibody against the 17-kDa Light Chain

Monoclonal antibodies against gizzard smooth muscle myosin were generated and characterized. One of these antibodies, designated MM-2, recognized the 17-kDa light chain and modulated the ATPase activities and hydrodynamic properties of smooth muscle myosin. Rotary shadowing electron microscopy showed that MM-2 binds 51 (+/- 25) A from the head-rod junction. The depression of Ca2+- and Mg2+-ATPase activities of myosin and Ca2+-ATPase activity of heavy meromyosin at low KCl concentration were abolished by MM-2. Viscosity measurement indicated that MM-2 inhibits the transition of 6 S myosin to 10 S myosin. While the rate of the production of subfragment-1 by papain proteolysis of 6 S myosin was inhibited by MM-2, the rate of proteolysis of the heavy chain of 10 S myosin was enhanced by MM-2 and reached the same rate as that of 6 S myosin plus MM-2. These results suggest that MM-2 inhibits the formation of 10 S myosin by binding to the 17-kDa light chain which is localized at the head-neck region of the myosin molecule. MM-2 increased the Vmax of actin-activated Mg2+-ATPase activities of both dephosphorylated myosin and dephosphorylated heavy meromyosin about 10- and 20-fold, respectively. MM-2 also activated the actin-activated Mg2+-ATPase activity of phosphorylated myosin at a low MgCl2 concentration and thus abolished the Mg2+-dependence of acto phosphorylated myosin ATPase activity. These results suggest that MM-2 inhibits the formation of 10 S myosin, and this results in the activation of actin-activated Mg2+-ATPase activity even in the absence of phosphorylation.

Phosphorylation of liver plasma membrane-bound calmodulin.

In highly purified rat liver plasma membrane preparations, membrane-bound calmodulin was phosphorylated by a membrane-bound protein kinase using [gamma-32P]ATP as phosphate donor. Maximum phosphorylation of calmodulin occurred in the absence of calcium ion, but was significantly decreased in its presence. Plasma membrane-bound calmodulin was identified by the following criteria: (i) extraction from the membrane by EGTA, (ii) stimulation of the activity of the Ca2+-calmodulin-dependent enzyme, (3':5'AMP)-phosphodiesterase, by the EGTA extract, and (iii) electrophoretic comigration of EGTA-extracted protein with standard bovine brain calmodulin, both in the presence and the absence of Ca2+. Phosphorylation of the plasma membrane-bound calmodulin was shown by electrophoretic comigration of the 32P-labelled molecule with bovine brain calmodulin, the absence of phosphorylation of this protein band in calmodulin-depleted membranes, and a Western blot of the phosphorylated band using a calmodulin antibody. Treatment of plasma membrane preparations with sheep anticalmodulin serum prevented the phosphorylation of the calmodulin band. Phosphocalmodulin, which could be partially extracted from the membrane by EGTA, comigrated with bovine brain calmodulin in polyacrylamide gel electrophoresis.

Phosphorylation of a ribosomal protein in invertebrate cells infected with ultraviolet-inactivated iridovirus type 6

Summary Chilo iridescent virus (CIV) infection induces a rapid shut-off of macromolecular synthesis in insect host cells. We have previously shown that CIV infection induced the appearance of new phosphoproteins in permissive cells of Choristoneura fumiferana (Cf-124) [15]. Among them, phosphorylation of a 31 Kd ribosomal protein (very similar to the S6 protein) was observed 3 h after infection of Cf-124 cells with UV-inactivated CIV. However, such phosphorylation could not be detected in cells infected native CIV in the same conditions. As previously postulated [15], these results could be interpreted either as an absence of phosphorylation of this ribosomal protein, or as a dephosphorylation step occurring very rapidly during the early stages of the replication cycle, in cells infected with native CIV, as reported for the infection of BHK21 cells by vaccinia virus [1].

Radiation inactivation of (Na + + K +)-ATPase a small target size for the K +-occluding mechanism

Radiation inactivation of partially purified (Na + + K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pig kidney outer medulla shows that the target size for Rb + occlusion by the enzyme (in the absence of phosphorylation) is much smaller than the target size for p- nitrophenyl phosphatase activity , which is itself smaller than the reported target size for (Na + + K +)-ATPase activity.

Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation.

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.

Conformational transitions between Na +-bound and K +-bound forms of (Na + + K +)-ATPase, studied with formycin nucleotides

1. 1. Fluorescence measurements have shown that formycin triphosphate (FTP) or formycin diphosphate (FDP) bound to (Na + + K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in Na +-containing media can be displaced by the following ions (listed in order of effectiveness): Tl +, K +, Rb +, NH 4 +, Cs +. 2. 2. The differences between the nucleotide affinities displayed by the enzyme in predominantly Na + and predominantly K + media in the absence of phosphorylation, are thought to reflect changes in enzyme conformation. These changes can therefore be monitored by observing the changes in fluorescence that accompany net binding or net release of formycin nucleotides. 3. 3. The transition from a K +-bound form (E 2 · (K)) to an Na +-bound form (E 1 · Na) is remarkably slow at low nucleotide concentrations, but is accelerated if the nucleotide concentration is increased. This suggests that the binding of nucleotide to a low-affinity site on E 2 · (K) accelerates its conversion to E 1 · Na; it supports the hypothesis that during the normal working of the pump, ATP, acting at a low affinity site, accelerates the conversion of dephosphoenzyme, newly formed by K +-catalysed hydrolysis of E 2P, to a form in which it can be phosphorylated in the presence of Na +. 4. 4. The rate of the reverse transformation, E 1 · Na to E 2 · (K), varies roughly linearly with the K + concentration up to the highest concentration at which the rate can be measured (15 mM). Since much lower concentrations of K + are sufficient to displace the equilibrium to the K-form, we suggest that the sequence of events is: (i) combination of K + with low affinity (probably internal) binding sites, followed by (ii) spontaneous conversion of the enzyme to a form, E 2 · (K), containing occluded K +. 5. 5. Mg 2+ or oligomycin slows the rate of conversion of E 1 · Na to E 2 · (K) but does not significantly affect the rate of conversion of E 2 · (K) to E 1 · Na. 6. 6. In the light of these and previous findings, we propose a model for the sodium pump in which conformational changes alternate with trans-phosphorylations, and the inward and outward fluxes of both Na + and K + each involve the transfer of a phorpharyl group as well as a change in conformation between E 1 and E 2 forms of the enzyme or phosphoenzyme.

In vitro phosphorylation of chicken erythrocyte histone by various protein kinases: Absence of phosphorylation from F1 histone

In spite of the presence of nucleus, genetic activity and mitosis are totally depressed in avian erythrocytes. If phosphorylation of histone is involved in such genetic depression, a comparative study of phosphorylation of avian erythrocyte histone can be expected to furnish information about the mechanism of gene control. The present study is the examination of susceptibility of chicken erythrocyte histone to histologically different (liver and muscle) and phylogenically different (avian, mammalian and ichthic) protein kinases. It was found that chicken erythrocyte F1 histone was phosphorylated not only by heterologous (rat and trout liver) but also by homologous (chicken liver and muscle) protein kinases. Addition of cAMP could not elicit phosphorylation of this histone, while phosphorylation of other histones was significantly enhanced by this drug. Avian erythrocyte-specific histone, F2c, was markedly phosphorylated not only by avian enzymes but also by mammalian enzyme. All the enzymes tested phosphorylated F2b histone. F3 histone was phosphorylated at least by avian and mammalian enzymes. F2a1 and F2a2 histones were poor substrate to all the enzymes tested.

Studies of oxidative energy deficiency: I. Achondroplasia in the rabbit

Defective oxidative energy formation with the absence of phosphorylation at the cytochrome oxidase region (site III) of the terminal respiratory system has been demonstrated to be present in isolated mitochondrial preparations from the livers of newborn achondroplastic rabbits. Normal-appearing littermates did not exhibit the defect. Terminal electron transport and oxidative activities of the terminal respiratory system as well as mitochondrial ATPase activity (coupling factor I) were similar in both normal and achondroplastic tissue preparations. No significant differences were detectable in the morphology of liver mitochondria from normal and achondroplastic littermates by electron microscopy examination.

Electron Transport and Photophosphorylation in Chloroplasts as a Function of the Electron Acceptor

Electron acceptors may be divided into three distinct classes on the basis of the nature of the electron transport and phosphorylation processes which accompany their reduction by illuminated chloroplasts. Class I acceptors are reduced slowly in the absence of phosphorylation. The addition of ADP and Pi usually increases the rate 2- to 3-fold and up to 1.3 molecules of ATP are formed for each pair of electrons transferred to the acceptor. Uncouplers such as methylamine increase the rate of reduction still further. Most of the electron acceptors heretofore employed in studies of noncyclic photophosphorylation (ferredoxin, ferricyanide, viologens, and flavins) belong to this class. All are ionic or otherwise polar substances with negligible lipid solubilities. Class II acceptors are reduced rapidly whether or not phosphorylation occurs and the addition of uncouplers often does not increase the rapid rate of reduction. Little or no ATP is formed. Substances such as phenolindophenols belonging to this class have dual functions, serving both as electron acceptors and as uncouplers. They are weak acids, lipid-soluble in their nonionized, protonated forms. Their uncoupling function is enhanced as the pH is lowered and the amount of lipid-soluble acid increases. Class III acceptors are also reduced rapidly whether or not phosphorylation occurs and uncouplers again cause little or no increase in the rate of reduction. However, when ADP and Pi are added in the absence of uncouplers a high rate of phosphorylation is observed. The phosphorylation associated with Class III acceptors is usually more rapid than is the phosphorylation associated with Class I acceptors, especially at suboptimal pH. Nevertheless, over a wide range of conditions, the efficiency of phosphorylation in terms of electrons transported (P:e2) is rather precisely half of the efficiency observed when Class I acceptors are reduced. All nonionic, lipid-soluble acceptors we have investigated belong to Class III. Thus the lipid-soluble p-benzoquinone is a Class III acceptor but the lipid-insoluble p-benzoquinone sulfonate is a Class I acceptor. From these observations we conclude that electrons are transported to Class I acceptors through two sites of phosphorylation whereas the transport of electrons to Class III acceptors utilizes only one of the sites. Presumably lipid-soluble acceptors have access to and accept electrons from carriers which normally transfer electrons between the two sites of phosphorylation.

Changes in Internal Hydrogen Ion Concentration Associated with Photophosphorylation in Intact and Sonically Treated Chloroplasts

Abstract By using different pH indicators it is possible to demonstrate, in the presence or absence of phosphorylation, the existence of two compartments in chloroplasts, one of which becomes more acidic and the other more basic during electron flow. The observed magnitude of the pH differential between these two compartments is over 1.5 pH units. Cations, such as NH4+, N-methylphenazonium methosulfate, and protonated neutral red, which compete with H+ ions for entry into chloroplasts, inhibit the development of pH gradients within chloroplasts. Diffusible anions, such as acetate and orthophosphate, also block formation of these internal pH gradients. All of the above inhibitors of formation of internal pH gradients also inhibit phosphorylation, in a parallel manner. Sonic treatment of chloroplasts causes progressive and parallel inhibition of phosphorylation and formation of internal pH gradients. These data support the concept that chloroplasts contain a proton-translocating oxidation-reduction chain and that the resultant efflux of H+ ions through chloroplast membranes supplies the energy for phosphorylation.

Structural basis for phosphorylation of adenosine congeners.

DURING the past few years the metabolism by mammalian cells of a number of natural and synthetic nucleosides structurally related to adenosine has been investigated in this1–5 and other laboratories6–10. In our studies with Ehrlich ascites cells, we have tried to establish a correlation between the structural variations and the utilization by cells of these nucleosides and have observed the following salient features: (a) In order to be phosphorylated to the 5′-triphosphate level, the adenosine analogue must have a primary amino group at the C-6 position of the purine moiety. Compounds that belong in this category are 3′-deoxyadenosine2,6, 3′-amino-3′-deoxyadenosine4 and 2-fluoroadenosine3. These three nucleosides were also found to be potent inhibitors of nucleic acid synthesis in Ehrlich ascites cells. Work in other laboratories has shown that 7-deazaadenosine7, xylosyladenine8, arabinosyladenine9 and the bases 2-azaadenine and 2,6-diaminopurine10 were also phosphorylated to 5′-triphosphate. 2,6-Diaminopurine is also metabolized to the triphosphate level by microbial cells11,12. Removal of the 6-amino group as in 3′-deoxynebularine resulted in complete absence of phosphorylation by ascites cells*4. (b) 6-Methylaminopurine ribonucleoside—an adenosine analogue with a 6-methyl-amino group—was metabolized to the monophosphate level only4. This was also found to be so for 6-methyl-aminopurine - 2′-deoxyribonucleoside, 6-methylaminopurine-3′-deoxyribonucleoside and 6-methylaminopurine5. (c) Replacement of the 6-amino group by 3′-deoxyadenosine with an ethylamino or dimethylamino group completely suppressed the capacity of the nucleoside for phosphorylation. This relationship was exemplified by 6-ethylaminopurine-3′-deoxyribonucleoside and 6-dimethylaminopurine-3′-deoxyribonucleoside4. Furthermore, 6-dimethylamino-3′-amino-3′-deoxyribonucleoside was also inactive, while its parent compound, as already mentioned, was readily phosphorylated. Nucleosides that were not phosphorylated had almost no effect on nucleic acid synthesis.

Interaction of ethanol and thyroxine on mitochondria

Thyroxine can stimulate mitochondrial swelling and uncouple oxidative phosphorylation. It was observed that ethanol inhibits both spontaneous and thyroxineinduced mitochrondrial swelling. The antiswelling activity of ethanol is manifested during mitochondrial respiration both in the presence and absence of phosphorylation. Ethanol was observed only to block thyroxine-induced swelling; it could not reverse the swelling. Ethanol also blocked Ca ++ and phosphate-induced swelling. Therefore, the antiswelling activity of ethanol is more generalized than that of serum albumin. It is concluded that the ethanol antagonism of mitochrondrial swelling produced by various agents is probably dependent on the fact that ethanol alone inhibits swelling; ethanol is therefore a pharmacological antagonist of agents that induce swelling. In addition to inhibiting swelling, ethanol also blocks uncoupling of oxidative phosphorylation by thyroxine and Ca ++ but not by salicylate. These observations were interpreted to indicate that ethanol affects oxidative phosphorylation only indirectly by blocking mitochondrial swelling. The observed effects of ethanol on mitochrondria are discussed in terms of reports on the ability of thyroid hormone to antagonize acute alcohol intoxication in man, and the ability of ethanol to block effects of Ca ++ on smooth muscle.