The heterogeneity of bovine growth hormone. Extraction from the pituitary of components with different biological and immunological properties.

Evidence is available to suggest that Ca2+-calmodulin and cyclic nucleotides are involved in the regulation of ion transport in rabbit ileum. Since both Ca2+-calmodulin and cyclic nucleotides exert many of their effects by phosphorylation, the effects of Ca2+-calmodulin and cyclic nucleotides on phosphorylation of purified microvillus membrane from rabbit ileal mucosa were evaluated. Ca2+-calmodulin increased phosphorylation of five microvillus-membrane peptides, with Mr values of 137000, 77000, 58000, 53000 and 50000. The increases in phosphorylation caused by Ca2+-calmodulin were: Mr-137000 peptide, 111 +/- 26%; Mr-77000 peptide, 71 +/- 17%; Mr-58000 peptide, 51 +/- 8%; Mr-53000 peptide, 113 +/- 20%. These increases were maximal at 1 microM-calmodulin and 0.3-0.9 microM free Ca2+; concentrations of Ca2+ causing half-maximal effects on phosphorylation for the different peptides were 0.06-0.12 microM. Cyclic AMP and cyclic GMP increased phosphorylation of two peptides, of Mr 137000 and 85000. The concentrations of cyclic nucleotides giving half-maximal phosphorylation of the Mr-137000 peptide were 0.3 microM-cyclic AMP and 4.6 microM-cyclic GMP, and for the Mr-85000 peptide, 3.9 microM-cyclic AMP and 0.05 microM-cyclic GMP. The maximal increase in phosphorylation of the Mr-137000 peptide was 200% for cyclic AMP and 95% for cyclic GMP, and that of the Mr-85000 peptide was 220% for cyclic AMP and 120% for cyclic GMP. These studies demonstrate the existence of Ca2+-calmodulin-, cyclic AMP- and cyclic GMP-dependent protein kinases and substrate proteins in purified rabbit ileal microvillus membranes and that Ca2+ can regulate phosphorylation of these proteins over the presumed physiological concentration range of cytosol free Ca2+.     

Stimulation of Ca2+ uptake by cyclic AMP and protein kinase in sarcoplasmic reticulum-rich and sarcolemma-rich microsomal fractions from rabbit heart.

The effect of cyclic AMP on Ca2+ uptake by rabbit heart microsomal vesicular fractions representing mainly fragments of either sarcoplasmic reticulum or sarcolemma was investigated in the presence and absence of soluble cardiac protein kinase and with microsomes prephosphorylated by cyclic AMP-dependent protein kinase. The acceleration of oxalate-promoted Ca2+ uptake by fragmented sarcoplasmic reticulum following cyclic AMP-dependent membrane protein phosphorylation, observed by other authors, was confirmed. In addition it was found that the acceleration was greatest at pH 7.2 and almost negligible at pH 6.0 and pH 7.8. A very marked increase in Ca2+ uptake by cyclic AMP-dependent membrane protein phosphorylation was observed in the presence of boric acid, a reversible inhibitor of Ca2+ uptake. In addition to the microsomal fraction thought to represent mainly fragments of the sarcoplasmic reticulum, the effect of protein kinase and cyclic AMP on Ca2+ uptake was investigated in a cardiac sarcolemma-enriched membrane fraction. Ca2+ uptake by sarcolemmal vesicles, unlike Ca2+ uptake by sarcoplasmic reticulum vesicles, was inhibited by low doses of digitoxin. The acceleration of oxalate-promoted Ca2+ uptake by cyclic AMP and soluble cardiac protein kinase, however, was quite similar to what was seen in preparations of fragmented sarcoplasmic reticulum, which suggests that it may reflect an acceleration of active Ca2+ transport across the myocardial cell surface membrane.     

Effects of Ca2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rt islets of langerhans

Activation of Ca2+ -calmodulin- and cyclic AMP-dependent protein kinases has been suggested to be involved in stimulus-secretion coupling in the pancreatic beta-cell. To study the properties of suc kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [gamma-32P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and detected by autoradiography. The phosphorylation of certain proteins could be enhanced by Ca2+ plus calmodulin or by cyclic AMP. The major effect of Ca2+ and calmodulin was to stimulate the phosphorylation of a protein (P53) of molecular weight 53,100 +/- 500 (n = 15). Maximum phosphorylation of protein P53 occurred within 2 min with 2 micrometers free Ca2+ and 0.7 micrometers calmodulin. Incorporation of label into protein P53 was inhibited by trifluoperazine or W7 but not by cyclic AMP-dependent protein kinase inhibitor. Phosphorylation of a proteins of similar molecular weight could be enhanced to a lesser extent in the absence of Ca2+ but in the presence of cyclic AMP and 3-isobutylmethylxanthine: this phosphorylation was blocked by cyclic AMP-dependent protein kinase inhibitor. Cyclic AMP also stimulated incorporation of label into polypeptides of molecular weights 55,000 and 70-80,000. The results are consistent with the hypothesis that protein phosphorylation mechanisms may play a role in the regulation of insulin secretion.     

Protein kinase activity associated with pancreatic zymogen granules.

Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues.     

In Vitro Phosphorylation of Bovine Adrenal Chromaffin Cell Tyrosine Hydroxylase by Endogenous Protein Kinases

Under phosphorylating conditions, addition of Ca2+ or cyclic AMP to the 100,000 g supernatant of purified bovine adrenal chromaffin cells increases both the incorporation of 32P into tyrosine hydroxylase and the activity of the enzyme. Combining maximally effective concentrations of each of these stimulating agents produces an additive increase in both the level of 32P incorporation into tyrosine hydroxylase and the degree of activation of the enzyme. The increased phosphorylation by Ca2+ is due to stimulation of endogenous Ca2+-dependent protein kinase activity and not inhibition of phosphoprotein phosphatases. When the chromaffin cell supernatant is subjected to diethylaminoethyl (DEAE) chromatography to remove calmodulin and phospholipids, tyrosine hydroxylase is no longer phosphorylated or activated by Ca2+; on the other hand, phosphorylation and activation of tyrosine hydroxylase by cyclic AMP are not affected. Subsequent replacement of either Ca2+ plus calmodulin or Ca2+ plus phosphatidylserine to the DEAE-fractionated cell supernatant restores the phosphorylation, but not activation of the enzyme. Reverse-phase HPLC peptide mapping of tryptic digests of tyrosine hydroxylase from the 100,000 g supernatant shows that the Ca2+-dependent phosphorylation occurs on three phosphopeptides, whereas the cyclic AMP-dependent phosphorylation occurs on one of these peptides. In the DEAE preparation, either cyclic AMP alone or Ca2+ in the presence of phosphatidylserine stimulates the phosphorylation of only a single phosphopeptide peak, the same peptide phosphorylated by cyclic AMP in the crude supernatant. In contrast, Ca2+ in the presence of calmodulin stimulates the phosphorylation of three peptides having reverse-phase HPLC retention times that are identical to peptides phosphorylated by Ca2+ addition to the crude unfractionated 100,000 g supernatant. Rechromatography of the peaks from each of the in vitro phosphorylations, either in combination with each other or in combination with each of the seven peaks generated from phosphorylation of tyrosine hydroxylase in situ, established that cyclic AMP, Ca2+/phosphatidylserine, and Ca2+/calmodulin all stimulate the phosphorylation of the same reverse-phase HPLC peptide: in situ peptide 6. Ca2+/calmodulin stimulates the phosphorylation of in situ peptides 3 and 5 as well. Thus, tyrosine hydroxylase can be phosphorylated in vitro by protein kinases endogenous to the chromaffin cell. Phosphorylation occurs on a maximum of three of the seven in situ phosphorylated sites, and all three of these sites can be phosphorylated by a Ca2+/calmodulin-dependent protein kinase.     

The effect of cyclic nucleotides and protein phosphorylation on calcium permeability and binding in the sarcoplasmic reticulum.

In the absence of cyclic nucleotides heart microsomes have two classes of calcium binding sites with binding constants of 0.69 and 0.071 micron-1 and capacities of 2.2 and 9.7 nmol/mg protein, respectively. Neither cyclic AMP nor monobutyryl cyclic AMP affect binding but cyclic GMP and monobutyryl cyclic GMP cause the complete loss of the high affinity calcium binding sites, Cyclic GMP (but not monobutyryl cyclic GMP) also causes a decrease in the binding constant of the low affinity binding sites. AMP, GMP and Tris-butyrate do not affect calcium binding. The effects of the cyclic nucleotides are direct and are not mediated by protein phosphorylation. Phosphorylation of microsomal proteins increases the binding constant but not the capacity of the high affinity calcium binding sites. The capacity and also, perhaps, binding constant of the low affinity sites is also increased by phosphorylation. In additon to their effects on calcium binding the cyclic nucleotides also affect the movements of calcium into and out of the microsomes. The effects are again direct and not mediated by protein phosphorylation. Cyclic GMP decreases the rate of Ca2+ efflux from preloaded cardiac microsomes and also appears to decrease the rate of uptake of Ca2+ by cardiac microsomes though this effect is less clear cut than the action on efflux. The cyclic nucleotide has a half maximal effect at a concentration of 100 microns. By contrast cyclic AMP increases the rate of influx of Ca2+ into heart microsomes and the rate of efflux of Ca2+ from preloaded preparations. The effect is, however, rather slight. It is suggested that the most obvious interpretation of these results is that cyclic GMP decreases the Ca2+ permeability of the cardiac microsomal membrane while cyclic AMP increases the permeability. In contrast to the results found with membrane preparations from certain other tissues phosphorylation of cardiac microsomal proteins does not appear to alter Ca2+ efflux or influx out of, or into, cardiac microsomal preparations. It is thus concluded that phosphorylation of cardiac microsomal proteins does not affect the Ca2+ permeability of the microsomal membrane.     

Calcitonin-induced phosphorylation of rat liver cytosolic proteins

Calcitonin (CT) stimulated phosphorylation of two liver cytosolic proteins whose molecular weights are 67,000 and 93,000. Stimulation of 67,000-Mr protein phosphorylation began shortly after subcutaneous injection of CT, reaching a maximum at 5 min and decreasing to below the control level at 30 min. The reaction was independent of cyclic AMP or Ca2+, and was not influenced by a calmodulin antagonist, W7. Stimulation of 93,000-Mr protein phosphorylation became evident by 30 min. This reaction was also stimulated by administration of vasopressin or epinephrine, which is known to cause increased phosphorylation of glycogen phosphorylase having the same molecular weight. The phosphorylation of 93,000-Mr protein, stimulated by CT, was dependent on Ca2+ but not on cyclic AMP, and appeared to be inhibited by W7. In addition, CT did not influence the phosphorylation of 61,000-Mr protein, a major protein phosphorylated in a cyclic AMP-dependent manner. These results suggest that CT may exert its effect on liver cells through protein phosphorylation, most probably in a cyclic AMP-independent manner.     

Calcium ion-stimulated phosphorylation of myelin proteins.

Myelin isolated from the central and peripheral nervous system contains a Mg2+-dependent protein kinase that catalyses phosphorylation of myelin-specific proteins. This phosphorylation is markedly stimulated by Ca2+ but not by cyclic AMP. Evidence was obtained that suggested an involvement of calmodulin-like protein in the stimulatory effects of Ca2+ on myelin phosphorylation.     

Functional shift from muscarinic to nicotinic cholinergic receptors involved in inositol trisphosphate and cyclic GMP accumulation during the primary culture of adrenal chromaffin cells.

The incorporation of 32P from [gamma-32P]ATP into intracellular proteins was studied in electrically permeabilized rat islets of Langerhans. Ca2+ (10 microM), cyclic AMP (100 microM) and a protein kinase C-activating phorbol ester, phorbol 13-myristate 12-acetate (PMA; 100 nM) produced marked changes in the phosphorylation state of a number of proteins in permeabilized islets after incubation for 1 min at 37 degrees C. Ca2+ modified the effects of cyclic AMP and PMA on protein phosphorylation. Noradrenaline (10 microM) had no detectable effects on Ca2+-dependent protein phosphorylation, but significantly inhibited Ca2+-induced insulin secretion from electrically permeabilized islets. These results suggest that electrically permeabilized islets offer a useful model in which to study rapid events in protein phosphorylation as a mechanism of stimulus-secretion coupling. If the rapid Ca2+-induced effects on protein phosphorylation are involved in the control of insulin secretion, the results of this study also imply that part of the catecholamine inhibition of insulin secretion occurs at a stage in the secretory pathway beyond the activation of the regulated protein kinases.     

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.     

The involvement of protein phosphorylation in stimulus-secretion coupling in the mouse exocrine pancreas

Secretagogue-induced protein phosphorylation was studied in the mouse pancreas in vitro, by using polyacrylamide-gel electrophoresis to separate the labelled proteins. Muscarinic cholinergic agonists increased the phosphorylation of a single band, which corresponded to Mr 32000, when the tissue was incubated with Ca2+ present in the extracellular medium, but not in Ca2+-free Krebs solution. In the presence of Ca2+, ionophore A23187 stimulated phosphorylation of the same band. The dose-response curve for carbachol-induced phosphorylation was biphasic, with maximum response at 1.0 microM-carbachol, and lesser responses when greater concentrations were used. This resembles the dose-response curve for carbachol-induced amylase secretion. The data suggest that the muscarinic-agonist-induced protein phosphorylation is stimulated secondarily to elevation of cytosol [Ca2+] and do not support the idea that diacylglycerol formed from hydrolysis of phosphatidylinositol is the activator of the protein kinase. Derivatives of cyclic AMP stimulated phosphorylation of bands corresponding to Mr 95500, 32000 and 20000. The effects of dibutyryl cyclic AMP and bethanechol on the protein of Mr 32000 were not additive, suggesting that the two agents produced phosphorylation of the same site(s) on this protein. Since derivatives of cyclic AMP, which are not very effective secretagogues in the exocrine pancreas, stimulate phosphorylation of the protein of Mr 32000, it is difficult to argue that phosphorylation of this particular protein leads to protein secretion.     

Effects of dehydrouramil on protein phosphorylation and insulin secretion in rat islets of Langerhans.

Dehydrouramil hydrate hydrochloride (DHU), a stable analogue of alloxan, inhibited the phosphorylation of an endogenous protein of Mr 53,000 catalysed by a Ca2+-calmodulin-dependent protein kinase in extracts of islets of Langerhans. The concentration of DHU required for 50% inhibition was 0.09 mM. DHU did not inhibit islet cyclic AMP-dependent protein kinase and caused only slight inhibition of Ca2+-phospholipid-dependent protein kinase. Inhibition of Ca2+-calmodulin-dependent protein kinase was neither prevented nor reversed by dithiothreitol. DHU did not affect the ability of calmodulin to activate cyclic AMP phosphodiesterase. In intact islets, pre-exposure to DHU impaired the insulin-secretory response to glucose and blocked the potentiatory effect on insulin secretion of forskolin, an activator of adenylate cyclase, and of tetradecanoylphorbol acetate (TPA), an activator of Ca2+-phospholipid-dependent protein kinase. The increase in islet cyclic AMP elicited by forskolin was not affected by DHU. The data are consistent with the hypothesis that protein phosphorylation catalysed by a Ca2+-calmodulin-dependent protein kinase may play a central role in the regulation of insulin secretion.     

Agonist-induced phosphorylation of an immunologically ras-related protein in human erythroleukemia cells

Monoclonal antibody M90 recognizes a specific epitope of the ras-encoded p21 protein. This region comprises amino acids 107-130 containing the residues 116-119, which are related to GTP binding. This antibody strongly reacts on Western Blots with a 22kDa protein from human erythroleukemia (HEL) cells. Treatment of HEL cells with iloprost, an agonist that increases cellular cyclic AMP levels, produces the appearance of a protein with an apparent molecular mass of 24kDa. This protein is also recognized by antiserum M90 on Western Blots; its appearance parallels a decrease of the 22kDa protein, and it can be labeled with 32P. This effect is also observed with dibutyryl cyclic AMP, which indicates phosphorylation of the 22kDa protein by cyclic AMP-dependent protein kinase. This phosphorylation produces an electrophoretic mobility change of the 22kDa protein to a 24kDa region on gels. The change of mobility of the 22kDa protein induced by iloprost in HEL cells is also observed when the protein is labeled with [35S]methionine and immunoprecipitated with antiserum M90. This information indicates a coupling mechanism involving phosphorylation of an oncogene product in HEL cells.     

Stimulation of calcium accumulation in cardiac sarcolemma by protein kinase.

Sarcolemmal membranes isolated from guinea pig heart ventricles contained an ATP-dependent calcium-sequestering activity. Sarcolemmal calcium accumulation but not binding was enhanced by preincubation of membranes with exogenous protein kinase, with cyclic AMP, or with isoproterenol. Protein kinase (EC 2.7.1.37) increased the V of Ca2+ accumulation by sarcolemma without any significant effect on the affinity for Ca2+. The endogenous protein kinase activity present in isolated sarcolemma affected membrane phosphorylation. Cyclic AMP increased the endogenous kinase activity modestly, whereas histone increased it significantly. Exogenous protein kinase also catalyzed phosphorylation of these membranes. Endogenous and exogenous kinase-catalyzed phosphorylation of sarcolemma was hydroxylamine-insensitive. Ca2+-dependent ATPase (EC 3.6.1.3) (extra ATPase) activity of sarcolemma was also increased by protein kinase.     

Effects of adenosine 3' : 5'-monophosphate and guanosine 3' : 5'-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle.

The effects of adenosine 3' : 5'-monophosphate (cyclic AMP), guanosine 3' : 5'-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P). While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10(-5) M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP. Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10(-8) M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10(-8) M, while with cyclic AMP a concentration of 10(-5) M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P. These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.     

Adenosine 3':5'-monophosphate-mediated inhibition of myosin light chain phosphorylation in bovine aortic actomyosin.

Ca2+-dependent phosphorylation of the myosin light chains in bovine aortic native actomyosin is markedly depressed in the presence of cyclic AMP and its dependent protein kinase. This inhibition occurs with either cardiac, skeletal, or aortic protein kinase plus cyclic AMP, while little or no inhibition occurs with either cyclic AMP or protein kinase alone. The extent of inhibition is related to the concentration of protein kinase and approaches a maximum of approximately 50%. Concomitant with the inhibition of myosin light chain phosphorylation is (a) an increased phosphorylation of a 100,000-dalton moiety which possibly corresponds to the myosin light chain kinase present in the native actomyosin preparation and (b) a decrease in the actomyosin Mg2+-ATPase activity. These findings suggest that modulation of actin-myosin interactions by the cAMP system directly at the level of the contractile proteins may represent a mechanism by which beta adrenergic relaxation occurs in mammalian vascular smooth muscle.     

Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump

Mild trypsin treatment of canine cardiac microsomes consisting largely of sarcoplasmic reticulum vesicles produced a severalfold activation of oxalate-facilitated calcium uptake. The increase in calcium uptake was associated with an increase in ATP hydrolysis. Proteases other than trypsin were also effective although to a lesser degree. Trypsin produced a shift of the Ca2+ concentration dependency curve for calcium uptake toward lower Ca2+ concentrations, which was almost identical with that produced by phosphorylation of microsomes by cyclic AMP dependent protein kinase when the trypsin and the protein kinase were present at maximally activating concentrations. The Hill numbers (+/- SD) of the Ca2+ dependency after treatment of microsomes with trypsin (1.5 +/- 0.1) or protein kinase (1.7 +/- 0.1) were similar and were not significantly different from those for untreated control microsomes (1.6 +/- 0.1 and 1.8 +/- 0.1, respectively). Autoradiograms of sodium dodecyl sulfate-polyacrylamide electrophoretic gels indicate that 32P incorporation into phospholamban (Mr 27.3K) or its presumed monomeric subunit (Mr 5.5K) was markedly reduced when trypsin-treated microsomes were incubated in the presence of cyclic AMP dependent protein kinase and [gamma-32P]ATP compared to control microsomes incubated similarly but pretreated with trypsin inhibitor inactivated trypsin. The activation of calcium uptake by increasing concentrations of trypsin was paralleled by the reduction of phosphorylation of phospholamban. Trypsin treatment of microsomes previously thiophosphorylated in the presence of cyclic AMP dependent protein kinase and [gamma-35S]thio-ATP did not result in a loss of 35S label from phospholamban, which suggests that phosphorylation of phospholamban protects against trypsin attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of the activity of the (Ca2+ + Mg2+)-dependent adenosine triphosphatase of the human erythrocyte

We previously reported that the activity of the (Ca2+ + Mg2+)-dependent adenosine triphosphatase (ATPase) of the human erythrocyte membrane is inhibited by micromolar or nanomolar concentrations of cyclic AMP. Our further studies have now indicated that the inhibition of (Ca2+ + Mg2+)-dependent phosphohydrolase activity requires the participation of a membrane-associated cyclic AMP-dependent protein kinase and a membrane-associated protein substrate that is distinct from the ATPase itself. We have furthermore, identified a 20 kDa membrane protein which undergoes phosphorylation that is promoted by micromolar, but not millimolar, concentrations of cyclic AMP and which, when phosphorylated, undergoes dephosphorylation that is promoted by Ca2+. We suggest that this membrane component can participate in the modulation of the activity of the (Ca2+ + Mg2+)-dependent ATPase of the human erythrocyte.     

Cholinergic Receptor-Mediated Phosphorylation and Activation of Tyrosine Hydroxylase in Cultured Bovine Adrenal Chromaffin Cells

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phosphorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+-dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+-dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.