Donal A. Walsh

Multiple pathway signal transduction by the cAMP-dependent protein kinase.

Since its discovery a quarter century ago, the cAMP‐dependent protein kinase has been a central model for study of the mode of transduction of second messenger signals; more than 300 protein kinases are now known to play keys roles in cellular control. Multiple cellular events are initiated by the activation of the cAMP‐dependent protein kinase and correlated with these events has been the identification of a broad spectrum of protein substrates. From model substrates and inhibitors an excellent understanding has been obtained of the “optimum” sequence for protein phosphorylation by the cAMP‐dependent protein kinases, and now, from pioneering crystal structure studies, we are beginning to understand exactly how an optimum substrate can interact with and be efficiently phosphorylated by the kinase. The next important step is for us to understand the full sequence of events that occurs within the cell upon activation of the protein kinase, and it is abundantly evident that this is indeed a complex process. It is not sufficient to simply know which proteins are phosphorylated but it is critical that we understand the dynamics of the events surrounding the phosphorylation of multiple proteins, what factors dictate those dynamics, and what might happen when the sequence of events is perturbed. This review focuses on the first simple question that must be addressed, namely, how might proteins vary as substrates for the cAMP‐dependent protein kinase and what ramifications might such variations have for the consequential events within the cell?—Walsh, D. A., Van Patten, S. M. Multiple pathway signal transduction by the cAMP‐dependent protein kinase, FASEB J. 8, 1227‐1236 (1994)

A cyclic 3′,5′-AMP-stimulated protein kinase from cardiac muscle

Abstract A protein kinase that is stimulated by cyclic 3′,5′-AMP has been purified 250-fold from beef heart. The purified enzyme catalyzes the phosphorylation of casein or histone. Of several nucleotides tested, only the dibutyryl derivative of cyclic 3′,5′-AMP had a significant stimulatory effect. When the heart protein kinase was aged, it lost the cyclic 3′,5′-AMP effect but this could be partially restored with mercaptoethanol. Cyclic 3′,5′-AMP decreased the stability of the kinase.

Protein Kinases: Aspects of Their Regulation and Diversity

Publisher Summary This chapter discusses the protein kinases, aspects of their regulation, and their diversity. Studies of protein kinases have become intimately associated with an understanding of the function of cyclic AMP. In the past few years, it has been observed that hormones, interacting with specific receptors on the cell surface, modulate adenyl cyclase activity. The resultant changes in intracellular cyclic AMP concentrations evoke the physiological responses distinctive of each hormone-target cell interaction. The cyclic nucleotide binds to a specific receptor protein that interacts at the promoter region of the operon and, in consequence, modulates the rate of synthesis of specific species of messenger RNA. Moreover, glycogenolysis is a common feature of most mammalian tissues and, therefore, caution must be used in expanding the interpretation of the enzyme distribution data to account for the hormonal regulation of other metabolic processes. Recently, no correlation was found in the developmental patterns in skeletal muscle, liver, and heart, between cyclic AMP-dependent protein kinase and the glycogenolytic enzymes phosphorylase and phosphorylase kinase.

A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts and its application to the study of 32Pi uptake in perfused rat heart

Abstract (i) A new, rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts in the presence of other 32P-containing compounds is described. The deproteinized extract is incubated with phosphorylase b and phosphorylase kinase, and the incorporation of 32P into protein from [γ-32P]ATP is measured by precipitation on filter paper in trichloroacetic acid. No separation of ATP or other treatment of the extracts is required for the assay. (ii) 32Pi uptake in perfused rat heart was found to be a relatively slow process, with a Km of 0.084 m m , whereas equilibration between intracellular 32Pi and [γ-32P]ATP occurred rapidly.

Histone 1 phosphotransferase activities during the maturation of oocytes of Xenopus laevis

Abstract A characterization has been initiated of histone 1 (H1) phosphotransferases in oocytes, progesterone-treated oocytes, unfertilized eggs, and A-23187 ionophore-treated eggs of Xenopus laevis . With the exception of one activity that was detected only in the oocytes and which was characterized as free catalytic subunit of the cAMP-dependent protein kinase, the profile of H1 phosphotransferases activities was qualitatively identical in all four types of cells. The only cAMP-dependent protein kinase detected was the Type II isozyme. All four types of cells contain a H1 phosphotransferase which would appear to be very similar to the growth-associated H1 kinase identified in other eukaryotic cells on the basis of the criteria of (a) molecular size, (b) the phosphorylation of both serine and threonine residues in the C-terminal portion of H1, and (c) the phosphorylation of the synthetic peptides LysSerProValLys and LysThrProValLys. All four types of cells contain at least two other histone phosphotransferase activities, one of which exhibits a high degree of selectivity for H1.

Decrease in force potentiation and appearance of α-adrenergic mediated contracture in aging rat skeletal muscle

The effect of increasing age on contractile performance and catecholamine receptor activity was investigated in a distal, predominantly fast twitch oxidative glycolytic (FOG) muscle from the plantar surface of the rat hindfoot. The ability of the flexor digitorum brevis (FDB), isolated from anesthetized rats and maintained in vitro, to undergo post-tetanic potentiation and a staircase response declined with age. Potentiation following repetitive stimulation was reduced by 50% in 2 year old rats and eliminated in 3 year old animals. The rate of muscle fatigue during intermittent tetanic stimulation also increased in aging muscles. FDB, regardless of age, did not develop a positive inotropic response to 10−6 M epinephrine applied in vitro, but 3 year old FDB generated a prolonged contracture. Contracture tension was approximately 25% of twitch tension and was maintained for 2–10 min in the continued presence of catecholamine. Contractures were eliminated by pretreatment with α-adrenergic antagonists or by removing Ca2+ from the bathing medium. In addition to decreased contractile capacity, aging muscles acquire a population of α-adrenergic receptors which may underlie some of the metabolic and structural changes associated with increasing age.

Multiplicity of the β Form of the cAMP-dependent Protein Kinase Inhibitor Protein Generated by Post-translational Modification and Alternate Translational Initiation

Two distinct species of the thermostable inhibitor of the cAMP-dependent protein kinase, PKIα and PKIβ, exist that are the products of separate genes. The PKIβ form, as first isolated from rat testis, is a 70-amino acid protein, but the genomic sequence suggested that an alternate form might exist, arising as a consequence of alternate translational initiation. This species, now termed PKIβ-78, has been synthesized by bacterial expression, demonstrated to be equipotent with PKIβ-70, and also now demonstrated to occur in vivo. By Western blot analyses, six additional species of PKIβ are also evident in tissues. Two of these represent the phospho forms of PKIβ-78 and PKIβ-70. The other four represent phospho and dephospho forms of two higher molecular mass PKIβ species. These latter forms are currently termed PKIβ-X and PKIβ-Y, awaiting the full elucidation of their molecular identity. In adult rat testis and cerebellum, PKIβ-70, PKIβ-X, and PKIβ-Y constitute 39, 23, and 32% and 15, 29, and 54% of the total tissue levels, respectively. In adult rat testis, 35–42% of each of these three species is present as a monophospho form, whereas no phosphorylation of them is evident in cerebellum. PKIβ-78 is present at much lower levels in both rat testis and cerebellum (∼6 and 2% of the total, respectively) and almost entirely as a monophospho species. PKIβ-78, like PKIβ-70, is a high affinity and specific inhibitor of the cAMP-dependent protein kinase. PKIβ-Y and PKIβ-X, in contrast, also significantly inhibit the cGMP-dependent protein kinase.

Metabolism of N6,O2′-[3H]dibutyryl cyclic adenosine 3′,5′-monophosphate and macromolecular interactions of the products in perfused rat liver☆

Abstract The perfusion of rat liver with N6,O2′-[3H]dibutyryl cyclic adenosine 3′,5′-monophosphate (Bt2-cAMP) has been used as a method to evaluate the site(s) of action of cyclic adenosine 3′,5′-monophosphate (cAMP). The metabolites produced from [3H]Bt2-cAMP have been determined by two-dimensional thin layer chromatography and electrophoresis. The accumulation of [3H]Bt2-cAMP within the cell was associated with the formation of N6-monobutyryl cyclic adenosine 3′,5′-monophosphate (N6-Bt-cAMP) and a product tentatively identified as either N6-monobutyryl AMP or N6,O2′-dibutyryl AMP. The latter product achieved a higher concentration in the liver than any other metabolite of [3H]Bt2-cAMP. Gel filtration of cytosol performed rapidly after termination of perfusion identified binding of [3H]N6-Bt-cAMP and [3H]cAMP to the macromolecular fraction. This binding was only decreased slightly if all extraction procedures were performed in the presence of cAMP, thus indicating that the binding observed occurred while the cells were intact. The only binding species detected in cytosol could be attributed to the regulatory subunit of the cAMP-dependent protein kinase. This assignment was made on the basis of: (i) sedimentation characteristics (5.2 S), (ii) the interaction with the catalytic subunit of skeletal muscle cAMP-dependent protein kinase, (iii) the equivalent degree of dependence of this interaction with the catalytic subunit on ATP-Mg2+ in correlation with the isozyme composition of the protein kinase in rat liver, (iv) the binding to nitrocellulose membranes, and (v) the correlation of the extent of binding of [3H]N6-Bt-cAMP and [3H]cAMP with the amount of 3H-labeled nucleotides produced within the cell and with the respective Ka values of the two cyclic nucleotides for the cAMP-dependent protein kinase. Treatment of the fraction with a 1:500 ratio of trypsin at 0 °C for 5 min results in the conversion of the binding species to a form that sediments at 3.2 S. A similar form was identified in extracts from frozen tissues, possibly due to the release of lysosomal proteases.

A reinvestigation of the kinetic parameters of phosphoenolpyruvate carboxykinase

Abstract A new assay for phosphoenolpyruvate carboxykinase has been devised whose sensitivity is sufficient to detect the formation by crude hepatic extracts of 1 × 10−11 moles of phosphoenolpyruvate. By the utilization of this assay it has been determined that the apparent Km of oxaloacetate is in the range of 1 to 5 × 10−6 M for the hepatic enzymes from rat and guinea pig. These values are approximately two orders of magnitude lower than all previous data and indicate that the enzyme is sensitive to modulations of oxaloacetate concentrations that occur physiologically.

10 Phosphorylase Kinase

Publisher Summary Protein phosphorylation and dephosphorylation are well recognized as major metabolic control mechanisms in eukaryotic cells. By the 1940s, through work in the laboratories of Con and others, the basic biochemistry of glycogen breakdown had been established with the characterization of the phosphorylase-catalyzed conversion of glycogen to glucose 1-phosphate. This chapter discusses the extensive network of regulatory phenomena that impinge upon the control of phosphorylase kinase activity. Phosphorylase kinase activity can be modulated by a number of effectors that interact in a noncovalent and specific manner and presumably modulate activity by affecting the enzymes conformation. Included in this group are Ca 2+ , Mg 2+ , calmodulin, and glycogen; each is discussed in the chapter. It is suggested that the understanding of the control of glycogenolysis has served well for many years as a guideline for how other metabolic control systems may function. Similarly, phosphorylase kinase can serve as a model to understand the properties of other regulatory enzymes. An attempt is made in this chapter to provide this understanding.