Abscisic acid-induced gene-expression requires the activity of protein(s) sensitive to the protein-tyrosine phosphatase inhibitor phenylarsine oxide

Abscisic acid-induced gene-expression requires the activity of protein(s) sensitive to the protein-tyrosine phosphatase inhibitor phenylarsine oxide. It is generally accepted that phosphorylation/dephosphorylation of proteins plays an important role in signal transduction cascades. evidence is now accumulating that for plants the same holds true. To study the role of phosphorylation in ABA signal transduction, we used six different compounds which were reported to inhibit phosphatase action. Three of these inhibitors: phenylarsine oxide (PAO), Calyculin A (CA) and Okadaic Acid (OA) appeared capable of inhibiting ABA-induced gene-expression. The same three inhibitors are shown to bring about hyperphosphorylation of two approximately 40 kDa proteins, present in the membrane-bound fraction of barley aleurone cells. The other three inhibitors had no visible effect on the phosphorylation status of the barley proteins. The hyperphosphorylation of the two 40 kDa proteins coincided with an increase of tyrosine-phosphorylation of two 40 kDa proteins with different pI, as determined with anti-phosphotyrosine antibodies.     

Heterologous expression of the cytochrome P450cam hydroxylase operon and the repressor gene of Pseudomonas putida in Escherichia coli

Abstract Cytoplasmic proteins from the antarctic psychrotrophic bacterium Pseudomonas syringae showed two phosphorylated proteins of molecular mass 66 kDa and 62 kDa. The phosphorylation of 66 kDa protein was enhanced in the presence of Triton X-100 solubilised membrane proteins at a higher temperature (30°C) only. Western blot analysis and phosphoamino acid analysis indicated that the 66 kDa protein is phosphorylated at a tyrosine residue. Surprisingly, sodium orthovanadate, which is a known phosphotyrosine phosphatase (PTPase) inhibitor, inhibited the phosphorylation of the protein. The possible importance of this tyrosine phosphorylated protein to growth at low temperature is suggested.     

Thiophosphorylation and phosphorylation of saponin-permeabilized cultured chromaffin cells

There is increasing evidence that phosphorylation of cellular proteins plays a role in the control of events surrounding secretion in neurons and chromaffin cells. In previous studies, we have used thiophosphorylation of cell proteins as a means of fixing cellular phosphorylation reactions in the phosphorylated state. Thiophosphorylation of permeabilized chromaffin cells with adenosine-5′-O-(3-thiotriphosphate) results in irreversible inhibition of secretion. Thiophosphate is incorporated primarily by two cellular proteins of 58 and 47 kDa. Calcium enhanced thiophosphorylation of the 47 kDa protein but not the 54 kDa protein. This pattern of thiophosphorylation differed markedly from that for phosphorylation under similar treatment conditions. The phosphoprotein composition of the cells depended upon the medium calcium and ATP concentration. In the absence of exogenous ATP, fewer phosphoproteins were seen in calcium stimulated cells than in unstimulated cells. Proteins labelled with ³²P or ³⁵S migrated to the same position on polyacrylamide gels containing sodium dodecyl sulfate. In the presence of exogenous ATP, ³²P incorporation was similar for both control and calcium-stimulated cells and was found primarily in a 64 kDa protein. Incorporation of [³²P]phosphate by calcium-stimulated cells was reduced to the same extent by pretreatment of the cells with either adenosine-5′-O-(3-thiotriphosphate) or ATP.The different electrophoretic banding patterns for thiophosphorylation and phosphorylation are likely due to the irreversibility of the thiophosphorylation reaction and reversibility of the phosphorylation reaction. The inability to turn over thiophosphate groups, in association with changes in secretion, may permit identification of those phosphoproteins that are putatively involved in secretion.     

Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the beta-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle.

When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5'-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5'-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5'-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed.     

Cyclic nucleotide- and calcium-independent phosphorylation of proteins in rat brain polyribosome: Effects of ACTH,spermine, and hemin

The incorporation of [-³²P]ATP into proteins of rat brain polyribosomes was studied in vitro. The effects of cyclic nucleotides, calcium, hemin, ACTH, GTP, and spermine were examined. The incorporation of phosphate into proteins increased with time and phosphatase activity was very low; thus, the extent of phosphorylation was predominantly a reflection of protein kinase activity. Phosphorylation of proteins was not sensitive to Ca²⁺ in the presence or absence of either calmodulin or phosphatidylserine. Phosphorylation was also unaffected by cyclic nucleotides in the absence of exogenous enzymes. However, addition of a cMAP-dependent protein kinase together with cAMP resulted in a stimulation of the incorporation of phosphate into 4 phosphoproteins (pp70, pp58, pp43, and pp32); phosphorylation of pp32 was completely dependent on the addition of the kinase. ACTH (1–24), (11–24), and spermine inhibited the endogenous phosphorylation of one protein band (pp30). The phosphorylation of this 30 kD band was also selectively increased by hemin (5 M). Higher concentrations of hemin exerted an inhibitory effect on the majority of the phosphoproteins. Protein phosphatase activity was not influenced by ACTH or spermine. The specific inhibition of pp30 phosphorylation by ACTH or spermine is most probably explained by an interaction with a cyclic nucleotide- and Ca²⁺-independent protein kinase.     

Platelet-activating factor induces tyrosine phosphorylation in human neutrophils

The addition of platelet-activating factor (PAF) to human neutrophils increases the levels of the tyrosine phosphorylation in several proteins. These proteins have molecular weights of 41 (pp41), 54 (pp54), 66 (pp66), 104 (pp104), and 116 (pp116) kDa. The effect of PAF was dose-dependent and could be seen at concentrations as low as 1 nM. The nonmetabolizable bioactive PAF analog, C-PAF, caused an increase in the level of phosphorylation of the same proteins in a time- and dose-dependent manner. On the contrary, lyso-PAF, enantio-PAF, and L-beta,gamma-dihexadecyl-alpha-lecithin failed to stimulate the phosphorylation of any of the aforementioned proteins. The response to PAF was prevented by the PAF antagonist BN-52021. The PAF-induced increases in tyrosine phosphorylation in pp66, pp116, and pp104 were selectively inhibited by pertussis toxin. In contrast, the level of pp41 phosphorylation remained unchanged after the pertussis toxin treatment. The calcium chelator EGTA significantly inhibited the PAF-produced phosphorylation of the pp41 protein. The intracellular calcium chelator 1,2-bis-(O-aminophenoxil)ethane-N,N,N',N'-tetraacetic acid (BAPTA) potentiated the PAF-enhanced levels of tyrosine phosphorylation on the pp41 protein. On the other hand, the PAF-induced phosphorylations of pp66, pp104, and pp116 were inhibited in BAPTA-treated cells. The calcium ionophore A23187 selectively potentiated the phosphorylation of the pp41 protein and reduced the phosphorylation in the pp54 protein. This phosphorylation was dependent on the extracellular calcium and was inhibited in toxin-treated cells. The results suggest that PAF is able to affect either directly or indirectly tyrosine kinase and/or phosphotyrosine phosphatase activities. The phosphorylation of the high and low molecular weight proteins are mediated by two different sets of kinases and/or phosphatases.     

Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis.

The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site.     

Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system.

We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.     

Phosphorylation/dephosphorylation of the beta light chain of clathrin from rat liver coated vesicles

The phosphorylation in vitro, on serine residues by endogenous casein kinase 2, of the clathrin beta light chain (33 kDa) of rat liver coated vesicles requires the presence of poly(L-lysine) which acts through binding to the beta light chain. The phosphorylation of other proteins is also increased in the presence of poly(L-lysine) and casein kinase 2. In contrast, the phosphorylation of the upper band of the 50-kDa protein doublet from rat liver coated vesicles is inhibited. Rat liver coated vesicles display a protein phosphatase activity which preferentially dephosphorylates clathrin beta light chain. This activity is different from the protein phosphatase which dephosphorylates the 50-kDa protein. This enzyme seems to be unrelated to the ATP/Mg-dependent protein phosphatase, or the polycation-stimulated protein phosphatases, which dephosphorylate the 50-kDa protein and beta light chain very efficiently, but with a different specificity. After dissociation of coated vesicles the beta-light-chain phosphatase activity is recovered in the membrane fraction. This phosphatase activity is inhibited by 50 microM orthovanadate and 5 mM p-nitrophenyl phosphate but not by 10 mM EDTA.     

Defective sarcolemmal phosphorylation associated with noninsulin-dependent diabetes

Noninsulin-dependent diabetes is associated with a decrease in the activity of sarcolemmal phosphatase 1, but no change in the activities of phosphatase 2A, 2B, or 2C. Also unaffected by diabetes were the activities of protein kinase C, cAMP-dependent protein kinase and calcium-calmodulin protein kinase. Because of the decrease in phosphatase 1 activity, 32P incorporation into sarcolemmal phosphoproteins catalyzed by either intrinsic protein kinases or extrinsic cAMP-dependent protein kinase was elevated in the diabetic. Among the proteins whose phosphorylation was elevated in diabetes was the phospholamban-like protein, which has been implicated in the regulation of ATP-dependent calcium transport. The phosphate-linked increase could be prevented by exposing the membranes to a phosphatase inhibitor and either extrinsic cAMP-dependent protein kinase or alamethicin. In addition to the phosphatase-linked effects, analysis of individual sarcolemmal phosphoproteins by SDS-polyacrylamide gel electrophoresis indicated that diabetes caused a specific elevation in membrane phosphorylation of some proteins (43 kDa and 78 kDa), but a decrease in the phosphorylation state of other phosphoproteins (31 kDa and 49 kDa). The data indicate that membrane phosphorylation is dramatically altered by diabetes. The possibility that this contributes to altered myocardial function is discussed.     

Phosphorylable proteins and alkaline phosphatase of brush border membranes from different parts of the rat small intestine

1. The distribution along the small intestine of phosphorylable proteins from the brush border has been studied by gel electrophoresis. 2. Four proteins, with apparent Mr of 190, 160, 140 and 120 kDa were distributed unequally along the gut, which incorporated 32P from gamma 32P (ATP) to different degrees. 3. Alkaline phosphatase activity has been shown to follow the same distribution. 4. Under denaturing conditions 90, 85 and 65 kDa proteins were observed, whilst the proteins of 190, 160, 140 and 120 kDa had disappeared. 5. All these proteins, with the exception of the 190 kDa protein, had also been labelled with 32Pi. Furthermore, a difference in the phosphorylation of the 65 kDa and the 90-85 kDa proteins was observed. 6. The 65 kDa protein like commercial calf alkaline phosphatase had a ratio of phosphorylation from ATP to phosphorylation from Pi less than the 90 and 85 kDa proteins. 7. Mg2+ (2.5-10 mM) decreased phosphorylation of only the 65 kDa protein whilst beta-glycerophosphate inhibited phosphorylation of all forms of alkaline phosphatase. 8. Incorporation of gamma 32P (ATP) into the proteins was enhanced in the presence of 5 mM theophylline or EDTA. 9. The nature of the phosphorylation of these different proteins is discussed.     

CK2 phosphorylation weakens 90 kDa MFP1 association to the nuclear matrix in Allium cepa

MFP1 is a conserved plant coiled-coil protein located on the stroma side of the chloroplast thylakoids, as well as in the nuclear matrix. It displays species-specific variability in the number of genes, proteins, and expression. Allium cepa has two nuclear proteins antigenically related to MFP1 with different M(r), pI, distribution, and expression, but only the 90 kDa MFP1 protein is a nuclear matrix component that associates with both the nucleoskeletal filaments and a new category of nuclear bodies. The 90 kDa AcMFP1 migrates in two-dimensional blots as two sets of spots. The hypo-phosphorylated forms (pI approximately 9.5) are tightly bound to the nuclear matrix, while high ionic strength buffers release the more acidic hyper-phosphorylated ones (pI approximately 8.5), suggesting that the protein is post-translationally modified, and that these modifications control its attachment to the nuclear matrix. Dephosphorylation by exogenous alkaline phosphatase and phosphorylation by exogenous CK2, as well as specific inhibition and stimulation of endogenous CK2 with heparin and spermine and spermidine, respectively, revealed that the protein is an in vitro and in vivo substrate of this enzyme, and that CK2 phosphorylation weakens the strength of its binding to the nuclear matrix. In synchronized cells, the nuclear 90 kDa AcMFP1 phosphorylation levels vary during the cell cycle with a moderate peak in G2. These results provide the first evidence for AcMFP1 in vivo phosphorylation, and open up further research on its nuclear functions.     

Protein tyrosine phosphorylation in the cyanobacterium Anabaena sp. strain PCC 7120.

Components of a protein tyrosine phosphorylation/dephosphorylation network were identified in the cyanobacterium Anabaena sp. strain PCC 7120. Three phosphotyrosine (P-Tyr) proteins of 27, 36, and 52 kDa were identified through their conspicuous immunoreactions with RC20H monoclonal antibodies specific for P-Tyr. These immunoreactions were outcompeted completely by free P-Tyr (5 mM) but not by phosphoserine or phosphothreonine. The P-Tyr content of the three major P-Tyr proteins and several minor proteins increased with their time of incubation in the presence of Mg-ATP and the protein phosphatase inhibitors sodium orthovanadate and sodium fluoride. Incubation of the same extracts with [gamma-32P]ATP but not [alpha-32P]ATP led to the phosphorylation of five polypeptides with molecular masses of 20, 27, 52, 85, and 100 kDa. Human placental protein tyrosine phosphatase 1B, with absolute specificity for P-Tyr, liberated significant quantities of 32Pi from four of the polypeptides, confirming that a portion of the protein-bound phosphate was present as 32P-Tyr. Alkaline phosphatase and the dual-specificity protein phosphatase IphP from the cyanobacterium Nostoc commune UTEX 584 also dephosphorylated these proteins and did so with greater apparent efficiency. Two of the polypeptides were partially purified, and phosphoamino analysis identified 32P-Tyr, [32P]phosphoserine, and [32P]phosphothreonine. Anabaena sp. strain PCC 7120 cell extracts contained a protein tyrosine phosphatase activity that was abolished in the presence of sodium orthovanadate and inhibited significantly by the sulfhydryl-modifying agents p-hydroxymercuriphenylsulfonic acid and p-hydroxymercuribenzoate as well as by heparin. In Anabaena sp. strain PCC 7120 the presence and/or phosphorylation status of P-Tyr proteins was influenced by incident photon flux density.     

Interferon gamma bound to endothelial cells is phosphorylated by ecto-protein kinases

The presence of protein kinase activity and its phosphorylated products has been demonstrated on the outer surface of the plasma membrane of endothelial cells. Extracellular phosphorylation was detected by incubation of primary endothelial cells (HUVEC's) and endothelial cell line EA.hy 926 with [gamma-32P]ATP. The reaction products were subjected to SDS/PAGE, autoradiography and scanning densitometry. Under the experimental conditions, five proteins with apparent molecular masses of 19, 23, 55, 88, and 110 kDa were prominently phosphorylated in both types of cells. Phosphorylation of the 19 kDa protein was the most rapid reaching maximum after 60 s and then the protein became dephosphorylated. Ecto-protein kinases responsible for the surface labeling of membrane proteins were characterized by using (a) protein kinase C inhibitors: K-252b, chelerythrine chloride, and [Ala113] myelin basic protein (104-118), (b) protein kinase A inhibitor Kemptide 8334, and (c) casein kinase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB). Stimulation of endothelial cells with tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) is associated with 20-80% reduction of extracellular phosphorylation of all membrane proteins. IFN gamma bound to membrane receptors becomes rapidly phosphorylated. Only in the case of IFN gamma it was associated with the appearance of a strongly phosphorylated band of 17 kDa corresponding to IFN gamma itself. Phosphorylation of this 17 kDa exogenous substrate was prevented by an ecto-kinase inhibitor K-252b. The existence of ecto-phosphoprotein phosphatase activity in endothelial cells was evidenced by testing the effect of microcystin LR--a membrane impermeable reagent that inhibits both PP-1 and PP-2a phosphoprotein phosphatases. The extent of phosphorylation of 19 kDa and 110 kDa phosphoproteins significantly increased in the presence of microcystin. Our results suggest the presence of at least two ecto-kinase activities on endothelial cells that may play a significant role(s) in the regulation of cytokines function.     

Phosphorylation of numatrin and other nuclear proteins by cdc2 containing CTD kinase cdc2/p58

Numatrin is a nuclear matrix phosphoprotein whose synthesis and abundance were shown to be regulated during the cell cycle in mitogen-stimulated lymphocytes (Feuerstein, N., and Mond, J. (1987) J. Biol. Chem. 262, 11389-11397). We examined the effect of (a) CTD-kinase, which contains the cdc2 catalytic component (p34) in a complex with a p58 subunit (cdc2/p58) and (b) the M phase-specific histone H1 kinase, which contains the cdc2 kinase in association with a p62 subunit (cdc2/p62), on phosphorylation of numatrin. We show that both cdc2 kinase complexes can phosphorylate numatrin. However, cdc2/p58 at conditions that caused a similar effect to cdc2/p62 on phosphorylation of histone H1 (dpm/micrograms of substrate/micrograms of enzyme) was found to have a 5-25-fold higher catalytic activity in the phosphorylation of numatrin. Analysis of the tryptic phosphopeptide map of numatrin phosphorylated by these cdc2 kinase complexes showed that both kinase complexes phosphorylated two major identical peptides, but minor additional peptides were differentially phosphorylated by each of these kinases. This indicates that under certain experimental conditions cdc2/p58 and cdc2/p62 may express some differences in their catalytic activity. In vitro phosphorylation by CTD kinase of a whole nuclear protein extract from murine fibroblasts showed that numatrin is the most prominent substrate for CTD kinase in this nuclear extract. CTD kinase cdc2/p58 was found to induce significantly the phosphorylation of five other discrete nuclear substrates. Particularly, two nuclear proteins at 75 kDa/pI approximately 6.5 and 85 kDa/pI approximately 5.3, which were not Coomassie Blue stainable, were found to be markedly phosphorylated by CTD kinase. The results of this study call for further study of the role of CTD kinase cdc2/p58 in the phosphorylation of numatrin under physiological conditions and to further characterization of the other nuclear substrates for CTD kinase.     

Topographic regulation of phosphorylation in giant neurons of the squid, Loligo pealei: role of phosphatases

In previous studies of phosphorylation in squid stellate ganglion neurons, we demonstrated that a specific multimeric phosphorylation complex characterized each cellular compartment. Although the endogenous protein profile of cell body extracts (giant fiber lobe, GFL), as determined by Coomassie staining, was similar to that of axoplasm from the giant axon, in this study we show that the protein phosphorylation profiles are qualitatively different. Whereas many axoplasm proteins were phosphorylated, including most cytoskeletal proteins, virtually all phosphorylation in perikarya was confined to low molecular weight compounds (<6 kDa). Because phosphorylation of exogenous substrates, histone and casein, was equally active in extracts from both compartments, failure to detect endogenous protein phosphorylation in cell bodies was attributed to the presence of more active phosphatases. To further explore the role of phosphatases in these neurons, we studied phosphorylation in the presence of serine/threonine and protein tyrosine phosphatase (PTP) inhibitors. We found that phosphorylation of axonal cytoskeletal proteins was modulated by okadaic acid-sensitive ser/thr phosphatases, whereas cell body phosphorylation was more sensitive to an inhibitor of protein tyrosine phosphatases, such as vanadate. Inhibition of PTPs by vanadate stimulated endogenous phosphorylation of GFL proteins, including cytoskeletal proteins. Protein tyrosine kinase activity was equally stimulated by vanadate in cell body and axonal whole homogenates and Triton X-100 free soluble extracts, but only the Triton X soluble fraction (membrane bound proteins) of the GFL exhibited significant activation in the presence of vanadate, suggesting higher PTP activities in this fraction than in the axon. The data are consistent with the hypothesis that neuronal protein phosphorylation in axons and cell bodies is modulated by different phosphatases associated with compartment-specific multimeric complexes.     

Phosphorylation of a pancreatic zymogen granule membrane protein by endogenous calcium/phospholipid-dependent protein kinase

The occurrence of phospholipid-sensitive calcium-dependent protein kinase (referred to as C kinase) and its endogenous substrate proteins was examined in a membrane preparation from rat pancreatic zymogen granules. Using exogenous histone H1 as substrate, C kinase activity was found in the membrane fraction. The kinase was solubilized from membranes using Triton X-100 and partially purified using DEAE-cellulose chromatography. An endogenous membrane protein (Mr approximately equal to 18 000) was found to be specifically phosphorylated in the combined presence of Ca2+ and phosphatidylserine. Added diacylglycerol was effective in stimulating phosphorylation of exogenous histone by the partially purified C kinase, but had no effect upon phosphorylation of the endogenous 18 kDa protein by the membrane-associated C kinase. Phosphorylation of the 18 kDa protein was rapid (detectable within 30 s following exposure to Ca2+ and phosphatidylserine), and highly sensitive to Ca2+ (Ka = 4 microM in the presence of phosphatidylserine). These findings suggest a role for this Ca2+-dependent protein phosphorylation system in the regulation of pancreatic exocrine function.     

A cAMP-binding phosphoprotein in Drosophila heads is similar to the regulatory subunit of the mammalian type II cAMP-dependent protein kinase

Cyclic AMP (cAMP)-dependent regulation of in vitro phosphorylation of several proteins including a cAMP-binding protein was studied with crude membrane and cytosol fractions from Drosophila heads. Phosphorylation of at least seven distinct proteins was enhanced in the presence of cAMP. Interestingly, however, the phosphorylation of a 56 kDa protein was apparently reduced by cAMP in the membrane but not in the cytosol fraction. The following data strongly indicate that the 56 kDa phosphoprotein in both membrane and cytosol fractions is a cAMP-binding protein, very similar to the regulatory subunit (RII) of a mammalian cAMP-dependent protein kinase, and that its binding to cAMP makes this protein very susceptible to the action of phosphatases: (i) cAMP highly stimulated the dephosphorylation of the 56 kDa phosphoprotein by the endogenous phosphatase in the membrane fraction. (ii) The dephosphorylation of a similar 56 kDa phosphoprotein in the cytosol fraction by an exogenous, cAMP-independent, alkaline phosphatase was also highly stimulated by cAMP. (iii) The 56 kDa phosphoprotein was covalently bound to cAMP by u.v. irradiation. (iv) The alkaline-phosphatase treatment reversibly converted this phosphoprotein to a 53 kDa non-phosphorylated protein. (v) The 53 kDa protein was selectively bound to cAMP-agarose and subsequently eluted by cAMP and high salt. (vi) This protein served as a substrate for the catalytic subunit of a mammalian cAMP-dependent protein kinase.