Phosphatidylinositol stimulates phosphorylation of protein components I and II in rod outer segments of frog photoreceptors

We studied the effect of phosphoinositides on the phosphorylation of endogenous proteins in the soluble fraction of the frog photoreceptor rod outer segments (ROS). Phosphatidylinositol (PI) stimulated the phosphorylation of two low molecular weight proteins, components I and II (12 and 11 kDa) which are known to be the preferential substrates of the cyclic GMP (cGMP)-dependent protein kinase in the ROS. Polyphosphoinositides (PPI) specifically inhibited the PI-dependent phosphorylation of these two components. On the other hand, PPI stimulated the phosphorylation of 38, 48 and 52 kDa proteins in the absence of PI. These data suggest that PI and PPI may function in the ROS by regulating the phosphorylation of some enzymes or regulator proteins in the transduction mechanism in the ROS.     

Vanadate stimulates tyrosine phosphorylation of two proteins in Raji human lymphoblastoid cell membranes

A membrane fraction from Raji human lymphoblastoid cells exhibited tyrosine-specific kinase activity. Vanadate increased tyrosine phosphorylation up to 5-fold; serine and threonine phosphorylation were unchanged. The stimulation was detectable within 15 s at 0 degrees C and at concentrations of vanadate (0.3 and 1.0 microM) present in normal tissues and blood. The tyrosine phosphorylation of two substrates, M1 61 000 and 55 000, was dependent upon vanadate and incorporation into these substrates represented the majority of the vanadate-sensitive tyrosine phosphorylation.     

The phosphorylation and isolation of two erythrocyte membrane proteins in vitro

Two proteins of bovine erythrocyte ghost membrane have been phosphorylated with γ-³²P-ATP and isolated by SDS polyacrylamide gel electrophoresis. One of the two proteins (MW 98,000) has been identified here as the phosphorylated intermediate of the Na⁺ + K⁺ activated ATPase. The other phosphorylated protein (MW 220,000) is apparently unrelated to the Na-K ATPase, but may be involved in other energy requiring membrane processes.     

Effect of barium and tetraethylammonium on membrane circulation in frog retinal photoreceptors

We studied the influence of altered ionic conditions on the recycling of synaptic vesicle membrane in frog retinal photoreceptors using horseradish peroxidase to monitor synaptic activity and trace the fate of internalized membrane. The addition of 1.2 mM barium or 20 mM tetraethylammonium to isolated retinas maintained in Ringer's solution, changes the usual balance of membrane circulation in the rod cells; the cone cells are much less affected. Retrieval of synaptic vesicle membrane in the rods, which normally regenerates small vesicles, becomes mediated predominantly by large sacs and vacuoles ("cisternae"). Because these cisternae can be labeled with peroxidase, they appear to arise from endocytized membrane. Morphometric analysis suggests strongly that the cisternae are formed of circulating synaptic vesicle membrane. The effects of barium and tetraethylammonium can be inhibited by high extracellular potassium, by high intensity light, and by 5 mM cobalt. They seem likely to depend on potassium channels, though additional more complex mediation may also be involved. The alterations in membrane retrieval that we find are of interest in terms of the multiple pathways of membrane cycling now being uncovered. They open potential experimental approaches to the controls of this circulation. In addition, the findings extend our previous ones demonstrating that rod cells and cone cells differ in their responses to divalent cations in ways that seem likely to be of physiological importance.     

Chloroform markedly stimulates the phosphorylation of myelin basic proteins

We have investigated the effect of chloroform on the phosphorylation of myelin basic proteins because tumor-promoting agents such as phorbol esters and chloroform are known to enhance the activity of protein kinase C. We report that the presence of chloroform, at a concentration known to enhance protein kinase C activity, stimulated the phosphorylation of myelin basic proteins 15-17 fold over control conditions. The phosphorylation of a 50 kiloDalton myelin protein was also stimulated but to a lesser extent. The concentration of chloroform required for the maximal phosphorylation of myelin basic proteins and the 50 kiloDalton protein was approximately 2% (v/v).     

Interleukin 2 stimulates tyrosine phosphorylation in T cell membrane fractions

Interleukin 2 is a growth factor secreted by T lymphocytes upon antigenic stimulation and inducing the proliferation of T cells bearing at their surface the heterodimeric high-affinity form of its receptor. No enzymatic function has so far been demonstrated in the receptor subunits. In an attempt to elucidate the biochemical pathway of signal transduction, we investigated the capacity of interleukin 2 to modulate tyrosine phosphorylation in T cell membranes. Membrane-rich fractions from T cells were tested for their ability to phosphorylate tyrosine in the presence or absence of added recombinant interleukin 2. Using as substrate a synthetic polymer of glutamic acid and tyrosine, we demonstrated a 3-4-fold stimulation of tyrosine phosphorylation in the presence of interleukin 2; this stimulating effect appeared to be well correlated with interleukin 2 function since (a) it was not observed in insensitive cells, (b) it required the presence of the high-affinity form of the receptor and (c) it was dose-dependent. Confirmatory results were obtained by phosphorylating membrane-rich fractions with [gamma-32P]ATP and by analysing the resulting phosphoproteins: only in fractions from cells with the high-affinity form of the receptor were several membrane proteins specifically phosphorylated on tyrosine residues in response to interleukin 2. At least two proteins of 115 and 58 kDa were consistently hyperphosphorylated on tyrosine in an interleukin-2-dependent manner. This stimulation was strongly dependent on the presence of the protein tyrosine phosphatase inhibitor, sodium orthovanadate. Thus, we propose that interleukin 2 enhances tyrosine phosphorylation by stimulating a tyrosine kinase activity. The nature of the enzyme involved remains to be determined.     

Light-induced decreases in cGMP concentration precede changes in membrane permeability in frog rod photoreceptors

This study examines whether changes in cGMP concentration initiated by illumination of frog rod photoreceptors occur rapidly enough to implicate cGMP as an intermediate between rhodopsin activation in the disc membrane and permeability changes in the plasma membrane. Previous studies using whole retinas or isolated outer segments have provided conflicting evidence on the role of cGMP in the initial events of phototransduction. The rod photoreceptor preparation employed in this work consists of purified suspensions of outer segments still attached to the mitochondria-rich ellipsoid portion of the inner segment. These photoreceptors are known to retain normal electrophysiological responses to illumination and have cGMP levels comparable to those measured in the intact retina. When examined under several different conditions, changes in cGMP concentrations were found to occur as rapidly or more rapidly than the suppression of the membrane dark current. Subsecond changes in cGMP concentration were analyzed with a rapid quench apparatus and confirmed by comparison with a rapid freezing technique. In a 1 mM Ca2+ Ringer's solution, cGMP levels decrease to 65% of their final extent within 200 ms after bright illumination; changes in membrane dark current follow a similar time course. When the light intensity is decreased to 8000 rhodopsins bleached per rod per s, the light-induced cGMP decrease is completed within 50 ms, with 7 X 10(5) cGMP molecules hydrolyzed per rhodopsin bleached. During this time the dark current has not yet begun to change. Thus, under physiological conditions it is clear that changes in cGMP concentration precede permeability changes at the plasma membrane. The correlation of rapid changes in cGMP levels with changes in membrane current leave open the possibility that changes in cGMP concentration may be an obligatory step in the reaction sequence linking rhodopsin activation by light and the resultant decrease in sodium permeability of the plasma membrane.     

Insulin stimulates the phosphorylation level of v-Ha-ras protein in membrane fraction

Insulin was found to stimulate the phosphorylation of the 21,000-dalton protein encoded by the ras oncogene of Harvey murine sarcoma virus in membrane fraction both in vivo and in vitro. When the human ras proteins expressed in E. coli were reconstituted with purified human insulin receptor, GTPase activity of normal or its mutated oncogenic ras protein was not stimulated by the addition of insulin. Likewise, tyrosine kinase activity or insulin binding capacity of the receptor was not influenced when assayed in the presence of the ras proteins. These results suggest that ras proteins may be coupled with the insulin receptor system through some unidentified membrane factors.     

Endogenous phosphorylation of platelet membrane proteins.

The characteristics of the phosphorylating activity of platelet membranes have been studied. Plasma membranes of human platelets isolated by the glycerol lysis technique were shown to incorporate significant amounts of [³²P]phosphate into specific membrane proteins. This activity was only partially cyclic 3′:5′-monophosphate (cyclic AMP)-dependent but had most of the other characteristics of protein kinases derived from other sources. Maximal stimulation of endogenous phosphorylation was obtained at 1 × 10^?7, m cyclic AMP and exceeded by approximately 30% the [³²P]phosphate incorporation in the absence of this cyclic nucleotide. The platelet membrane protein kinase was able to phosphorylate exogenous proteins, e.g., histone, fibrinogen etc., as well as endogenous membrane proteins. The latter solubilized by sodium dodecyl sulfate and separated by dodecyl sulfate-polyacrylamide gel electrophoresis incorporated [³²P]phosphate into three polypeptides of apparent molecular weights 52,000, 31,000, and 20,000. The phosphorylation of the polypeptide of molecular weight 52,000 was cyclic AMP-dependent.     

The phosphorylation of coated membrane proteins in intact neurons

To complement studies that have demonstrated the prominent phosphorylation of a 50-kD coated vesicle polypeptide in vitro, we have evaluated the phosphorylation of coated membrane proteins in intact cells. A co-assembly assay has been devised in which extracts of cultured rat sympathetic neurons labeled with [32P]-Pi were combined with unlabeled carrier bovine brain coat proteins and reassembled coat structures were isolated by gradient centrifugation. Two groups of phosphorylated polypeptides, of 100-110 kD (pp100-110) and 155 kD (pp155) apparent molecular mass, were incorporated into reassembled coats. The neuronal pp100-110 are structurally and functionally related to the 100-110-kD component of the bovine brain assembly protein (AP), a protein complex that also contains 50-kD and 16.5-kD components and is characterized by its ability to promote the reassembly of clathrin coat structures under physiological conditions of pH and ionic strength (Zaremba, S. and J. H. Keen, 1983, J. Cell Biol., 97:1337-1348). The neuronal pp155 detected in reassembled coat structures was readily observable in total extracts of [32P]-Pi-labeled neurons dissolved in SDS-containing buffer. A bovine brain counterpart to the neuronal pp155 was also observed when brain coated vesicles were subjected to two-dimensional gel electrophoresis. Phosphoserine was the predominant phosphoaminoacid found in both the pp100 and pp155. A structural and functional counterpart to the 50-kD brain assembly polypeptide (AP50) was also identified in these neurons. Although the brain AP50 is prominently phosphorylated by an endogenous protein kinase in isolated coated vesicle preparations, the neuronal AP50 was not detectably phosphorylated in intact cells as assessed by two-dimensional non-equilibrium pH gradient gel electrophoresis of labeled cells dissolved directly in SDS-containing buffers. These results demonstrate that the bovine brain assembly polypeptides of 50 kD and 100-110 kD that we have previously described, as well as a novel 155-kD polypeptide reported here, have structural and functional counterparts in cultured neurons. They also indicate that phosphorylation of the 100-110-kD AP may be involved in the regulation of coated membrane structure and function. The extent of phosphorylation of the AP50 in intact cells and in isolated coated vesicles is strikingly different: it has been suggested that the latter process reflects an autophosphorylation reaction (Campbell C., J. Squicciarini, M. Shia, P. F. Pilch, and R. E. Fine, 1984, Biochemistry, 23:4420-4426).(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytochemical analysis of oligosaccharide processing in frog photoreceptors

Summary Lectin cytochemistry, together with exoglycosidase enzyme digestion, has been used to characterize partially glycoconjugates of several intracellular compartments in frog photoreceptors. In order to obtain uniform access of reagents to all intracellular compartments, the experiments were performed directly on semi-thin sections ofXenopus laevis retinal tissue embedded in a hydrophilic plastic resin. In the rod, the major photoreceptor intracellular binding sites for wheat germ agglutinin (WGA) are the outer segment, the Golgi complex, and other inner segment organelles which are probably involved in the transport of glycoconjugates from the Golgi complex to the outer segment. In addition, shed outer segment tips (phagosomes) are uniformly labelled with WGA. The WGA-binding sites of the outer segment and of the presumed transport organelles are resistant to neuraminidase digestion. This is consistent with the possibility that glycoconjugates (primarily opsin) are transported from the Golgi complex to the outer segment without further oligosaccharide processing. Specific staining of rod outer segments and of phagosomes is also obtained with theN-acetylglucosamine-specific lectin, succinyl-WGA (S-WGA). Outer segments and phagosomes stain the same with WGA, S-WGA and a variety of other lectins tested suggesting that no major post-Golgi oligosaccharide processing accompanies the shedding-phagocytosis event. Concanavalin A (Con A) staining of intracellular sites in rod inner segments reveals a striking difference compared to WGA staining in that the Con A binding sites are concentrated in the photoreceptor axon and presynaptic terminal. These results, and results from previous studies, indicate that the photoreceptor may utilize different mechanisms of oligosaccharide processing from the level of a single Golgi complex to the opposite ends of this cell. Furthermore, those glycoconjugates destined for the presynaptic terminal may undergo post-Golgi processing at or near their sites of insertion into the presynaptic plasma membrane.     

1,25-dihydroxyvitamin D3 stimulates the phosphorylation of two acidic membrane proteins of 42,000 and 48,000 daltons in rat colonocytes: An effect modulated by vitamin D status

Our laboratory has recently demonstrated that 1,25-dihydroxyvitamin D₃(1,25(OH)₂D₃) rapidly stimulated membrane polyphosphoinositide breakdown and increased intracellular calcium, as well as activated protein kinase C (PKC) in vitamin D-sufficient rat colonocytes. These effects of 1,25(OH)₂D₃ were, however, lost in vitamin D-insufficient rats and restored by the in vivo repletion of 1,25(OH)₂D₃. In the present studies we have examined the ability of 1,25(OH)₂D₃ to stimulate the phosphorylation of colonic membrane proteins in intact D-sufficient cells. In addition, we investigated the effects of vitamin D status on the phosphorylation of these membrane proteins in broken cell preparations. These studies demonstrated that 1,25(OH)₂D₃ increased the phosphorylation of at least two colonic membrane proteins with apparent molecular weights of 42,000 (pp42) and 48,000 (pp48) in intact cells of vitamin D-sufficient rats. Moreover, in vitamin D-sufficient rats, treatment of colonocytes with 1,25(OH)₂D₃ or 12-Otertradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, significantly increased the phosphorylation of pp42 and pp48 in broken cell preparations. The kinetics of these phosphorylations in response to 1,25(OH)₂D₃ were both rapid and transient. In addition, PKC_19–36, a specific PKC inhibitor, decreased the phosphorylation of pp42 and pp48, whereas okadaic acid (OA), a type 1 and 2A protein phosphatase inhibitor, further augmented their phosphorylation in response to 1,25(OH)₂D₃. The isoelectric points of pp42 and pp48 were 5.79 and 5.97, respectively, and both were predominantly phosphorylated on threonine residues. In contrast to our findings in colonocytes from vitamin D-sufficient animals, basal phosphorylation of pp42 and pp48 were increased in membranes prepared from vitamin D-insufficient rats. Moreover, these phosphorylations failed to change in response to 1,25(OH)₂D₃-treatment of colonocytes from vitamin D-insufficient rats. The basal phosphorylation of each of these proteins was restored to control levels, as was their ability to respond to the direct addition of 1,25(OH)₂D₃ following the in vivo repletion of vitamin D-insufficient rats with this secosteroid. In summary, we have identified two acidic membrane proteins from rat colonocytes that are phosphorylated in both intact and broken cell preparations in response to 1,25(OH)₂D₃ treatment, an event modulated by vitamin D status and mediated, at least in part, by PKC. © 1995 Wiley-Liss, Inc.     

Epidermal growth factor stimulates tyrosine phosphorylation of specific proteins in permeabilized human fibroblasts

We have investigated the epidermal growth factor (EGF)-stimulated tyrosine-specific protein kinase activity in quiescent cultures of diploid human fibroblasts that have a well characterized mitogenic response to EGF. We developed a method of permeabilizing cells with digitonin or other agents that permitted the rapid labeling of cellular proteins with exogenously added [gamma-32P]ATP while allowing only about 25% of marker cytosolic enzymes to escape from the cells. When phosphatases were inhibited with zinc and vanadate, EGF induced up to 8-fold stimulation of the incorporation of radioactivity from [gamma-32P]ATP into a 35-kDa band on sodium dodecyl sulfate gels. Alkali treatment of gels showed that EGF stimulated the phosphorylation of bands with apparent molecular masses of 170, 45, 35, 26, 22, and 21 kDa. Phosphoamino acid analysis was performed on the 170- and 35-kDa bands and revealed that the EGF-stimulated phosphorylation was on tyrosyl residues. The 35-kDa band was resolved into four spots by two-dimensional gel electrophoresis. The most acidic form was the most prominent and it was precipitated by an antiserum against a 35-kDa protein from A-431 cells; heretofore, this protein has only been reported to be phosphorylated in an EGF-dependent manner by A-431 membranes in vitro (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). This antiserum also precipitated a 35-kDa phospho-protein from extracts of intact [32P]orthophosphate-labeled fibroblasts which was phosphorylated on tyrosine in an EGF-dependent manner. None of the forms of the 35-kDa phosphoproteins labeled in permeabilized cells were immunologically related to the 34-kDa protein that is a substrate for the tyrosyl kinase encoded by Rous sarcoma virus. Other mitogens (serum, insulin, platelet-derived growth factor, and thrombin) did not detectably stimulate phosphorylation in permeabilized cells.     

Light and gtp effects on the turbidity of frog visual membrane suspensions

Summary The time course of turbidity changes of frog visual membranes, dependent on osmotic shocks, on light and on nucleotide substrates or effectors of enzyme activities, were measured as absorption changes in a rapid mixing stopped-flow spectrophotometer.As a result of studies on different preparations, it is concluded that light can cause both rapid (within 50 msec) and slow (within 90 sec) changes in the turbidity of visual membranes, not associated with permeability changes, and that they are affected by GTP or its analog guanyl-5-yl imidodiphosphate; however, the light and GTP effects are lost when a water soluble fraction containing the light-sensitive enzyme cGMP-phosphodiesterase, is removed from the rod outer segments membranes.It is suggested that the fast light and GTP-sensitive response is related to the activation of cGMP-phosphodiesterase.Abbreviation used ROS rod outer segments     

Light as a signal influencing the phosphorylation status of plant proteins

The phosphorylation-status of a number of plant enzymes has been shown to be altered in response to light. Phosphoenolpyruvate carboxylase is phosphorylated (more active) in C₄ plants in the light but CAM phosphoenolpyruvate carboxylase is phosphorylated (more active) in the dark. C₄ plant pyruvate, Pi dikinase is dephosphorylated (activated) in the light and sucrose phosphate synthase is less phosphorylated (more active) in the light. The mitochondrial pyruvate dehydrogenase is inactivated (phosphorylated) in the light. The reversal of these events occurs in the dark or when photosynthesis is inhibited. Phytochrome and blue light receptors also alter the phosphorylation-status of proteins. The evidence is rapidly increasing in support of signal transduction networks in plants that involve light reception.     

Histamine stimulates phosphorylation of adherens junction proteins and alters their link to vimentin

Histamine increases microvascular permeability by creating small transitory (100-400 nm) gaps between adjacent endothelial cells at sites of vascular endothelial (VE)-cadherin-based adhesion. We examined the effects of histamine on the proteins within the VE-cadherin-based adherens junction in primary human umbilical vein endothelial cells. VE-cadherin is linked not only by beta- and alpha-catenin to cortical actin but also by gamma-catenin to the intermediate filament vimentin. In mature human umbilical vein cultures, the VE-cadherin immunoprecipitate contained equivalent amounts of alpha- and beta-catenin, 130% as much beta- as gamma-catenin, and 50% as much actin as vimentin. Within 60 s, histamine decreased the fraction of VE-cadherin in the insoluble portion of the cell lysate by 35 +/- 1.5%. At the same time, histamine decreased the amount of vimentin that immunoprecipitated with VE-cadherin by 50 +/- 6%. Histamine did not affect the amount of actin or the amount of alpha-, beta-, or gamma-catenin that immunoprecipitated with VE-cadherin. Within 60 s, histamine simulated a doubling in the phosphorylation of VE-cadherin and beta- and gamma-catenin. The VE-cadherin immunoprecipitate contained kinase activity that phosphorylated VE-cadherin and gamma-catenin in vitro.     

Effects of kinetin on phosphorylation of leaf membrane proteins.

Isolated Chinese cabbage leaf membranes were phosphorylated by membrane-associated protein kinase(s) in the presence or [gamma-32P]ATP. Membrane-associated 32P radioactivity appeared to be bound to membrane proteins. Both smooth cell membranes and chloroplast lamellae reacted with ATP. Phosphorylation of the membranes was inhibited by Ca2+ and partially inhibited by kinetin or 6-benzyladenine. The possibility that cytokinin effects on membrane phosphorylation might increase ion availability was investigated in vivo. It was found that Ca2+ could substitute for kinetin in the leaf disc expansion assay.