Phosphorylation of an actin · tropomyosin · troponin complex from human skeletal muscle

A human skeletal actin · tropomyosin · troponin complex was phosphorylated in the presence of [γ-³²P]ATP, Mg²⁺, adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 μM cyclic AMP. In the presence of 10⁻⁷ M Ca²⁺ and protein kinase 0.1 mole of [³²P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [³²P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca²⁺, 5 · 10⁻⁵ M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [³²P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [³²P]phosphate. Increasing the Ca²⁺ concentration did not significantly decrease the [³²P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstituted human skeletal actomyosin made with the [³²P]phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca²⁺.     

Phosphorylation of cardiac troponin by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

The purpose of this investigation was to characterize the phosphorylation of bovine cardiac troponin by cyclic AMP-dependent protein kinase. The purified troponin-tropomyosin complex from beef heart contained 0.78 +/- 0.15 mol of phosphate per mol of protein. Analysis of the isolated protein components indicated that the endogenous phosphate was predominately in the inhibitory subunit (TN-I) and the tropomyosin-binding subunit (TN-T) of troponin. When cardiac troponin or the troponin-tropomyosin complex was incubated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, the rate of phosphorylation was stimulated by cyclic AMP and inhibited by the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase. The 32P was incorporated specifically into the TN-I subunit with a maximal incorporation of 1 mol of phosphate per mol of protein. The maximal amount of phosphate incorporated did not vary significantly between troponin preparations that contained low or high amounts of endogenous phosphate. The Vmax of the initial rates of phosphorylation with troponin or troponin-tropomyosin as substrates was 3.5-fold greater than the value obtained with unfractionated histones. The rate or extent of phosphorylation was not altered by actin in the presence or absence of Ca2+. The maximal rate of phosphorylation occurred between pH 8.5 and 9.0. At pH 6.0 and 7.0 the maximal rates of phosphorylation were 13 and 45% of that observed at pH 8.5, respectively. These results indicate that cyclic AMP formation in cardiac muscle may be associated with the rapid and specific phosphorylation of the TN-I subunit of troponin. The presence of endogenous phosphate in TN-T and TN-I suggests that kinases other than cyclic AMP-dependent protein kinase may also phosphorylate troponin in vivo.     

Phosphorylation of filamin and other proteins in cultured fibroblasts.

Incubation of subcellular fractions of fibroblasts with [³²P]ATP demonstrated 10 phosphoproteins whose phosphorylation can be increased by cyclic AMP or cyclic AMP-dependent protein kinase. One of these phosphoproteins, MW 240,000, resembles the actin binding protein, filamin, and can be selectively precipitated by antibodies to chicken gizzard filamin. Furthermore chicken gizzard filamin can be phosphorylated by skeletal muscle protein kinase and cyclic AMP stimulates this reaction.     

Effect of phosphorylation of porcine cardiac troponin I by 3':5'-cyclic AMP-dependent protein kinase on the actomyosin ATPase activity

1. Porcine cardiac native tropomyosin was phosphorylated by bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. Most of the phosphate incorporation was observed in troponin I, the maximum of which was 0.7 mol of Pi per mol of troponin I. 2. In the presence of phosphorylated native tropomyosin, actomyosin ATPase activity was 15-40% lower than that in the presence of the unphosphorylated preparation at all calcium ion concentrations (1.5 x 10(-8) M-2.4 x 10(-5) M). Half-maximum activation of ATPase was obtained with a concentration of 7 x 10(-7) M Ca2+ (unphosphorylated) and 1.3 x 10(-6) M Ca2+ (phosphorylated), respectively. Maximum ATPase activity was reached with 3 x 10(-6) M Ca2+ (unphosphorylated) and 1.0 x 10(-5) M Ca2+ (phosphorylated). 3. Porcine cardiac troponin I isolated by affinity chromatography inhibited ATPase activity of desensitized actomyosin in the presence of tropomyosin. There was little difference between phosphorylated troponin I and a control preparation with regard to the inhibitory effect of ATPase activity. 4. Troponin C from rabbit skeletal muscle neutralized the inhibitory effect of troponin I. The minimum amount of troponin C required for complete neutralization was approximately equimolar to troponin I. The inhibitory effect of phosphorylated troponin I was neutralized by troponin C less effectively than that of unphosphorylated preparation.     

The in vitro phosphorylation of actin from rat cerebral cortex

Actin was phosphorylated by a cyclic AMP-stimulated protein kinase in a lysed synaptosomal fraction incubated with [gamma-32P]ATP, while calcium had no effect on endogenous labeling of the protein. Incubation of an intact synaptosomal fraction with 32P-inorganic phosphate did not lead to any detectable phosphorylation of actin in the presence or absence of dibutyryl-cyclic AMP, or chemical depolarization. It is suggested that actin is not phosphorylated in the physiologically relevant intact synaptosomes but gains access to protein kinases on lysis.     

Cyclic AMP-stimulated phosphorylation of bovine tracheal smooth muscle contractile and non-contractile proteins.

1. Various proteins isolated from bovine tracheal smooth muscle were examined as phosphate acceptor substrates for a cyclic AMP-dependent protein kinase isolated from the same tissue. A fraction prepared in a manner similar to that of skeletal muscle troponin was the best substrate of the presumptive contractile proteins isolate. Actomyosin and tropomyosin were relatively poor substrates. 2. An assay was developed for the rapid detection in a large number of samples of the muscle specific substrate for the protein kinase on which we reported previously. 3. Using this assay, the muscle specific substrate found in bovine tracheal smooth muscle was partially purified resulting in a preparation which when resolved by polyacrylamide gel electrophoresis showed a single peak of 32P incorporated, and which could be further characterized. 4. Our findings suggest that the substrate contains a protein subunit of molecular weight 19 000, which can be phosphorylated at serine and threonine residues, in the presence of cyclic AMP and protein kinase. The phosphate is in a covalent ester linkage with these residues. 5. A phosphoprotein phosphatase was isolated from the bovine tracheal smooth muscle. 6. Bovine tracheal smooth muscle contains cyclic AMP dependent protein kinase and phosphoprotein phospahatase activity as well as the muscle specific substrate, suggesting that these elements may be part of a mechanism which regulates smooth muscle tone.     

Two calcium regulation systems in squid (Ommastrephes sloani pacificus) muscle. Preparation of calcium-sensitive myosin and troponin-tropomyosin.

The Ca-regulatory system in squid mantle muscle was studied. The findings were as follows. (a) Squid mantle myosin B (squid myosin B) was Ca-sensitive, and its Ca-sensitivity was unaffected by addition of a large amount of rabbit skeletal myosin (skeletal myosin) or rabbit skeletal F-actin (skeletal F-actin). (b) Squid myosin was prepared from the mantle muscle. It showed a heavy chain component and two light chain components in the SDS-gel electrophoretic pattern: the molecular weights of the latter two were 17,000 and 15,000. Actomyosin reconstituted from squid myosin and skeletal (or squid) actin showed Ca-sensitivity in superprecipitation and Mg-ATPase assays. EDTA- treatment had no effect on the Ca-sensitivity of squid myosin. (c) Squid mantle actin (squid actin) was prepared by the method of Spudich and Watt. Hybrid actomyosin reconstituted by using the pure squid actin preparation with skeletal myosin showed no Ca-sensitivity in Mg-ATPase assay, whereas that reconstituted using crude squid actin showed marked Ca-sensitivity. The crude squid actin contained four protein components which were capable of associating with F-actin in 0.1 M KCl, 1 mM MgCl2 and 20 mM Tris-maleate (pH7.5). (d) Native tropomyosin was prepared from squid mantle muscle, and it conferred Ca-sensitivity on skeletal actomyosin as well as on a hybrid actomyosin reconstituted from squid actin and skeletal myosin. (e) Squid native tropomyosin was separated into troponin and tropomyosin fractions by placing it in 0.4 M LiCl at pH 4.7. The troponin fraction was further purified by DEAE-cellulose chromatography. Squid troponin thus obtained was different in mobility from rabbit skeletal or carp dorsal troponin; three bands of squid troponin corresponded to molecular weights of 52,000, 28,000, and 24,000 daltons. It could confer Ca-sensitivity in the presence of tropomyosin on skeletal actomyosin as well as on a hybrid reconstituted from squid actin and skeletal myosin. (f) Squid myosin B, and two hybrid actomyosins were compared as regards Ca and Sr requirements for their Mg-ATPase activities. The myosin-linked regulatory system rather than the thin-filament-linked regulatory system was predominant in squid myosin B. Squid myosin B required higher Ca2+ and Sr2+ concentrations for Mg-ATPase activity; half-maximal activation of Mg-ATPase was obtained at 0.8 micron Ca2+ and 28 micron Sr2+ with skeletal myosin B, and at 2.5 micron Ca2+ and 140 micron Sr2+ with squid myosin B.     

Platelet glycoprotein Ib beta is phosphorylated on serine 166 by cyclic AMP-dependent protein kinase

Platelet responses are inhibited by agents such as prostaglandin E1 that increase the cytoplasmic concentration of cyclic AMP. Inhibition is thought to result from phosphorylation of specific proteins. One protein that becomes phosphorylated is glycoprotein (GP) Ib beta, a component of the GP Ib.IX complex. We have suggested that phosphorylation of GP Ib beta inhibits the collagen-induced polymerization of actin. The aim of the present study was to identify the amino acid(s) in GP Ib beta that is phosphorylated. Purified GP Ib.IX complex was phosphorylated by the catalytic subunit of purified bovine cyclic AMP-dependent protein kinase in the presence of [gamma-32P]ATP. Phosphoamino acid analysis showed that in GP Ib beta, [32P]phosphate was incorporated only into serine and was in a single tryptic peptide. Amino acid sequencing showed that this peptide was from the cytoplasmic domain of GP Ib beta and encompassed residues 161-175. A single serine residue, serine 166, contained the radiolabel. To determine whether the same residue was phosphorylated in intact platelets, GP Ib beta was isolated from 32P-labeled platelets before or after their exposure to prostaglandin E1. In both cases, radiolabel was present in phosphoserine and was in a single tryptic peptide. This peptide was the same as that which was phosphorylated in the purified GP Ib.IX complex, as shown by its identical mobility on two-dimensional tryptic maps, the presence of a positively charged residue in the fourth position, and the presence of the radiolabel in the sixth position of the peptide. This study shows that when cyclic AMP concentrations rise in platelets, the cytoplasmic domain of GP Ib beta is phosphorylated on serine 166, probably by cyclic AMP-dependent protein kinase. We suggest that phosphorylation of this residue may contribute to the inhibitory actions of cyclic AMP by inhibiting collagen-induced polymerization of actin.     

Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[gamma-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143,000, 60,000, 42,000, 33,000, and 11,000. The 143,000 MW substrate was identified as C-protein; the 42,000 MW substrate is probably actin; the 33,000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60,000 and 11,000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein as phosphorylated to the extent of approximately 0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5-6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of approximately 0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitivity of actomyosin ATPase to calcium and strontium ions. Effect of hybrid troponins

1. Hybrid or reconstituted troponins were prepared from troponin components of rabbit skeletal muscle and porcine cardiac muscle and their effect on the actomyosin ATPase activity was measured at various concentrations of Ca2+ or Sr2+. The Ca2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing cardiac troponin I was slightly higher than that with troponin containing skeletal troponin I. The Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing skeletal troponin C was higher than that with troponin containing cardiac troponin C. 2. Reconstituted cardiac troponin was phosphorylated by cyclic AMP-dependent protein kinase. The Ca2+ sensitivity of actomyosin ATPase with cardiac troponin decreased upon phosphorylation of troponin I; maximum ATPase activity was depressed and the Ca2+ concentration at half-maximum activation increased. On the other hand, phosphorylation of troponin I did not change Sr2+ sensitivity. 3. The inhibitory effect of cardiac troponin I on the actomyosin ATPase activity was neutralized by increasing the amount of brain calmodulin at high Ca2+ and Sr2+ concentrations but not at low concentrations. 4. ATPase activity of actomyosin with a mixture of troponin I and calmodulin was assayed at various concentrations of Ca2+ or Sr2+. The Ca2+ or Sr2+ sensitivity of actomyosin ATPase containing skeletal troponin I was approximately the same as that of actomyosin ATPase containing cardiac troponin I. Phosphorylation of cardiac troponin I did not change the Ca2+ sensitivity of the ATPase. 5. The Ca2+ or Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin I-T-calmodulin was higher than that of actomyosin ATPase with the mixture of troponin I and calmodulin. Maximum ATPase activity was lower than that with the mixture of troponin I and calmodulin.     

Ca2+-dependent protein phosphorylation associated with microsomal fraction of rat pancreas

Microsomes isolated from cat pancreas were incubated with [gamma-32P]ATP in the presence or absence of Ca2+. Following fractionation of phosphoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single microsomal protein with an apparent molecular mass of 77,000 dalton (77K) was found to be phosphorylated in a Ca2+-dependent mechanism. Maximal phosphate incorporation into the 77K protein was observed at 10(-6) mol/l [Ca2+] and was 4-fold higher than in the absence of Ca2+. The 77K phosphoprotein showed characteristic of a stable phosphoester rather than an acyl phosphate. Measurable phosphate incorporation into the 77K protein was noted 5 s following addition of [gamma-32P]ATP and reached maximum at 9-10th min. The lack of effect of exogenous cyclic AMP, cyclic AMP-dependent protein kinase, calmodulin, the calmodulin antagonist trifluoperazine, leupeptin and the suppression of phosphorylation by some phospholipid-interacting drugs suggested that the 77K protein is a substrate for cyclic AMP- and calmodulin-independent, Ca2+-activated phospholipid-sensitive kinase activity. Centrifugation of the pancreatic homogenate in a ficoll-sucrose density gradient indicated that both the 77K protein and enzyme were associated in a fraction enriched in rough endoplasmic reticulum.     

Phosphorylation of partially purified 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

A new improved method for purification of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) from rat spleen is described. The catalytic subunit of cyclic AMP-dependent protein kinase in the presence of MgATP stimulated about 3-fold the activity of this partially purified enzyme activity. When [gamma-32P]ATP was included in the assay mixture, the analysis of phosphoprotein products by SDS/polyacrylamide-gel electrophoresis and autoradiography showed the incorporation of [32P]phosphate into a single protein band of about 30 kDa. Analysis of the phosphorylated amino acids indicated that the phosphate was incorporated into a serine residue. Activation of the acetylation reaction by the protein kinase was reversible. The reversal of the activation was coincident with the loss of the [32P]phosphate incorporated into the 30 kDa protein band, which suggests that the acetyltransferase is regulated by a phosphorylation-dephosphorylation mechanism dependent on cyclic AMP.     

Relationship between phosphorylation of tyrosine aminotransferase and regulation of its synthesis by cyclic AMP and hormones.

Tyrosine adminotransferase (EC 2.6.1.5) has been found to be phosphorylated in intact rat hepatoma cells in culture. Incorporation of [32p]i into the enzyme is rapid and is exclusively found as phosphoserine. Cycloheximide treatment reduced phosphorylation of the aminotransferase only slightly and in the presence of three different inducers of this enzyme, dexamethasone, insulin, and dibutyryl cyclic AMP, [32P]I incorporation was increased. It is concluded that [32p]i incorporation into this enzyme probably reflects turnover of phosphate groups associated with pre-existing enzyme molecules catalyzed by a cyclic AMP-independent protein kinase.     

Cyclic AMP-dependent phosphorylation of filamin in mammalian smooth muscle.

Filamin is a high molecular weight actin-binding protein found in large quantities in smooth muscle and other non-muscle cells. We have studied the phosphorylation of filamin in a mammalian smooth muscle, the guinea pig vas deferens. Intact vas deferens incorporated [32P]orthophosphate into filamin. Incubation of particulate fractions of vas deferens with [gamma-32P]ATP resulted in 32P-labeling of filamin. Cyclic AMP stimulated this phosphorylation, whereas cyclic GMP and Ca2+ had no effect. Purified vas deferens filamin can be phosphorylated by purified cyclic AMP-dependent protein kinase. We have compared cyclic AMP and cyclic GMP effects on phosphorylation in smooth muscle. Cyclic GMP stimulated phosphorylation of two particulate proteins, G-I (Mr = 130,000) a protein previously described by Casnellie, J. E., and Greengard, P. (1974) Proc. Natl. Acad, Sci. U.S.A. 71, 1891-1895 and G-III (Mr = 240,000). Both proteins and the kinase responsible for their phosphorylation appear to be membrane-bound. Phosphorylation of both proteins is stimulated by cyclic GMP (Ka = 3 x 10(-8) M), cyclic AMP (Ka = 3 x 10(-7) M), and to a lesser degree by Ca2+. In contrast, filamin phosphorylation is due to a soluble kinase stimulated only by cyclic AMP (Ka = 3 x 10(-7) M) and not by cyclic GMP or Ca2+.     

Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation

Calponin isolated from chicken gizzard smooth muscle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system composed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phosphorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phosphorylation was unaffected. Furthermore, calponin inhibited the actin-activated MgATPase of fully phosphorylated or thiophosphorylated myosin. Although calponin is a Ca2(+)-binding protein, inhibition did not require Ca2+. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not dependent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, but not by cAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein retained calmodulin and tropomyosin binding capabilities, but actin binding was greatly reduced. The calponin-actin interaction, therefore, appears to be responsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be involved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Calponin function in turn is regulated by Ca2(+)-dependent phosphorylation.     

Myosin and actomyosin from human skeletal muscle.

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (+/-2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37 degrees C. Activation of the Mg-ATPase activity of Ca2+-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 muM Ca2+ concentration (CaEGTA binding constant equals 4.4 - 10(5) at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6-9 range, the Ca2+-ATPase activity of the subfragment 1 was 1.8- and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6-10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca2+-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca2+ is similar to that of rabbit fast or slow muscle.     

Phosphorylation of troponin and the effects of interactions between the components of the complex

1. The troponin complex from skeletal muscle contains approximately 1 mol of phosphate/80000g of complex, covalently bound to the troponin T component. 2. On prolonged incubation of the troponin complex or troponin T with phosphorylase kinase the phosphate content of troponin T was increased to approx. 3mol/mol. 3. On prolonged incubation of troponin I with phosphorylase kinase up to 1.6mol of phosphate/mol were incorporated. 4. Phosphorylation of troponin I was greatly inhibited by troponin C owing to the strong interaction between these proteins. Thus in the troponin complex troponin T was the main substrate for phosphorylase kinase. The phosphorylation of isolated troponin T was also inhibited by troponin C. 5. Troponin I was phosphorylated when the troponin complex was incubated with a bovine cardiac 3′:5′-cyclic AMP-dependent protein kinase. Troponin T either in its isolated form or in the troponin complex was not phosphorylated by bovine protein kinase to any significant extent under the conditions used. 6. If the troponin complex was dephosphorylated to 0.2mol/mol, or phosphorylated up to 2.5mol/mol there was no significant effect on the ability of normal concentrations to confer Ca²⁺ sensitivity on the adenosine triphosphatase of densensitized actomyosin.     

Insulin action inhibits insulin-like growth factor-II (IGF-II) receptor phosphorylation in H-35 hepatoma cells. IGF-II receptors isolated from insulin-treated cells exhibit enhanced in vitro phosphorylation by casein kinase II

Insulin caused a rapid, dose-dependent increase in the binding of 125I-insulin-like growth factor-II (IGF-II) to the surface of cultured H-35 hepatoma cells. The [32P]phosphate content of the IGF-II receptors, immunoprecipitated from extracts of H-35 cell monolayers previously incubated with [32P]phosphate for 24 h, was decreased after brief exposure of the cells to insulin. Analysis of tryptic digests of labeled IGF-II receptors by bidimensional peptide mapping revealed that the decrease in the content of [32P]phosphate occurred to varying degrees on three tryptic phosphopeptides. Thin layer electrophoresis of an acid hydrolysate of isolated IGF-II receptors revealed the presence of [32P] phosphoserine and [32P]phosphothreonine. Insulin treatment of cells caused a decrease in the labeled phosphoserine and phosphothreonine content of IGF-II receptors. The ability of a number of highly purified protein kinases (cAMP-dependent protein kinase, protein kinase C, phosphorylase kinase, and casein kinase II) to catalyze the phosphorylation of purified IGF-II receptors was examined. Casein kinase II was the only kinase capable of catalyzing the phosphorylation of the IGF-II receptor on serine and threonine residues under the conditions of our assay. Bidimensional peptide mapping revealed that the kinase catalyzed phosphorylation of the IGF-II receptor on a tryptic phosphopeptide which comigrated with the main tryptic phosphopeptide found in receptors obtained from cells labeled in vivo with [32P]phosphate. IGF-II receptors isolated by immunoadsorption from insulin-treated H-35 cells were phosphorylated in vitro by casein kinase II to a greater extent than the receptors isolated from control cells. Similarly, IGF-II receptors from plasma membranes obtained from insulin-treated adipocytes were phosphorylated by casein kinase II to a greater extent than the receptors from control adipocyte plasma membranes. Thus, the insulin-regulated phosphorylation sites on the IGF-II receptor appear to serve as substrates in vivo for casein kinase II or an enzyme with similar substrate specificity.     

Dependence on Ca2+ and tropomyosin of the actin-activated ATPase activity of phosphorylated gizzard myosin in the presence of low concentrations of Mg2+

Ca2+ and tropomyosin are required for activation of ATPase activity of phosphorylated gizzard myosin by gizzard actin at less than 1 mM Mg2+, relatively low Ca2+ concentrations (1 microM), producing half-maximal activation. At higher concentrations, Mg2+ will replace Ca2+, 4 mM Mg2+ increasing activity to the same extent as does Ca2+ and abolishing the Ca2+ dependence. Above about 1 mM Mg2+, tropomyosin is no longer required for activation by actin, activity being dependent on Ca2+ between 1 and 4 mM Mg2+, but independent of [Ca2+] above 4 mM Mg2+. Phosphorylation of the 20,000-Da light chain of gizzard myosin is required for activation of ATPase activity by actin from chicken gizzard or rabbit skeletal muscle at all concentrations of Mg2+ employed. The effect of adding or removing Ca2+ is fully reversible and cannot be attributed either to irreversible inactivation of actin or myosin or to dephosphorylation. After preincubating in the absence of Ca2+, activity is restored either by adding micromolar concentrations of this cation or by raising the concentration of Mg2+ to 8 mM. Similarly, the inhibition found in the absence of tropomyosin is fully reversed by subsequent addition of this protein. Replacing gizzard actin with skeletal actin alters the pattern of activation by Ca2+ at concentrations of Mg2+ less than 1 mM. Full activation is obtained with or without Ca2+ in the presence of tropomyosin, while in its absence Ca2+ is required but produces only partial activation. Without tropomyosin, the range of Mg2+ concentrations over which activity is Ca2+-dependent is restricted to lower values with skeletal than with gizzard actin. The activity of skeletal muscle myosin is activated by the gizzard actin-tropomyosin complex without Ca2+, although Ca2+ slightly increases activity. The Ca2+ sensitivity of reconstituted gizzard actomyosin is partially retained by hybrid actomyosin containing gizzard myosin and skeletal actin, but less Ca2+ dependence is retained in the hybrid containing skeletal myosin and gizzard actin.     

Comparison of Ca2+-dependent effects of caldesmon-tropomyosin-calmodulin and troponin-tropomyosin complexes on the structure of F-actin in ghost fibers and its interaction with myosin heads

Comparison of two types of Ca2+-regulated thin filament, reconstructed in ghost fibers by incorporating either caldesmon-gizzard tropomyosin-calmodulin or skeletal muscle troponin-tropomyosin complex, was performed by polarized microphotometry. The changes in actin structure under the influence of these regulatory complexes, as well as those upon the binding of the myosin heads, were followed by measurements of F-actin intrinsic tryptophan fluorescence and the fluorescence of phalloidin-rhodamine complex attached to F-actin. The results show that in the presence of smooth muscle tropomyosin and calmodulin, caldesmon causes Ca2+-dependent alterations of actin conformation and flexibility similar to those induced by skeletal muscle troponin-tropomyosin complex. In both cases, transferring of the fiber from '-Ca2+' to '+Ca2+' solution increases the number of turned-on actin monomers. However, whereas troponin in the absence of Ca2+ potentiates the effect of skeletal muscle tropomyosin, caldesmon-calmodulin complex inhibits the effect of smooth muscle tropomyosin. This difference seems to be due to the qualitatively different alterations in the structure and flexibility of F-actin in ghost fibers evoked by smooth and skeletal muscle tropomyosins. Troponin can bind to F-actin-smooth muscle tropomyosin-caldesmon complex and, in the presence of Ca2+, release the restraint by caldesmon for S-1-induced alterations of conformation, and reduce that for flexibility of actin in ghost fibers. This effect seems to be related to the abolishment by troponin of the potentiating effect of tropomyosin on caldesmon-induced inhibition of actomyosin ATPase activity.