Hormone-induced phosphorylation of a 16000 Dalton polypeptide following meiosis reinitiation in starfish oocytes

In vitro phosphorylation of endogenous proteins is increased in homogenates prepared from 1-methyladenine-treated starfish oocytes when compared with control oocytes, although addition of the hormone to homogenates from control oocytes has no such effect. Following hormonal stimulation the best endogenous substrate is by far a 16 000 dalton (D) protein, the content of which also seems to increase, perhaps through proteolysis of a 21 000 D precursor. cAMP-dependent protein kinases are not involved in either basal or hormone-stimulated phosphorylations, as demonstrated by the lack of effect of either cAMP or of the heat-stable inhibitor of cAMP-dependent protein kinase on the extent of phosphorylation of individual endogenous substrates. Addition of 0.1 mM Ca²⁺ decreases to some extent the protein kinase activity in starfish homogenates and specifically suppresses the phosphorylation of a 40 000 D membrane protein. Starfish oocytes appear to contain myosin light chain kinase activity, as shown by the ability of homogenates to catalyse phosphorylation of exogenous 20 000 D myosin light chains.     

Cyclic nucleotide-dependent endogenous phosphorylation of rabbit myometrium membranes

The membrane-bound protein kinase activity in plasma membranes (PM) and sarcoplasmic reticulum (SR) of rabbit myometrium was revealed, which catalyzes the synthesis of protein phosphoester products. cAMP had no effect on the phosphorylation of membrane substrates by soluble protein kinases I and II as well as by the membrane-bound enzyme of SR. At the same time, cAMP (10(-8) stimulated by 200% the phosphorylation of sarcolemmal components at functional rest (FR). In preparations obtained from pregnant animals, cAMP (10(-8) and 10(-5) M) stimulated the phosphorylation of PM 7- and 3-fold, respectively. cGMP had no effect on the phosphorylation of PM and SR proteins at FR. At 10(-5) and 10(-8) M, cGMP stimulated endogenous phosphorylation of PM and SR 7- and 4-fold, respectively. In pregnancy, the degree of endogenous phosphorylation of PM and SR increased by 70% and 260% as compared to that at FR; the activity of soluble protein kinases decreased two times under these conditions. At FR, the sarcolemmal proteins with Mr 35 000, 57 000, 89 000 and 174 000 underwent phosphorylation. The phosphorylation of the proteins with Mr 35 000 and 57 000 was cAMP-dependent. In pregnant animals sarcolemma, the phosphorylation affected the proteins with Mr 47 000, 57 000 and 174 000 and was cAMP-dependent for the former two proteins and cGMP-dependent for the latter protein. At FR, two SR proteins with Mr 47 000 and 168 000, while in pregnant animals the proteins with Mr 47 000, 132 000 and 168 000 were phosphorylatable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of endogenous proteins of cilia from Paramecium tetraurelia in vitro

Several endogenous substrate proteins of cilia from axenically grown Paramecium tetraurelia were phosphorylated in vitro by inherent protein kinases (PKs). Labeling was stimulated by cAMP and to a lesser extent by cGMP. ATP breakdown was most rapid in cilia and subciliary fractions. Using multiple substrate additions during incubations it was shown that phosphorylation was almost completed within 30 s. Very little dephosphorylation by phosphoprotein phosphatases occurred during 5 min of incubation. Proteins of molecular weight of 103 000 and 46 000 were shown to be particularly associated with axonemal structures of the cilia. No distinct differences in phosphorylation patterns were apparent in ciliary membrane vesicles of low and high buoyant density, which exhibit differential enzyme patterns. cAMP receptor proteins were identified by use of the photoaffinity label 8-azido-[32P]cAMP. Receptor proteins with apparent molecular weights of 43 000, 39 000, 37 000, 31 000 and 30 000 were probably related to the regulatory subunits of cAMP-dependent protein kinases as evidenced by inhibition of incorporation of the photoaffinity label by low concentrations of cAMP. Tagging of a protein of 85 000 molecular weight was specifically inhibited by cGMP, thus in all likelihood it corresponded to a cGMP-dependent protein kinase. Corresponding autophosphorylated protein bands were observed with gamma-[32P]ATP. A functional role for protein phosphorylation in cilia of Paramecium remains to be established.     

Phosphorylation of proteins in the kidney papillary zone by endogenous cAMP-dependent protein kinases

Using SDS-PAAG electrophoresis with subsequent autoradiography, several proteins from plasma membranes and cell cytosol of rat kidney papillary zone were identified as substrates for endogenous cAMP-dependent protein kinases. The cAMP-dependent phosphorylation of plasma membrane proteins was made possible only after the destruction of membrane vesicles. Plasma membrane and cytosol fractions were found to contain a 58 kDa protein whose properties are similar to those of the regulatory subunit of cAMP-dependent protein kinase of the second type. It was shown also that the content of endogenous substrates of cAMP-dependent protein kinases in cell cytosol is higher than that in plasma membranes.     

Cyclic adenosine monophosphate-dependent and -independent protein kinase activity of renal brush border membranes.

Kinase(s) in brush border membranes, isolated from rabbit renal proximal tubules, phosphorylated proteins intrinsic to the membrane and exogenous proteins. cAMP stimulated phosphorylation of histone; phosphorylation of protamine was cAMP independent. cAMP-dependent increases in phosphorylation of endogenous membrane protein were small, but highly reproducible. Most of the ³²P incorporated into membranes represented phosphorylation of serine residues, with phosphorylthreonine comprising a minor component. cAMP did not alter the electrophoretic pattern of ³²P-labeled membrane polypeptides. The small cAMP-dependent phosphorylation of brush border membrane proteins was not due to membrane phosphodiesterase or adenylate cyclase activities. Considerable cAMP was found “endogenously” bound to the membranes as prepared. However, this did not result in preactivation of the kinase since activity was not inhibited by a heat-stable protein inhibitor of cAMP-dependent protein kinases. With intrinsic membrane protein as phosphate acceptor, the relationship between rate of phosphorylation and ATP concentration appeared to follow Michaelis-Menton kinetics. With histone the relationship was complex. cAMP did not affect the apparent K_m for histone. One-half maximal stimulation of the rate of histone phosphorylation was obtained with 7 × 10^?8m cAMP. The K_a values for dibutyryl cAMP, cIMP, and cGMP were one to two orders of magnitude greater. Treatment of brush border membranes with detergent greatly increased the dependency of histone phosphorylation on cAMP. Phosphorylations of intrinsic membrane protein and histone were nonlinear with time, due in part to the lability of the protein kinase, the hydrolysis of ATP, and minimally to the presence of phosphoprotein phosphatase in the border membrane. The membrane phosphoprotein phosphatase was unaffected by cyclic nucleotides. Protein kinase activity was also found in cytosolic and crude particulate fractions of the renal cortex. Activity was enriched in the brush border membrane relative to that in the crude membrane preparation. The kinase activities in the different loci were distinct both in relative activities toward different substrates and in responsiveness to cAMP.     

cAMP-dependent protein phosphorylation of membrane proteins in the parotid gland, platelets and liver. Comparison of a 22-kDa phosphoprotein from rat parotid microsomes (protein III) with phosphoproteins of similar molecular size from platelet and liver membranes

Stimulation of secretion in exocrine secretory glands leads to the phosphorylation of a 22-kDa membrane protein (protein III) whose function is still unknown [Jahn et al. (1980) Eur. J. Biochem. 112, 345-352; Jahn & S?ling (1980) Proc. Natl Acad. Sci. USA 78, 6903-6906]. This report describes the comparison of this protein with phosphorylated membrane proteins of similar molecular mass in platelets and liver. Incubation of platelets with agents which raise the intracellular cAMP concentration results in the phosphorylation of a 22-kDa protein which is also phosphorylated in membrane preparations by endogenous kinases or by exogenous cAMP-dependent protein kinase. It is shown that this protein is distinct from protein III although both proteins have the same molecular mass and are substrates of cAMP-dependent protein kinase. In contrast to platelets, protein III could be demonstrated in liver microsomes. This indicates that the function of protein III is not exclusively linked to the stimulus-secretion coupling in exocrine cells.     

Phosphorylation of Endogenous Proteins by Adenosine 3':5'-Monophosphate-Dependent Protein Kinase in Mouse Neuroblastoma Cells

Abstract: Increased intracellular adenosine 3':5'-monophosphate (cAMP) levels and activation of cAMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) in vivo were correlated in mouse neuroblastoma cells grown in the presence of 1 mM-⁶ N.O ²-dibutyryl 3':5'-monophosphate (Bt₂cAMP). The time course for activation showed that cAMP-dependent protein kinases were activated by 30 min. A heat-stable inhibitor protein inhibited a majority of activated cAMP-dependent protein kinase. Activation of cAMP—dependent protein kinase caused additional phosphorylation of proteins when compared with untreated control cells, as demonstrated by endogenous phosphorylation of proteins in vitro using [γ-³²P]ATP and analysis by two—dimensional polyacrylamide gel electrophoresis. The phosphorylation data show selective phosphorylation of specific proteins by cAMP-independent and cAMP-dependent protein kinase. Among the proteins in the postmitochondrial supernatant fraction phosphorylated by cAMP-dependent protein kinases, two proteins with a molecular weight of 43,000 were heavily phosphorylated. It is suggested that phosphorylation of cellular proteins by cAMP-dependent protein kinases might be involved in the cAMP-modulated biochemical changes in neuroblastoma cells.     

Regulation of plasma membrane protein phosphorylation in two mammalian cell types.

The appreciation of protein phosphorylation as a ubiquitous mechanism for the post-translational control of protein function has drawn our attention to the phosphorylation of plasma membrane proteins. We have studied this phenomenon in the human erythrocyte and rat adipocyte, and have observed several features, common to the two systems, which may be of general significance. In examining protein phosphorylation in intact cells incubated with 32Pi, it is evident that the 32P-polypeptides of the plasma membrane are among the most highly labelled species in the cell, despite their minor contribution to overall protein content. The addition of epinephrine (to adipocytes) or cAMP (to erythrocytes) increases the phosphorylation of certain peptides, whereas others are unaffected. The protein kinases mediating these phosphorylations are present in the plasma membrane as isolated, and can be divided into two groups--cAMP dependent and cAMP independent. These two classes of kinase differ markedly in their substrate specificity toward endogenous and exogenous polypeptide substrates. Two classes of protein kinases with similar properties can be detected in the cytoplasm. The relationship between the membrane-bound and cytoplasmic enzymes is uncertain. The potential roles of the plasma membrane cAMP dependent protein kinases are evident from the diverse effects of cAMP on surface properties; however, the prevalence of plasma membrane proteins phosphorylated via cAMP independent pathways is striking. Thus, elucidation of the regulatory properties of the plasma membrane cAMP independent protein kinases may give new insight into the control of a variety of surface phenomena not mediated by cAMP.     

cAMP-dependent phosphorylation in the pig brain

Protein phosphorylation in the cytosol, membranes and nuclei of pig brain cells was investigated. Endogenous cAMP-dependent phosphorylation was observed in all fractions studied. The degree of activation of this process by cAMP was different, depending on the fraction. The molecular weights of the endogenous protein substrates were found to be equal to 60 000, 45 000 and 28 000 for the nuclei, 80 000, 71 000, 31 000, 25 000, 16 000 and 11 000 for the membranes and 280 000, 80 000, 67 000, 53 000, 32 000, 25 000 and 22 000 for cytosol. It was shown that the majority of these proteins can be phosphorylated by the exogenous cAMP-dependent pig brain protein kinase.     

The role of Ca2+ and cyclic AMP in the phosphorylation of rat-liver soluble proteins by endogenous protein kinases

Rat liver soluble proteins were phosphorylated by endogenous protein kinase with [gamma-32P]ATP. Proteins were separated in dodecyl sulphate slab gels and detected with the aid of autoradiography. The relative role of cAMP-dependent, cAMP-independent and Ca2+-activated protein kinases in the phosphorylation of soluble proteins was investigated. Heat-stable inhibitor of cAMP-dependent protein kinase inhibits nearly completed the phosphorylation of seven proteins, including L-type pyruvate kinase. The phosphorylation of eight proteins is not influenced by protein kinase inhibitor. The phosphorylation of six proteins, including phosphorylase, is partially inhibited by protein kinase inhibitor. These results indicate that phosphoproteins of rat liver can be subdivided into three groups: phosphoproteins that are phosphorylated by (a) cAMP-dependent protein kinase or (b) cAMP-independent protein kinase; (c) phosphoproteins in which both cAMP-dependent and cAMP-independent protein kinase play a role in the phosphorylation. The relative phosphorylation rate of substrates for cAMP-dependent protein kinase is about 15-fold the phosphorylation rate of substrates for cAMP-independent protein kinase. The Km for ATP of cAMP-dependent protein kinase and phosphorylase kinase is 8 microM and 38 microM, respectively. Ca2+ in the micromolare range stimulates the phosphorylation of (a) phosphorylase, (b) a protein with molecular weight of 130 000 and (c) a protein with molecular weight of 15 000. The phosphate incorporation into a protein with molecular weight of 115 000 is inhibited by Ca2+. Phosphorylation of phosphorylase and the 15 000-Mr protein in the presence of 100 microM Ca2+ could be completely inhibited by trifluoperazine. It can be concluded that calmodulin is involved in the phosphorylation of at least two soluble proteins. No evidence for Ca2+-stimulated phosphorylation of subunits of glycolytic or gluconeogenic enzymes, including pyruvate kinase, was found. This indicates that it is unlikely that direct phosphorylation by Ca2+-dependent protein kinases is involved in the stimulation of gluconeogenesis by hormones that act through a cAMP-independent, Ca2+-dependent mechanism.     

ACTH-SENSITIVE PROTEIN KINASE FROM RAT BRAIN MEMBRANES

—The protein kinase which in rat brain synaptosomal plasma membranes is responsible for the phosphorylation of a protein band B-50 (MW 48, 000) was inhibited by the behaviorally active peptide ACTH_1–24 and not stimulated by cAMP. Treatment with 0.5% Triton X-100 in 75 mM-KCl solubilized 15% of the total B-50 protein kinase activity and preserved the sensitivity of the enzyme to ACTH_1–24. The rate of endogenous phosphorylation of protein band B-50 was different in intact SPM, solubilized fraction and residue. cAMP stimulated the endogenous phosphorylation of the solubilized fraction in a rather general manner. The solubilized membrane material also phosphorylated B-50 proteins which were previously extracted from membranes. Column chromatography of the solubilized material over DEAE-cellulose pointed to the presence of multiple protein kinase activities from rat brain synaptosomal plasma membranes, one of which was the ACTH-sensitive B-50 protein kinase.     

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.     

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

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

Renal brush border membrane phosphorylation: influence of pH, cAMP and ATP concentrations, parathyroid hormone status, and dietary phosphate

It is known that parathyroidectomy, administration of parathyroid hormone (PTH), and dietary phosphate depletion or excess result in variations in phosphaturia and in phosphate transport through brush border membrane vesicles isolated from the kidneys of various animals. Parathyroid hormone has been shown to ultimately phosphorylate some brush border membrane proteins and it has been postulated that the resulting phosphaturia is related to this phosphorylation. However, it is not known whether the regulation of phosphate transport by the diet is affected through similar pathways. Our experiments were designed to study the phosphorylation of brush border membrane with [gamma-32P]ATP using the intrinsic protein kinase of the membranes. Five groups of rats were used: normal, phosphate loaded, phosphate depleted, and thyroparathyroidectomized and acutely loaded with parathyroid hormone. In each series of animals, the proteins whose phosphorylation was cAMP dependent were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and their phosphorylation with various concentrations of ATP, in the presence or absence of cAMP in the incubation medium, was quantified. In the normal rat, 17 proteins were phosphorylated, the phosphorylation of two of them (Mr, 71 000 and 84 000) being cAMP dependent. Maximal response to cAMP for these two proteins was obtained with 10 microM cAMP. The peaks of phosphorylation were observed at pH 7 for protein 71 000 and pH 10 for protein 84 000. When brush border membranes from normal rats were incubated with 10-100 microM ATP, cAMP-dependent phosphorylation increased to reach a maximal phosphorylation of 4.44 +/- 0.90 pmol/mg protein for protein 71 000 and 1.32 +/- 0.15 pmol/mg protein for protein 84 000.(ABSTRACT TRUNCATED AT 250 WORDS)     

Minor participation of cAMP on the protein kinase phosphorylation of mitochondrial and cytosolic fractions from Ascaris suum: a comparative study with porcine heart muscle

In contrast to porcine heart muscle in which cAMP effectively activated the phosphorylation of cytosolic proteins, cAMP exerted a minor effect on the phosphorylation of proteins from the soluble fraction of Ascaris suum muscle. Similarly, cAMP did not enhance the kinase activity in the mitochondrial membranes from porcine heart and A. suum, although major differences in protein phosphorylation were observed between both fractions. However, cAMP-dependent protein kinases (PKA) were evidenced in the parasitic soluble mitochondrial fraction, since the phosphorylation of histone IIA and kemptide was augmented in this fraction, in the presence of cAMP. An increase in the phosphorylation of exogenously added A. suum phosphofructokinase was also obtained when cAMP was added to the parasite soluble mitochondrial fraction. The phosphorylation of phosphofructokinase by this fraction was inhibited when kemptide and cAMP were included in the reaction mixture, suggesting substrate competition for the same PKA. Although PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent PKAs, did not affect the endogenous phosphorylation of proteins in the various A. suum fractions, an inhibition on the phosphorylation of exogenously added kemptide and phosphofructokinase was observed when PKI (6-22) was incubated with the parasite mitochondrial soluble fraction.     

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

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

In vivo phosphorylation of distinct domains of the 70-kilodalton neurofilament subunit involves different protein kinases

A combination of in vivo and in vitro approaches were used to characterize phosphorylation sites on the 70,000-kilodalton (kDa) subunit of neurofilaments (NF-L) and to identify the protein kinases that are likely to mediate these modifications in vivo. Neurofilament proteins in a single class of neurons, the retinal ganglion cells, were pulse-labeled in vivo by injecting mice intravitreously with [32P]orthophosphate. Radiolabeled neurofilaments were isolated after they had advanced along optic axons, and the individual subunits were separated on sodium dodecyl sulfate-polyacrylamide gels. Two-dimensional alpha-chymotryptic phosphopeptide map analysis of NF-L revealed three phosphorylation sites: an intensely labeled peptide (L-1) and two less intensely labeled peptides (L-2 and L-3). The alpha-chymotryptic peptide L-1 was identified as the 11-kDa segment containing the C terminus of NF-L. The ability of these peptides to serve as substrates for specific protein kinases were examined by incubating neurofilament preparations with [gamma-32P]ATP in the presence of purified cAMP-dependent protein kinase or appropriate activators and/or inhibitors of endogenous cytoskeleton-associated protein kinases. The heparin-sensitive, calcium- and cyclic nucleotide-independent kinase associated with the cytoskeleton selectively phosphorylated L-1 and L-3 but had little, if any, activity toward L-2. When this kinase was inhibited with heparin, cAMP addition to the neurofilament preparation stimulated the phosphorylation of L-2, and addition of the purified catalytic subunit of cAMP-dependent protein kinase induced intense labeling of L-2. At higher labeling efficiencies, the exogenous kinase also phosphorylated L-3 and several sites at which labeling was not detected in vivo; however, L-1 was not a substrate. Calcium and calmodulin added to neurofilament preparations in the presence of heparin modestly stimulated the phosphorylation of L-1 and L-3, but not L-2, and the stimulation was reversed by trifluoperazine. The selective phosphorylation of different polypeptide domains on NF-L by second messenger-dependent and -independent kinases suggests multiple functions for phosphate groups on this protein.     

Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens

We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.     

Endogenous phosphorylation of sarcoplasmic reticulum fragments of rabbit fast skeletal muscles

The purified membrane fragments of sarcoplasmic reticulum (SR) of rabbit fast skeletal muscles were found to incorporate 32P from[gamma-32P]ATP in endogenous membrane substrates and in histone H1. The existence of membrane-bound protein kinase of SR was demonstrated by steady state binding of [3H]-cAMP to the SR membranes. The constant of [3H]cAMP binding to the membranes is 2.5 +/- 0.003 x 10(6) M-1, the number of binding sites is 6.1 +/- 0.8 pmol per 1 mg of protein. The endogenous phosphorylation of SR components was inhibited by cAMP and cGMP at concentrations of 10(-7)-10(-6) and depended on Mg2+ and Ca2+. The thermostable protein inhibitor of cAMP-dependent protein kinase inhibited the endogenous phosphorylation of SR membranes by 30-40%. The protein phosphoproduct of SR membranes revealed the properties of a phosphoester. The membrane-bound protein kinase was active towards the exogenous substrate--histone H1. Phosphorylation in the presence of histones was independent of cyclic nucleotides, Mg2+ and Ca2+. Fractionation of 32P-labelled solubilized membranes in polyacrylamide gel in the presence of Na-SDS showed that the radioactivity is bound to protein zones with molecular weights of 95 000 and 6000.     

Mechanism of the stimulation of Ca2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase

Sarcoplasmic reticulum isolated from moderately fast rabbit skeletal muscle contains intrinsic adenosine 3',5'-monophosphate (cAMP)-independent protein kinase activity and a substrate of 100 000 Mr. Phosphorylation of skeletal sarcoplasmic reticulum by either endogenous membrane bound or exogenous cAMP-dependent protein kinase results in stimulation of the initial rates of Ca2+ transport and Ca2+-ATPase activity. To determine the molecular mechanism by which protein kinase-dependent phosphorylation regulates the calcium pump in skeletal sarcoplasmic reticulum, we examined the effects of protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Skeletal sarcoplasmic reticulum vesicles were preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated sarcoplasmic reticulum) and absence (control sarcoplasmic reticulum) of adenosine 5'-triphosphate (ATP). Control and phosphorylated sarcoplasmic reticulum were subsequently assayed for formation (5-100 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase. Protein kinase mediated phosphorylation of skeletal sarcoplasmic reticulum resulted in pronounced stimulation of initial rates and levels of E approximately P in sarcoplasmic reticulum preincubated with either ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) prior to assay (Ca2+-free sarcoplasmic reticulum), or with calcium/EGTA buffer (Ca2+-bound sarcoplasmic reticulum). These effects were evident within a wide range of ionized Ca2+. Phosphorylation of skeletal sarcoplasmic reticulum by protein kinase also increased the initial rate of E approximately P decomposition. These findings suggest that protein kinase-dependent phosphorylation of skeletal sarcoplasmic reticulum regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the active calcium transport observed at steady state.