Phosphate Dependent Protein Kinase Research Articles

Mapping of the two intrachain cyclic nucleotide binding sites of adenosine cyclic 3',5'-phosphate dependent protein kinase I.

Mapping of the two intrachain cyclic nucleotide binding sites of adenosine cyclic 3',5'-phosphate dependent protein kinase I.

Adenosine cyclic 3',5'-phosphate-dependent protein kinase from bovine brain: inactivation of the catalytic subunit and holoenzyme by 7-chloro-4-nitro-2,1,3-benzoxadiazole

Adenosine cyclic 3',5'-phosphate-dependent protein kinase from bovine brain: inactivation of the catalytic subunit and holoenzyme by 7-chloro-4-nitro-2,1,3-benzoxadiazole

Binding of 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] by the regulatory subunits of adenosine cyclic 3',5'-phosphate dependent protein kinase.

Binding to the regulatory subunits of types I and II adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase (RI and RII, respectively) produces large distinctive increases in fluorescence and optical activity of 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] [bis(ANS)]. Both specific and nonspecific interactions are involved. Association of the regulatory subunits with either the catalytic subunit or cAMP results in dissociation of a major portion of the bound bis(ANS) as detected by changes in fluorescence and circular dichroism. The results are consistent with the accepted cAMP binding properties of RI and RII, showing cooperativity in case of RI and two heterologous binding sites for RII. cGMP has the same overall effect on bis(ANS) binding as cAMP. However, very high concentrations are required for complete dissociation of bis(ANS) from RII, consistent with the observation that cGMP is inefficient in bringing about the dissociation of the type II holoenzyme. Magnesium binding to sites having dissociation constants of ca. 12 mM increases the interaction of bis(ANS) with both of the isolated regulatory subunits. Experiments involving the 37 000-dalton fragment of RII indicate that the limited proteolytic cleavage was heterogeneous, with only 24-39% of the resulting population interacting strongly with the catalytic subunit.

Reconstitution of types I and II adenosine cyclic 3',5'-phosphate dependent protein kinase.

Fluorescence intensity and anisotropy measurements using the fluorescent adenosine cyclic 3',5'-phosphate (cAMP) analogue 1,N6-ethenoadenosine cyclic 3',5'-phosphate (epsilon-cAMP) are sensitive to the dissociation of epsilon-cAMP which occurs when either the type I or the type II regulatory subunit (RI or RII) of cAMP-dependent protein kinase associates with the catalytic subunit. Studies using epsilon-cAMP show that MgATP has opposite effects on the reconstitution of both types of protein kinase: MgATP strongly stabilizes the type I holoenzyme while it slightly destabilizes the type II holoenzyme. The synthetic substrate Kemptide has a small inhibitory effect on the reconstitution of both holoenzymes when tested at 10 microM concentration. The protein kinase inhibitor has a larger effect which is especially pronounced in the reassociation of the type I enzyme. The diminished relative ability of the type I regulatory subunit to compete with the protein kinase inhibitor suggests that the combined effects of the two opposing equilibria (epsilon-cAMP and catalytic subunit binding) are different for the two types of regulatory subunits. Displacement experiments show that cAMP and epsilon-cAMP bind about equally well to the type I subunit. Slow conformational changes accompanying the binding of epsilon-cAMP by both regulatory subunits are greatly accelerated with the holoenzymes, suggesting that dissociation of the holoenzymes occurs via ternary complexes. The time courses of epsilon-cAMP binding also show the heterogeneity of binding characteristics of RII. The 37 000-dalton fragment of type II subunit retains the epsilon-cAMP binding properties of the native subunit. However, only a fraction of the fragment preparation (approximately 32% estimated from sedimentation measurements) binds the catalytic subunit well, suggesting heterogeneity of cleavage.

Functional interactions between smooth muscle myosin light chain kinase and calmodulin.

Calmodulin (CaM) binding by turkey gizzard myosin light chain kinase (MLCK) causes subtle changes in the fluorescence emission and polarization excitation spectra of the enzyme. Fluorescence experiments using 9-anthroyl-choline (9AC), which competes with ATP in binding, demonstrate mutually stabilizing interactions between the CaM and ATP binding sites corresponding to delta G = -0.6 to -0.7 kcal/mol. Fluorescence titrations in the presence of 9AC or 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] confirm the stoichiometry of 1 mol of CaM/MLCK. Phosphorylation of MLCK has no effect on either the protein fluorescence or the binding of ATP and 9AC. The dissociation constant for the MLCL-CaM complex is increased approximately 500-fold on phosphorylation. Values of Kd for the phosphorylated enzyme range from 0.5 to 1.1 microM in 0.2 N KCl, pH 7.3, 25 degrees C. We showed competition between MLCK and other CaM binding proteins and peptides by using both fluorescence and catalytic activity measurements. Competition for CaM occurs with ACTH, beta-endorphin, substance P, glucagon, poly(L-arginine), myelin basic protein, troponin I, and histone H2A. Phosphorylation of the last three proteins by the adenosine cyclic 3',5'-phosphate dependent protein kinase diminishes their ability to compete. Phosphorylation of MLCK by the protein kinase gives 0.95 +/- 0.04 and 2.2 +/- 0.4 mol of incorporated 32P in the presence and absence of CaM, respectively. These stoichiometries agree with those recently reported [Conti, M. A. & Adelstein, R. S. (1981) J. Biol. Chem. 256, 3178].

Salt-induced conformational changes in the catalytic subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase. Use for establishing a connection between one sulfhydryl group and the gamma-P subsite in the ATP site of this subunit.

Salt-induced conformational changes in the catalytic subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase. Use for establishing a connection between one sulfhydryl group and the gamma-P subsite in the ATP site of this subunit.

Phosphorylase phosphatase complex from skeletal muscle. Activation of one of two catalytic subunits by manganese ions.

Sarcoplasmic phosphorylase phosphatase extracted from ground skeletal muscle was recovered in a high molecular weight from (Mr = 250000). This enzyme has been purified from extracts by anion-exchange and gel chromatography to yield a preparation with three major protein components of Mr 83000, 72000, and 32000 by sodium dodecyl sulfate gel electrophoresis. The phosphorylase phosphatase activity of the complex form was activated more than 10-fold by Mn2+, with a K0.5 of 10(-5) M, but not by Mg2+ or Ca2+. Manganese activation occurred over a period of several minutes and resulted primarily in an increase in Vmax of a phosphatase that was sensitive to trypsin. Activation persisted after gel filtration, and the active form of the enzyme did not contain bound manganese measured by using 54Mn2+. A contaminating p-nitrophenylphosphatase was activated by either Mn2+ (K0.5 of 10(-4) M) or Mg2+ (K0.5 of 10(-3) M). Unlike the protein phosphatase this enzyme was inactive following removal of the metal ions by gel filtration. The phosphatase complex could be dissociated into its component subunits by precipitation with 50% acetone at 20 degrees C in the presence of an inert divalent cation, reducing agent, and bovine serum albumin. Two catalytic subunits were quantitatively recovered; one of Mr 83000 was a trypsin-sensitive manganese-activated phosphatase and the second of Mr 32000 was trypsin-stable and metal ion dependent. Both enzymes were effective in catalyzing the dephosphorylation of either phosphorylase a or the regulatory subunit of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase, but neither subunit possessed p-nitrophenylphosphatase activity.

Characterization of calmodulin effects on calcium transport in cardiac microsomes enriched in sarcoplasmic reticulum.

Calmodulin prepared from red cell hemolysates was found to significantly increase Ca2+ uptake into cardiac microsomal preparations enriched in sarcoplasic reticulum in a dose-dependent manner. The stimulation of calcium uptake by calmodulin was additive to that stimulation produced by maximal stimulatory concentrations of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase and cAMP, indicating separate mechanisms of action and potentially different modulatory roles for these two systems in the control of calcium transport. K+ significantly decreased calmodulin stimulation of calcium uptake, while in the absence of calmodulin, K+ increased Ca2+ uptake. In the absence of K+, calmodulin increased Ca2+ uptake to levels observed at maximal K+ concentrations without calmodulin present. Na+ produced effects similar to those of K+ in this preparation both in the presence and absence of calmodulin. The effect of calmodulin on the intermediate steps of the (Mg2+,Ca2+)ATPase in cardiac sarcoplasmic reticulum was also investigated. Calmodulin was found to reduce the steady-state level of the Ca2+-dependent phosphoprotein (ECaP) and increase the (Mg2+,Ca2+)ATPase activity of this preparation. Dephosphorylation of ECaP in the presence of Tris-ATP (0.5 mM) was significantly stimulated by calmodulin. These studies indicate that calmodulin stimulates Ca2+ transport in cardiac sarcoplasmic reticulum by increasing the turnover rate of the transport process.

Ontogeny of the Adenosine‐3′:5′‐Phosphate‐Dependent Protein‐Kinase System during Early Uterine Development

The development of protein kinase and estrogen‐binding proteins was studied in the nuclear and cytosol fractions of rat uteri as a function of postnatal age.Analysis of cyclic‐AMP‐dependent protein kinase activity, with protamine sulfate as substrate in the presence of 3 μM cyclic AMP, revealed that the cytosol as well as the nuclear cAMP‐dependent protein kinase activity increased markedly between the third and fifth postnatal days. The total cytosol protein kinase activity remained constant after the age of 5 days (850 pmol [32P]phosphate incorporated × min−1× mg cytosol protein−1), but nuclear cAMP‐dependent protein kinase activity decreased sharply (60%), coinciding with a time period during which nuclei acquire considerable estrogen‐binding activity.Analysis of the multiplicity of cyclic‐AMP‐dependent protein kinases by chromatography of uterine cytosol on DEAE‐cellulose revealed the presence of type I and type II cyclic‐AMP‐dependent protein kinase. Type I isoenzyme levels decreased relative to the type II isozyme from the time of birth to the age of 20 days, type II isozyme did not undergo any major developmental changes.The specific estrogen binding activity in utertine cytosol reached a maximum in 12‐day‐old rats (1.7 pmol of 17β‐estradiol bound/mg protein). It declined thereafter and reached constant levels in 17‐day‐old and older rats. Concomitantly nuclear levels of estrogen binding protein increased progressively from very low levels (0.1 pmol) in 5‐day‐old rats to 1.2 pmol of 17β‐estradiol bound/mg of nuclear protein in 12‐day‐old and 20‐day‐old rats.The results indicate the early existence of a functional uterine cyclic‐AMP/protein kinase system in the neonatal rat and suggest that the protein kinase system and specific estrogen‐binding activity develop in a reciprocal manner.

Characterization of two adenosine 3′:5′-phosphate-dependent protein kinase species from Chinese hamster ovary cells

Chinese hamster ovary cells exhibit several characteristic morphological and physiological responses upon treatment with agents which increase the intracellular level of adenosine 3′:5′-phosphate (cyclic AMP). To better understand the mechanism of these cyclic AMP-mediated responses, we separated two cyclic AMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) (protein kinase I and protein kinase II) from the cytosol of Chinese hamster ovary cells by DEAE-cellulose chromatography and studied their properties. Protein kinase I is eluted at a lower salt concentration than protein kinase II and is stimulable to 10 times its basal catalytic activity, while protein kinase II is stimulable only 2-fold. Both kinases are completely dissociated by cyclic AMP and inhibited by specific cyclic AMP-dependent protein kinase inhibitor. They have similar K m values for magnesium (approximately 1 mM), cyclic AMP (approximately 60 nM), and ATP (approximately 0.1 mM), and the dissociation constant ( K dis) for cyclic AMP (approximately 13 nM) is the same for both enzymes. However, they appear to have different substrate preferences and cyclic AMP-binding properties in that cyclic AMP bound to protein kinase II exchanges readily with free cyclic AMP, while that bound to protein kinase I is not exchangeable. The native enzymes have different sedimentation coefficients (6.4 S for protein kinase I and 4.8 S for protein kinase II), whereas those of the activated enzymes are the same (2.9–3.0 S). It appears that the two cyclic AMP-dependent protein kinases which differ from each other in their regulatory subunits may play different roles in the mediation of cyclic AMP action in Chinese hamster ovary cells.

Phosphorylation of the inhibitory subunit of troponin in perfused hearts of mice deficient in phosphorylase kinase. Evidence for the phosphorylation of troponin by adenosine 3′:5′-phosphate-dependent protein kinase in vivo

When hearts from control and phosphorylase kinase-deficient (I strain) mice were perfused with 0.1 micrometer-DL-isoprenaline, there was a parallel increase in contraction, cyclic AMP concentration and troponin I phosphorylation. However, there was no increase in phosphorylase a in the I-strain hearts, whereas the control hearts showed a large increase. Assays of I-strain heart extracts showed a normal cyclic AMP-dependent protein kinase activity but no phosphorylase kinase activity. It is concluded that troponin I is phosphorylated in intact hearts by protein kinase and not phosphorylase kinase.

Studies on adenosine 3′,5′-phosphate-binding and adenosine 3′,5′-phosphate-dependent protein kinase activities associated with subcellular fractions of Chinese hamster ovary cells

Both cyclic AMP-binding and cyclic AMP-dependent protein kinase activities exists in Chinese hamster ovary cell extract. Competition experiments demonstrate that the binding is specific for cyclic AMP. All cellular elements including nucleus, mitochondria, plasma membrane, microsome, ribosome and cytosol contain both activities. Binding activity is highest in the cytosol and lowest in the nucleus. Each fraction contains endogenous protein kinase activity which is insensitive to cyclic AMP activation. When histone was used as a substrate, protein kinase activity in all fractions was stimulated by cyclic AMP (with the highest in cytosol and lowest in the nucleus) and inhibited by Walsh's protein kinase inhibitor.

Effect of thyroliberin on the concentration of adenosine 3′:5′-phosphate and on the activity of adenosine 3′:5′-phosphate-dependent protein kinase in prolactin-producing cells in culture

1. The effects of thyroliberin were studied in cultured rat pituitary-tumour cells that synthesize and secrete prolactin (the GH4C1 cell strain). 2. Prolactin and cyclic AMP were measured by radioimmunological methods, and a cyclic AMP-dependent protein kinase was characterized by using histone as substrate. 3. Prolactin release was studied after 5-60min of treatment, and synthesis after 48h of treatment with thyroliberin. One-half maximum stimulation of release and synthesis were observed at 0.25 and at 4nM respectively. 4. Cyclic AMP was temporarily increased in cell suspensions after treatment with thyroliberin, and one-half maximum stimulation was observed at 25nM. 5. Dibutyryl cyclic AMP increased prolactin release and synthesis, one-half maximum effects being obtained at 20 micronM. 6. A cyclic AMP-dependent protein kinase, which was one-half maximally stimulated at 30 nM-cyclic AMP, was demonstrated. 7. An increase in the activity ratio (-cyclic AMP/+cyclic AMP) of the cyclic AMP-dependent protein kinase was observed after treatment with thyroliberin. Total protein kinase activity in the presence of cyclic AMP was unaltered. The time-course of enzyme activation was similar to that of cyclic AMP formation and corresponded to the time when prolactin release was first observed. 8. It is concluded that thyroliberin induces cyclic AMP formation, resulting in the activation of a cyclic AMP-dependent protein kinase.

Formula omitted] activation of cyclic adenosine 3′:5′-phosphate-dependent protein kinase in Chinese hamster ovary cells treated with N 6, O 2′-dibutyryl cyclic adenosine 3′:5′-phosphate

Treatment of Chinese hamster ovary cells with dibutyryl cyclic AMP, which results in a net increase of the intracellular cyclic AMP level, converts the epithelial-like cells to a fibroblast-like shape. Protein kinase activity in cells treated with 1 mM dibutyryl cyclic AMP show a 3-fold increase in V max but no appreciable changes in the apparent K m for ATP. When cells are treated with dibutyryl cyclic AMP, there is a time-dependent conversion of cyclic AMP-stimulable protein kinase to cyclic AMP-independent catalytic subunits, as demonstrated by Sephadex G-100 gel filtration. These experiments demonstrate the activation of the cyclic AMP-dependent protein kinase in vivo . This activation may lead to phosphorylation of certain cellular constituent(s) and thus may be involved in the observed morphological transformation.

Synthesis and enzymic activity of 8-acyl and 8-alkyl derivatives of guanosine 3',5'-cyclic phosphate

Several new 8-alkyl and 8-acyl derivatives of quanosie 3',5'-cyclic phosphate (cGMP) and inosine 3',5'-cyclic phosphate (cGMP) were prepared by direct alkylation or acylation of the parent cyclic nucleotide via free radicals generated in situ. These compounds have been examined for their ability to stimulate a cGMP-dependent protein kinase, and several of the cGMP derivatives were as active in this regard as cGMP. These compounds proved to be quite ineffective when tested for their ability to activate an adenosine 3',5'-cyclic phosphate (cAMP) dependent protein kinase. In addition, these 8-substituted cGMP derivatives are not substrates for a phosphodiesterase preparation from rabbit kidney, but do show inhibition of the hydrolysis of cAMP by crude phosphodiesterase preparations from rabbit lung and beef heart.

Adenosine 3',5'-cyclic phosphate, lipolysis and oxygen consumption in brown adipose tissue from newborn rabbits. Effect of cervical sympathectomy and of noradrenalin and theophylline in vitro, and the partial purification of an adenosine 3',5'-cyclic phosphate-dependent protein kinase.

The rates of lipolysis and O 2 consumption and the concentration of cyclic AMP were measured in lipid-replete and lipid-depleted brown fat and isolate cells. A cyclic AMP-dependent protein kinase, with properties similar to those of other tissues, was partially purified. Noradrenalin caused an increase in the concentration of cyclic AMP which occurred before the fall in the concentration of ATP and was within the range that affected the activity of protein kinase. Theophylline potentiated the effect of noradrenalin upon lipolysis, the concentration of cyclic AMP and the rate of O 2 consumption in brown fat. Insulin and propranolol lowered the concentration of cyclic AMP in the presence of noradrenalin. There was a significant correlation between the concentration of cyclic AMP and submaximal lipolysis. Interruption of the cervical sympathetic nerve to brown fat prevented the increase in the concentration of cyclic AMP in the tissue when the animals were exposed to cold. Depletion of lipid from the tissue did not impair its ability to increase the concentration of cyclic AMP. It is concluded that cyclic AMP can mediate the effect of noradrenalin upon lipolysis in brown fat. The possibility that cyclic AMP also mediates at least some aspects of the stimulation of the heat production of the tissue is discussed.

The subunit structure of rabbit-skeletal-muscle phosphorylase kinase, and the molecular basis of its activation reactions.

Phosphorylase kinase was isolated from rabbit skeletal muscle in a state approaching homogeneity as judged by the criteria of ultracentrifugal analysis, ion‐exchange chromatography, and antigen‐antibody precipitation in agar. The purified enzyme showed four protein‐staining components upon acrylamide gel electrophoresis in the presence of sodium dodecylsulphate, termed α, α′, β and γ. A variety of precautions designed to eliminate the possible action of proteases and ensure complete reduction and denaturation of the protein had no influence on the gel pattern. The molecular weights of the four components determined by gel electrophoresis, and supported by sedimentation equilibrium in the presence of 6 M guanidinium chloride, following partial separation of the chains by gel filtration on Sephadex G‐200 in the presence of sodium dodecylsulphate, were:–α= 145000, α′= 140000, β= 128000 and γ= 45000. The α' component was present only in trace amounts and the evidence suggests it may be derived from the α component. The three subunits α, β, and γ, were found to exist in equimolar quantities by densitometric analysis of acrylamide gels, by gel filtration on Sephadex G‐200 in the presence of sodium dodecylsulphate, and by carboxymethylation with iodo[14C]acetate, suggesting a minimal binding molecular weight, αβγ, of 318000. The molecular weight of the native enzyme was determined as 1.28 × 106, demonstrating that phosphorylase kinase is composed of 4.0 αβγ units.Activation of the enzyme by incubation with adenosine‐3′:5′‐phosphate‐dependent protein kinase, adenosine 3′:5′‐phosphate and Mg‐ATP, was accompanied by the phosphorylation of one site on the β subunit, although a second site on the α subunit was phosphorylated at a slower rate. Activation of the enzyme by proteolysis resulted from a limited cleavage of the α subunit. The products of proteolytic attack suggest that the α and β subunits may be structurally related. The γ subunit was not phosphorylated, was resistant to proteolysis and distinct from the (α+β) subunits in its amino acid composition. The possible functions of the chains, and the implications of the activation reactions to the nervous and hormonal control of glycogenolysis in vivo are discussed.