Insulin-stimulated phosphorylation of calmodulin by rat liver insulin receptor preparations

Insulin stimulates autophosphorylation of the beta subunit of its receptor and activates the associated tyrosine kinase. This kinase, in turn, phosphorylates a number of specific protein substrates; however, the functional and structural identity of these substrates is largely unknown. In this study, we demonstrate that insulin also stimulates the phosphorylation of calmodulin by rat hepatocyte insulin receptors partially purified by wheat germ agglutinin affinity chromatography. Phosphorylation occurred predominantly on tyrosine residues and had an absolute requirement for insulin receptors, divalent cations, and certain basic proteins. Maximal 32P incorporation was observed at an insulin concentration of 5 X 10(-9) M, and the K0.5 for insulin was approximately 4 X 10(-10) M. Phosphorylation of calmodulin was dependent upon ATP, saturating at 100 microM ATP with a K0.5 of 30 microM. Insulin-stimulated phosphorylation of calmodulin was also dependent upon Mg2+ or Mn2+, but was approximately 12-fold greater in the presence of Mg2+. Maximal phosphorylation was observed in the absence of Ca2+ and was inhibited at Ca2+:EGTA ratios greater than 0.8 (0.16 microM free Ca2+). Certain basic proteins, such as polylysine, histone Hf2b, and protamine sulfate, were necessary to observe insulin-stimulated phosphorylation of calmodulin. The relative amount of insulin-stimulated phosphorylation of calmodulin observed in the presence of each of these proteins differed. Maximal insulin-stimulated phosphorylation was observed in the presence of polylysine. These data suggest that both Ca2+ and calmodulin may participate in the early post-receptor events in the cellular mechanism of insulin action in hepatocytes.     

Comparison of the phosphorylation events in membranes from proliferating vs. quiescent endothelial cells

In an attempt to elucidate the intracellular events regulating the proliferation of endothelial cells (EC), we have compared the phosphorylation events in membranes prepared from proliferating (sparse) and quiescent (confluent) EC. Triton-solubilized membranes from sparse and confluent EC were incubated at pH 6.5 in the presence of divalent cations and [32P]ATP. Membrane proteins were then separated by SDS-PAGE and the radiolabeled phosphoproteins visualized by autoradiography. The overall kinase activity per milligram protein was 1.7 +/- 0.2-fold greater in membranes prepared from proliferating than from quiescent cells. The extent of phosphorylation was dramatically elevated in sparse over confluent samples for four phosphoproteins having the following approximate molecular masses: 180, 100, 97, and 55 kDa. The 180 and 100 kDa phosphoproteins exhibited 3.6- and 7.4-fold higher labeling, respectively, in sparse than in confluent membranes and both were phosphorylated on serine residues exclusively. The 97 kDa phosphoprotein was 11.6-fold higher in sparse membranes and contained both phosphoserine (p-ser) and phosphotheronine (p-thr), the latter comprising 61% of the radioactivity. The 55 kDA phosphoprotein contained 62% p-ser, 16% p-thr, and 22% phosphotyrosine (p-tyr) and was 2.3-fold higher in sparse membranes. Of these four phosphoproteins, only the 55 kDa protein was phosphorylated in confluent samples to an appreciable degree. Whereas the p-ser and p-thr content of the 55 kDa band increased moderately in sparse vs. confluent sample (1.8-fold increase), the tyrosine residues of this protein in sparse membranes were radiolabeled to a much greater extent relative to confluent membranes (5.4-fold increase). Analysis of the cofactor requirements of the FC membrane kinase(s) revealed that Mn2+ is the optimum cofactor and that Mg2+ can replace Mn2+ only for the kinase acting on the 100 kDa band. This suggests the presence of multiple EC membrane kinases. In the presence of both cofactors, the phosphorylation pattern is similar to the pattern obtained with Mn2+ alone. The kinase activity acting on all four phosphoproteins was independent of Ca2+, cAMP, cGMP, and phorbol 12-myristate 13-acetate. The mechanism responsible for the difference in kinase activity of proliferating vs. quiescent cells was not due to an inhibitor or enhanced phosphatase activity in confluent cells; the phosphorylation patterns obtained with sparse solubilized membranes and a mixture of sparse and confluent solubilized membranes were similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of enzyme activities involved in protein phosphorylation-dephosphorylation of thyroid plasma membranes

Bovine thyroid tissue exhibited cAMP-dependent and Ca2+-dependent protein kinase activities as well as a basal (cAMP- and Ca2+-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kinase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent Km values of cAMP and Ca2+ for the membrane-bound protein kinase were 5 . 10(-8) M and 8.3 . 10(-4) M in the presence of 1 Mm EGTA), respectively. The apparent Km values of Mg2+ were 7.10-4M (without (in the cAMP and Ca2+), 5 . 10(-4) M (with cAMP) and 1.3 . 10(-3) M (with Ca2+), and those of ATP were 3.5 . 10(-5)M (with or without cAMP) and 8.5 . 10(-5) M (with Ca2+). The Ca2+-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using 32P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca2+, but was stimulated by a rather broad range (5-25 mM) of Mg2+ and Mn2+. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca2+-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.     

Inactivation of bovine kidney cytosolic protamine kinase by the catalytic subunit of protein phosphatase 2A

Incubation of highly purified preparations of the bovine kidney cytosolic protamine kinase in the presence of near homogeneous preparations of the catalytic subunit of protein phosphatase 2A (PrP2Ac) from bovine kidney resulted in time-dependent inactivation of the protamine kinase. By contrast, incubation of bovine kidney cytosolic casein kinase II with PrP2Ac had no effect on the activity of this casein kinase II. In the presence of 10 mM sodium fluoride, 10 mM inorganic orthophosphate, 1 mM pyrophosphate or 0.1 mM ATP, the inactivation of the protamine kinase by PrP2Ac was completely inhibited. Half-maximal inhibition by ATP occurred at about 20 microM. The rate of inactivation of the protamine kinase by PrP2Ac was unaffected by Mg2+, Mn2+, Ca2+, EDTA or EGTA at 1 mM. The results strongly indicate that the activity of the cytosolic protamine kinase is regulated by phosphorylation/dephosphorylation.     

Manganese-phospholipid-stimulated protein kinase activity of human leukemic cells

A protein kinase system with unusual characteristics was noted in extracts of HL-60 cells using endogenous proteins as substrates. This system exhibited a cation preference for manganese at an optimal concentration of 0.5 mM. Moderate activity was detectable with magnesium at an optimal concentration of 5.0 mM, but calcium was inactive. Activity was markedly stimulated by phospholipid, with phosphatidylglycerol and phosphatidylinositol exhibiting greater activity than phosphatidylserine. In isolated plasma membranes, the major substrate of this system was a 73-kDa protein, while cytoplasmic extracts exhibited larger amounts of a 42-kDa substrate. 73-kDa phosphorylating activity of membranes was comparably active at 0 and 31 degrees C, although in cytosol activity was greater at 31 degrees C. No 73-kDa protein phosphorylation was observed in the presence of Ca2+, Mg2+, and phosphatidylserine. Phosphoamino acid analysis of the 73-kD band revealed phosphothreonine and phosphoserine. The 42-kDa substrate was distinguishable from actin by two-dimensional gel electrophoresis, which disclosed that both major substrates were highly basic in the isoelectric focusing dimension. Protamine and histones (H2B greater than H1 greater than H3) exhibited phospholipid-stimulated phosphorylation in the presence of Mn2+, but phosvitin, casein, and vinculin were not substrates. High levels of phosphorylative activity involving the 73-kDa substrate were noted in nuclear extracts. Complex patterns of increase of this activity were noted in both cytosol and nuclear extracts following induction of differentiation with dimethyl sulfoxide, retinoic acid, or phorbol 12-myristate 13-acetate. This study thus demonstrated the presence of a previously undescribed type of protein kinase activity which exhibited alterations during leukemic cellular differentiation.     

Phosphorylated intermediates of (Ca2+ + Mg2+)-ATPase and alkaline phosphatase in renal plasma membranes

Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [gamma-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as (Na+ + K+)-ATPase and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius Mr of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 microM) in a similar way as the (Ca2+ + Mg2+)-ATPase from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte (Ca2+ + Mg2+)-ATPase. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal (Na+ + K+)-ATPase. In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.     

Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat beta-cell tumour.

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.     

Endogenous hyperphosphorylation in plasma membrane from an ascites hepatocarcinoma cell line

Plasma-membrane-bound kinases of AS-30D ascites from transplantable rat hepatocarcinoma were shown to extensively catalyze the phosphorylation of plasma membrane proteins and membrane lipids, using [gamma-32P]ATP or [gamma-32P]GTP as a phosphate donor. In contrast, plasma membranes from normal adult rat liver or fast-growing regenerating liver (24 h after partial hepatectomy) produce significantly less activity for protein phosphorylation and little phosphorylation of the lipids. However, neonatal (24 h old) rat liver plasma membrane preparations show levels of phosphorylation of proteins and lipids intermediate between those in the tumor cell line and normal adult plasma membrane preparations. Phosphatidic acid was identified as one of the 32P-labelled lipids in the tumor plasma membrane chloroform-methanol (2:1, v/v) extract. Phosphorylation of protein was not affected by cAMP or cGMP. However, calcium ion (in the presence or absence of calmodulin) significantly modifies the 32P labelling of a series of proteins in normal tissue but has little effect with the neoplastic preparations. Some plasma membrane proteins were capable of nucleotide binding, instead or in addition to being phosphorylated. Finally, the presence of membrane-bound phosphoprotein phosphatase(s) was also demonstrated in all the preparations examined by means of chase experiments with nonlabelled ATP or GTP, and (or) by the use of the phosphoprotein phosphatase inhibitor, orthovanadate.     

Characterization of the catalytic activities of the PhoQ histidine protein kinase of Salmonella enterica serovar Typhimurium

Studies of Escherichia coli membranes that were highly enriched in the Salmonella enterica serovar Typhimurium PhoQ protein showed that the presence of ATP and divalent cations such as Mg2+, Mn2+, Ca2+, or Ba2+ resulted in PhoQ autophosphorylation. However, when Mg2) or Mn2+ was present at concentrations higher than 0.1 mM, the kinetics of PhoQ autophosphorylation were strongly biphasic, with a rapid autophosphorylation phase followed by a slower dephosphorylation phase. A fusion protein lacking the sensory and transmembrane domains retained the autokinase activity but could not be dephosphosphorylated when Mg2+ or Mn2+ was present at high concentrations. The instability of purified [32P]phospho-PhoP in the presence of PhoQ-containing membranes indicated that PhoQ also possesses a phosphatase activity. The PhoQ phosphatase activity was stimulated by increasing the Mg2+ concentration. These data are consistent with a model in which Mg2+ binding to the sensory domain of PhoQ coordinately regulates autokinase and phosphatase activities.     

Acidic fibroblast growth factor receptor purified from bovine liver is a novel protein tyrosine kinase

Acidic fibroblast growth factor (aFGF) receptor was purified from plasma membranes of bovine liver using Triton X-100 extraction, wheat germ lectin-Sepharose 4B gel affinity chromatography, and DEAE-cellulose anion-exchange chromatography. As previously reported for the aFGF receptor in murine fibroblasts (Huang, S. S., and Huang, J. S. (1986) J. Biol. Chem. 261, 9568-9571), the purified aFGF receptor was also found to be a 135-kDa glycoprotein which showed an intrinsic and ligand-stimulated autophosphorylation activity. The 32P-labeled aFGF receptor was specifically immunoprecipitated by anti-FGF receptor (anti-flg/bek/cek gene product) antiserum. In contrast to other growth factor receptors/protein tyrosine kinases, the protein tyrosine kinase activity (autophosphorylation) of the aFGF receptor was stimulated (approximately 1.5-fold) by low concentrations of Mn2+, Mg2+, and Ca2+ (optimal concentrations of approximately 0.1, approximately 0.1, and 1 microM, respectively) but inhibited by higher concentrations of Mn2+, Mg2+, Ca2+, and pyrophosphate (greater than or equal to 20, greater than or equal to 50, greater than or equal to 10, and greater than or equal to 100 microM, respectively). However, addition of Mn2+ and pyrophosphate at a ratio of 1:1 not only reversed the inhibitory effect but also enhanced the kinase activity about 3-4-fold. The apparent Km of ATP for intrinsic and ligand-stimulated protein kinase activity of the aFGF receptor was estimated to be 25 microM. The preferred exogenous substrates for the protein tyrosine kinase activity of the aFGF receptor were found to be myelin basic protein and histone. Poly-L-arginine, an inhibitor for aFGF binding to the receptor, appeared to stimulate the mitogenesis or cell growth of responsive cells by mimicking aFGF activity.     

Endogenous phosphorylation of the GABA(A) receptor protein is counteracted by a membrane-associated phosphatase

Incubation of bovine brain membranes with [gamma-33P]ATP phosphorylated mainly a 51-kDa band. Electrophoretic co-migration was observed for 33P- and [3H]flunitrazepam-labeled bands in both membrane fractions and in affinity-purified GABA(A) receptor (GABAA-R) preparations. An alpha-subunit monoclonal antibody adsorbed most of the radiolabeled-band, suggesting that the labeled-membrane polypeptide corresponds to the GABA(A)-R alpha1-subunit, which is the only GABA(A)-R subunit with a molecular weight of 51 kDa. The phosphorylation rate was much faster in membranes than in purified receptor. Dephosphorylation was detected in membranes only. The membrane-bound phosphatase was potently inhibited by vanadate and Zn2+>Mn2+ , but was insensitive to okadaic acid (a phosphatase 1, 2 and 2B inhibitor), cyclosporin (specific calcineurin inhibitor) and phosphatase-1 inhibitor. Endogenous kinase was activated by divalent cations including calcium (Mg2- > Mn2+ > Ca2+), whilst dephosphorylation did not require the presence of Ca2+ ions. This suggests that at least one membrane-bound phosphatase counteracts the endogenous phosphorylation of the GABA(A)-R: the lack of dephosphorylation in the purified receptor preparation indicates that, in contrast to the endogenous kinase, no phosphatase is closely associated with the receptor protein complex.     

A phosphatidylinositol kinase in rat mast cell granules

Intact granules were isolated from sonicated purified rat serosal mast cells on a Percoll gradient. The granules were shown to contain a highly active phosphatidylinositol kinase that catalyzes the formation of diphosphoinositide from endogenous phosphatidylinositol in the granule membrane. The enzyme requires ATP and Mg2+ or Mn2+ for activity; Ca2+, fluoride and cyclic AMP are inhibitory. The Km for ATP is 25 microM. The initial reaction is rapid, but the response ceases within a few minutes. A comparison of the rate of phosphorylation of intact and broken membrane granules suggests that the phosphorylation occurs on the outer (cytoplasmic) surface of the granules.     

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.     

Identification of Zn2+-dependent and Mg2+/Triton X-100-dependent tyrosine protein kinases in ethylenediaminetetraacetate-treated P2 membrane fraction of monkey brain basal ganglia

Tyrosine phosphorylation of a 55- and 60-kDa protein was observed when EDTA-treated P2 membrane fraction from monkey basal ganglia was incubated with [gamma-32P]-ATP in the presence of Zn2+. Other metal ions were less effective in this phosphorylation. The effect of Zn2+ did not appear to be due to its inhibition of a tyrosine phosphatase. In the presence of Mg2+/Triton X-100 instead of Zn2+, phosphorylation on tyrosine residues of a 17-kDa protein and the external substrate poly(Glu, Tyr) 4:1 copolymer was observed. Both Mg2+ and Triton X-100 were essential for this and Zn2+ inhibited both of these phosphorylations. Convincing evidence for the existence of Zn2+-dependent and Mg2+/Triton X-100-dependent tyrosine protein kinases was obtained when the two kinases could be separated by extraction of the membranes by Triton X-100. The Zn2+-dependent phosphorylation was present exclusively in the Triton-solubilized supernatant whereas the Mg2+/Triton X-100-dependent phosphorylation was found associated with the Triton-insoluble membrane fractions. Externally added histone could also be phosphorylated on tyrosine residues in a Zn2+- or Mg2+/Triton X-100-dependent manner by the supernatant or membrane fraction, respectively.     

Protein phosphorylation and dephosphorylation in liver plasma membrane. Effect of inorganic phosphate

When a plasma membrane preparation isolated from rat liver was incubated with [gamma-32P]ATP and Mg2+, protein-bound 32P increased rapidly, followed by a gradual decrease. The time course suggested the existence of membrane-bound kinase(s) and phosphatase(s) phosphorylating and dephosphorylating endogenous proteins. The extent of phosphorylation was not affected by inclusion of cyclic AMP in the reaction mixture. The extent of the maximum phosphorylation was dependent on membrane concentration, owing to rapid hydrolysis of ATP by the membrane-bound ATPase activity. Thus, phosphorylation proceeded further on repeated addition of ATP. Both phosphorylation and dephosphorylation were stimulated by Mg2+, an effective rate of phosphorylation being obtained at 15 mM. Pi up to 20 mM stimulated phosphorylation with little effect on the rate of dephosphorylation. At higher phosphate concentrations, the maximum 32P-incorporation decreased again, and at 100 mM, dephosphorylation was prevented significantly. Autoradiography after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea revealed six main phosphorylated bands, two of which (Band 3 and 5) were partly extractable with 1 M NaCl. In the presence of 100 mM Pi, very strong phosphorylation of Band 5 (about 23,000 daltons) was noted, and a new strongly labeled band (Band P, about 20,000 daltons) was observed. It was concluded that the phosphoproteins in the membrane may be turned over at different rates and high concentrations of Pi may affect the turnover rate of some phosphoproteins, probably through interference with the phosphatase.     

The inositol lipids of Paramecium tetraurelia and preliminary characterizations of phosphoinositide kinase activity in the ciliary membrane

Inositol glycerolipids make up less than 10% of total phospholipids of Paramecium tetraurelia cells. Unlike inositol lipids found in mammalian and other cell types, these lipids from Paramecium lack arachidonic acid. It was demonstrated that kinase and possibly phosphatase enzymes that interconvert phosphatidylinositol (PI), phosphatidylinositol phosphate (PI-P) and phosphatidylinositol-bis-phosphate (PI-P2) exist in ciliary membranes of this ciliate. When exogenous soybean PI and [gamma-32P]ATP were provided as substrates, isolated cilia preparations exhibited PI and PI-P kinase activities as demonstrated by the incorporation of radiolabel into PI-P and PI-P2. Kinase activity was activated by millimolar [Mg2+] and inhibited by millimolar [Ca2+]. Significant inhibition of kinase activity in the presence of unlabeled excess ATP suggested that ATP is the preferred phosphate donor for this reaction. Of 4 suborganellar fractions of isolated cilia, the membrane fraction had the greatest kinase activity indicating that the enzyme(s) is membrane-associated.     

Phosphorylation sites in human erythrocyte band 3 protein

The human red cell anion-exchanger, band 3 protein, is one of the main phosphorylated proteins of the erythrocyte membrane. Previous studies from this laboratory have shown that ATP-depletion of the red blood cell decreased the anion-exchange rate, suggesting that band 3 protein phosphorylation could be involved in the regulation of anion transport function (Bursaux et al. (1984) Biochim. Biophys. Acta 777, 253-260). Phosphorylation occurs mainly on the cytoplasmic domain of the protein and the major site of phosphorylation was assigned to tyrosine-8 (Dekowski et al. (1983) J. Biol. Chem. 258, 2750-2753). This site being very far from the integral, anion-exchanger domain, the aim of the present study was to determine whether phosphorylation sites exist in the integral domain. The phosphorylation reaction was carried out on isolated membranes in the presence of [gamma-32P]ATP and phosphorylated band 3 protein was then isolated. Both the cytoplasmic and the membrane spanning domains were purified. The predominant phosphorylation sites were found on the cytoplasmic domain. RP-HPLC analyses of the tryptic peptides of whole band 3 protein, and of the isolated cytoplasmic and membrane-spanning domains allowed for the precise localization of the phosphorylated residues. 80% of the label was found in the N-terminal tryptic peptide (T-1), (residues 1-56). In this region, all the residues susceptible to phosphorylation were labeled but in varying proportion. Under our conditions, the most active membrane kinase was a tyrosine kinase, activated preferentially by Mn2+ but also by Mg2+. Tyrosine-8 was the main phosphate acceptor residue (50-70%) of the protein, tyrosine-21 and tyrosine-46 residues were also phosphorylated but to a much lesser extent. The main targets of membrane casein kinase, preferentially activated by Mg2+, were serine-29, serine-50, and threonine(s)-39, -42, -44, -48, -49, -54 residue(s) located in the T-1 peptide. A tyrosine phosphatase activity was copurified with whole band 3 protein which dephosphorylates specifically P-Tyr-8, indicating a highly exchangeable phosphate. The membrane-spanning fragment was only faintly labeled.     

Diacylglycerol kinase in plasma membranes from wheat.

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg2+ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl2. The Mg2+ requirement could be substituted only partially by Mn2+ and not at all by Ca2+. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.     

Guanine nucleotides induce tyrosine phosphorylation and activation of the respiratory burst in neutrophils.

Activation of the NADPH oxidase was examined in electrically permeabilized human neutrophils exposed to non-hydrolysable guanine nucleotides. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induced a marked increase in the rate of O2 consumption, which was partially resistant to staurosporine, an inhibitor of protein kinase C, under conditions where the response to diacylglycerol was virtually abolished. The respiratory burst elicited by GTP[S] was dependent on the presence of ATP and Mg2+, suggesting involvement of phosphorylation reactions. Accordingly, phosphoprotein formation was greatly stimulated by the guanine nucleotide. The polypeptide phosphorylation pattern induced by GTP[S] was similar to, but not identical with, that observed with diacylglycerol, indicating the activation of kinases other than protein kinase C by the guanine nucleotide. The possible involvement of tyrosine kinases was assessed by immunoblotting using anti-phosphotyrosine antibodies. Treatment of electroporated cells with GTP[S] stimulated the accumulation of tyrosine-phosphorylated proteins. This effect was not induced by diacylglycerol, indicating that tyrosine phosphorylation is not secondary to stimulation of protein kinase C. The results indicate that, in neutrophils, activated G-proteins can stimulate tyrosine kinase and/or inhibit tyrosine phosphatase activity. Changes in the amounts of tyrosine-phosphorylated proteins may signal activation of the respiratory burst.     

Specific association of calmodulin-dependent protein kinase and related substrates with the junctional sarcoplasmic reticulum of skeletal muscle

A systematic study of protein kinase activity and phosphorylation of membrane proteins by ATP was carried out with vesicular fragments of longitudinal tubules (light SR) and junctional terminal cisternae (JTC) derived from skeletal muscle sarcoplasmic reticulum (SR). Following incubation of JTC with ATP, a 170,000-Da glycoprotein, a 97,500-Da protein (glycogen phosphorylase), and a 55,000-60,000-Da doublet (containing calmodulin-dependent protein kinase subunit) underwent phosphorylation. Addition of calmodulin in the presence of Ca2+ (with no added protein kinase) produced a 10-fold increase of phosphorylation involving numerous JTC proteins, including the large (approximately 450,000 Da) ryanodine receptor protein. Calmodulin-dependent phosphorylation of the ryanodine receptor protein was unambiguously demonstrated by Western blot analysis. The specificity of these findings was demonstrated by much lower levels of calmodulin-dependent phosphorylation in light SR as compared to JTC, and by much lower cyclic AMP dependent kinase activity in both JTC and light SR. These observations indicate that the purified JTC contain membrane-bound calmodulin-dependent protein kinase that undergoes autophosphorylation and catalyzes phosphorylation of various membrane proteins. Protein dephosphorylation was very slow in the absence of added phosphatases, but was accelerated by the addition of phosphatase 1 and 2A (catalytic subunit) in the absence of Ca2+, and calcineurin in the presence of Ca2+. Therefore, in the muscle fiber, dephosphorylation of SR proteins relies on cytoplasmic phosphatases. No significant effect of protein phosphorylation was detected on the Ca2(+)-induced Ca2+ release exhibited by isolated JTC vesicles. However, the selective and prominent association of calmodulin-dependent protein kinase and related substrates with junctional membranes, its Ca2+ sensitivity, and its close proximity to the ryanodine and dihydropyridine receptor Ca2+ channels suggest that this phosphorylation system is involved in regulation of functions linked to these structures.