Recombinant baculoviruses were created and used to coexpress rat phosphorylase kinase (Phk) alpha, gamma, and delta subunits and rabbit beta subunit in insect cells. Coexpression allowed creation of the (alphabetagammadelta)(4) hexadecamer, the alphagammadelta heterotrimer, and the gammadelta heterodimeric subcomplexes. Neither the individual alpha, beta, or gamma subunit nor any complex containing the beta subunit other than the hexadecameric holoenzyme was obtained in soluble form. The expressed complexes exhibited pH- and [Ca2+]-dependent specific activities that were similar to those of the Phk holoenzyme purified from rabbit skeletal muscle (SkM Phk). SkM Phk, expressed Phk, and the alphagammadelta subcomplex were activated by exogenous calmodulin and underwent Ca2+-dependent autophosphorylation. In some of these features there were subtle differences that could likely be attributed to differences in the covalent modification state of the baculovirus-driven expressed protein. Our results provide an important avenue to probe the detailed characterization of the structure of Phk and the function of the individual domains of the subunits using baculovirus-mediated expression of Phk and Phk subcomplexes.

The amount of phosphorylase kinase in skeletal muscle is exquisitely sensitive to developmental signals such as differentiation and innervation, and is clearly regulated in such a manner so as to always maintain the gamma catalytic subunit under the control of its regulatory alpha, beta and gamma subunits. To identify how the transcription of the gamma subunit is regulated, we have analysed 3.8 kb of the upstream regulatory region using a luciferase reporter system. A complex sequence of interdependent regulations is evident. The gamma catalytic subunit gene contains two inhibitory controls with very dominant features. Also evident are an array of multiple positive regulatory elements, prominent amongst which are four E-boxes, of which two are downstream, one is upstream and one is in the middle of the CAAT-TATA core promoter. Differentiation-dependent positive regulation arises as a consequence of both E-box regulation and the activation of at least one other regulatory element. The primary mode of transcriptional regulation of the gamma catalytic subunit gene appears to occur by the relief of regulation of an otherwise default inhibitory status. It is noteworthy that such a mode of regulation mirrors the regulation of the enzymic activity of many protein kinases, including phosphorylase kinase. With phosphorylase kinase, both its transcriptional regulation as well as the regulation of the protein itself, are primed to maintain the gamma catalytic subunit either unexpressed or inactivate respectively, until a positive signal occurs to override an otherwise dominant default inhibitory condition.

We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

Previous neutron scattering studies elaborated the topographical relationship of the regulatory (RIIα) and catalytic (Cα) subunits of the cAMP-dependent protein kinase. We present here the results of a set of computations that lead to an atomic model of the cAMP-dependent protein kinase heterodimer, Δ1–91RIIα-Cα. The first step in the modeling utilized the crystal structures for the porcine Cα and bovine Δ1–90RIαor rat Δ1–111RIIβ, to homology-model structures of the species and isoforms that had been used in the neutron scattering experiments (bovine Cαsubunit and murine Δ1–91RIIαsubunit, respectively). A docking procedure, constrained by the dimensions and positions of the ellipsoids in the neutron-derived R-C model as well as mutagenesis data, was used to develop “best fit” models for the heterodimer. Simulated annealing, molecular dynamics, and energy minimization were then used to refine the side chain packing at the heterodimer interface. For comparison, the calculations were done using the homology models derived from both the RIα and RIIβ crystal structures. Both resultant models had many similarities. Each predicted similar interfaces. The RIα-based model has 25% more hydrogen bonds than that based on RIIβ, with seven of these potential bonds in common. The distribution of hydrophobic, polar, and charged residues at the interface was similar for both models, with a distribution more characteristic of the exposed surface residues than those in the protein interior. The calculated interface area in each is relatively small (<2000 Å2). The RIα-based model, however, has a significantly better fit with the scattering data and is therefore the one of distinctly higher probability. With its small interface area that has a high proportion of charged and polar residues, the complex appears poised for dissociation, and each subunit existing as a stable entity. This result is consistent with the known physiological events required for cAMP-dependent activation of the kinase.

Flavopiridol has been shown to induce cell cycle arrest and apoptosis in various tumor cells in vitro and in vivo. Using immobilized flavopiridol, we identified glycogen phosphorylases (GP) from liver and brain as flavopiridol binding proteins from HeLa cell extract. Purified rabbit muscle GP also bound to the flavopiridol affinity column. GP is the rate-limiting enzyme in intracellular glycogen breakdown. Flavopiridol significantly inhibited the AMP-activated GP-b form of the purified rabbit muscle isoenzyme (IC50 of 1 microM at 0.8 mM AMP), but was less inhibitory to the active phosphorylated form of GP, GP-a (IC50 of 2.5 microM). The AMP-bound GP-a form was poorly inhibited by flavopiridol (40% at 10 microM). Increasing concentrations of the allosteric effector AMP resulted in a linear decrease in the GP-inhibitory activity of flavopiridol suggesting interference between flavopiridol and AMP. In contrast the GP inhibitor caffeine had no effect on the relative GP inhibition by flavopiridol, suggesting an additive effect of caffeine. Flavopiridol also inhibited the phosphorylase kinase-catalyzed phosphorylation of GP-b by inhibiting the kinase in vitro. Flavopiridol thus is able to interfere with both activating modifications of GP-b, AMP activation and phosphorylation. In A549 NSCLC cells flavopiridol treatment caused glycogen accumulation despite of an increase in GP activity, suggesting direct GP inhibition in vivo rather than inhibition of GP activation by phosphorylase kinase. These results suggest that the cyclin-dependent kinase inhibitor flavopiridol interferes with glycogen degradation, which may be responsible for flavopiridol's cytotoxicity and explain its resistance in some cell lines.

Asymptomatic elevations of serum creatine kinase (CK) may be observed in patients undergoing routine health assessment or evaluation for an acute illness. Diverse myopathic conditions may cause asymptomatic hyper-CK-emia, including polymyositis, congenital myopathies, and mitochondrial myopathies.1 Elevations of CK are common in patients with myophosphorylase deficiency (McArdle's disease) not only during episodes of exertional myalgia and cramps, but also during rest.2 We report an asymptomatic patient with negligible muscle glycogen phosphorylase (MGP) activity and marked elevation of CK. Genetic analyses showed that the patient is heterozygous for an uncommon mutation at codon 396 of the MGP gene; the genetic defect affecting the other allele remains undetermined.Case report. A 15½-year-old girl who had extensively participated in competitive athletics and aerobics without neuro-muscular symptoms experienced an acute episode of gastrointestinal discomfort, emesis, and dehydration, unassociated with muscle cramps, myalgia, or pigmenturia, which resolved quickly with the administration of IV fluids. Abnormal laboratory studies included a serum CK of 11,649 U/L, which remained elevated for 14 days with a maximum of 41,000 U/L. Before neurologic evaluation, she refrained from athletic activities for 1 month. Results of her neurologic and general physical examinations were normal, but serum …

Chimeric molecules of the cAMP-dependent protein kinase (PKA) holoenzyme (R2C2) and of a Δ1–91RC dimer were reconstituted using deuterated regulatory (R) and protiated catalytic (C) subunits. Small angle scattering with contrast variation has revealed the shapes and dispositions of R and C in the reconstituted complexes, leading to low resolution models for both forms. The crystal structures of C and a truncation mutant of R fit well within the molecular boundaries of the RC dimer model. The area of interaction between R and C is small, seemingly poised for dissociation upon a conformational transition within R induced by cAMP binding. Within the RC dimer, C has a "closed" conformation similar to that seen for C with a bound pseudosubstrate peptide. The model for the PKA holoenzyme has an extended dumbbell shape. The interconnecting bar is formed from the dimerization domains of the R subunits, arranged in an antiparallel configuration, while each lobe contains the cAMP-binding domains of one R interacting with one C. Our studies suggest that the PKA structure may be flexible via a hinge movement of each dumbbell lobe with respect to the dimerization domain. Sequence comparisons suggest that this hinge might be a property of the RII PKA isoforms.

We have previously demonstrated that there exist two distinct genes for the thermostable inhibitor protein of the cAMP-dependent protein kinase, PKIα and PKIβ (Van Patten, S. M., Howard, P., Walsh, D. A., and Maurer, R. A. (1992)Mol. Endocrinol. 6, 2114–2122). We have also shown that in the testis, at least eight forms of PKIβ exist, differing as a result of at least post-translational modification and alternate translational initiation (Kumar, P., Van Patten, S. M., and Walsh, D. A. (1997)J. Biol. Chem. 272, 20011–20020). We now report that in the testis, there is a unique cellular distribution of protein kinase inhibitor forms, with PKIβ being essentially (if not exclusively) a germ cell protein and PKIα being expressed primarily in Sertoli cells. Furthermore, there is a progressive change in the forms of PKIβ that are present within germ cells with development that is initiated in testis tubules and continues as the germ cells migrate through the epididymis. These conclusions are derived from studies with isolated cell populations and with the at/atgerm cell-deficient mouse line, by in situ hybridization, and by following the developmental expression of these proteins in both testis and epididymis. We have also shown that follicle-stimulating hormone (FSH) can increase the expression of both PKIα and PKIβ. The FSH-regulated expression of PKIα in the Sertoli cell likely occurs via the normal route of second messenger signal transduction. In contrast, the FSH-dependent PKIβ expression must arise by some form of Sertoli cell-germ cell intercommunication.

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.

The high-affinity interaction between protein kinase inhibitor (PKI) (6-22)amide (Thr(6)-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn- Ala-Ile(22)-NH2) and the catalytic subunit of cAMP-dependent protein kinase requires both the N-terminal Thr(6) to Ile(11) sequence of the inhibitor peptide and its C-terminal pseudosubstrate site comprised of Arg(15) to Ile(22). Small angle X-ray scattering data indicate that PKI(6-22)amide has a compact, rather than extended, structure in solution (Reed J et al., 1989, Biochem J264:371-380). CD spectroscopic analysis of the PKI peptide led to the suggestion that a beta-turn structure might be located in the-Ala(12)-Ser-Gly-Arg(15)-connecting sequence in the middle of the molecule (Reed J, Kinzel V, Cheng HC, Walsh DA, 1987, Biochemistry 26: 7641-7647). To investigate this possibility further, conformationally constrained and flexible analogs of PKI(6-22)amide were synthesized and used to study the structure-function relationships of this central portion of the inhibitor. (Des 12-14)PKI(6-22) amide exhibited over a 200-fold loss in inhibitory activity. Replacement of the omitted -Ala(12)-Ser-Gly(14)-sequence with aminocaprylic acid yielded an analog that regained more than 90% of the lost binding energy. The d-alanine(14) PKI analog was as potent as the parent peptide, whereas the beta-alanine(14) and the sarcosine(14) analogs were only 10-fold less active. Several peptides that promoted a beta-turn structure at residues 12-15 showed about 200-fold decreases in inhibitory activity. Two constrained analogs that could not assume a beta-turn conformation were only 30-fold less potent than PKI(6-22)amide. Thus, the structure of the central connecting portion of the PKI peptide, encompassing residues 12-15, greatly influences its ability to effectively bind to and inhibit the catalytic subunit. We conclude, however, that a formal beta-turn at this position is not required and is actually detrimental for a high-affinity interaction of PKI(6-22)amide with the enzyme. These results are interpreted in light of the Fourier-transform infrared spectra of the peptide analogs and the crystal structure of the peptide bound at the active site of the protein kinase (Knighton DR et al., 1991b, Science 253:414-420).

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?

Phosphorylase kinase, a key enzyme in glycogen metabolism, has a subunit composition of (alphabetagammadelta)4, in which the alpha and beta subunits are regulatory, delta is calmodulin, and the gamma subunit is catalytic. As one segment of our studies on the regulation of the expression of phosphorylase kinase subunits, we present in this report the structure of the gene for the catalytic gamma subunit. The gene extends over 16 kilobase pairs (kb) of DNA, and contains eight introns within the coding region plus one 3.3-kb intron upstream in the 5'-untranslated region. Within this first intron, and also upstream of the transcription start site, are sequences homologous to defined regulatory elements, including some found in other muscle-specific genes. The positions of intron splice junctions for this gene have been compared with similar data for other protein kinase genes. A somewhat unexpected finding for the gamma subunit is that two of the splice junctions fall in the midst of highly conserved strings of amino acids, both of which have been nominally defined as functional domains for the protein kinases and appear to make key contributions to substrate binding and phosphotransferase catalysis.

Small-angle X-ray scattering and Fourier transform infrared (FTIR) spectroscopy experiments have been completed on the catalytic subunit of the cAMP-dependent protein kinase. Measurements were made both with and without the protein kinase inhibitor peptide, PKI alpha(5-22)amide. Binding of the peptide results in an overall contraction of the structure that is characterized by a decrease of 9% in radius of gyration and about 16% in the maximum linear dimension. Both the secondary structure content of the protein/peptide complex, as determined by FTIR, and the solution structure of this binary complex, as determined by X-ray scattering, agree well with the structural characteristics of this complex as elucidated by the crystal structure [Knighton, D.R., Zheng, J., Ten Eyck, L. F., Ashford, V.A., Xuong, N.H., Taylor, S.S., & Sowadsi, J. M. (1991a) Science 253, 407-414]. Further, the contraction of the structure observed by X-ray scattering upon inhibitor peptide binding is not accompanied by any detectable change in secondary structure content of the kinase. We have modeled the contraction of the kinase upon inhibitor peptide binding as a simple rotation of the large and small lobes seen in the crystal structure such that the cleft between them is closed. For a substrate these changes would then allow catalysis to ensue. The hinge for this movement occurs around a glycine that is one of the protein kinase family consensus amino acids.

The developmental expression of the alpha, beta, and gamma subunits of skeletal muscle phosphorylase kinase has been examined in regenerating muscle. Rat extensor digitorum longus (EDL) muscles, treated with bupivacaine, promptly undergo a rapid degeneration of the muscle, followed by regeneration and recovery of essentially normal morphology and physiology by 3-4 weeks post-treatment (Hall-Craggs, E. C. B., and Seyan, H. S. (1975) Exp. Neurol. 46, 345-354). Phosphorylase kinase activity dropped to approximately 10% of control within 3 days of bupivacaine treatment and remained at this low level for several days but had attained at least 60% of normal levels by day 21. The pH 6.8/8.2 activity ratio was unusually high during the period of low activity, suggesting that the catalytic activity was not under normal regulation at this time. The subunit mRNAs were readily detected in control EDL but were undetectable at day 3 post-bupivacaine treatment. Very small amounts of message for all three subunits were evident by day 6 and began to approach normal levels by day 12-15. The mRNA for both the alpha and alpha' subunits of phosphorylase kinase exhibited a similar pattern of recovery, as did also the mRNA for phosphorylase. In contrast to both phosphorylase kinase and phosphorylase, actin mRNA exhibited a quite a different pattern, with a nearly full recovery of message levels by day 6 post-bupivacaine. These data indicate that synthesis of phosphorylase and the alpha, beta, and gamma subunits of phosphorylase kinase appears to be coordinately regulated at the level of message accumulation and that the expression of phosphorylase kinase activity is likely to be also regulated post-transcriptionally.

Phosphorylase kinase is a calcium-regulated multimeric enzyme of composition (alpha-beta-gamma-delta)4, which contains calmodulin as the integral delta-subunit and also is activated further by addition of extrinsic calmodulin. Previous studies by Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. ((1989) J. Biol. Chem. 264, 1715617163) have identified gamma-302-326 and gamma-342-366 as two calmodulin binding regions. Using peptides that were synthesized based on alpha and beta-primary structure and that were predicted to contain the basic amphiphilic alpha-helix motif thought important for calmodulin binding, four additional potential calmodulin binding domains have now been identified: one of high affinity, beta-770-794; two of intermediate affinity, beta-5-28 and beta-920-946; and one with marginally low affinity, alpha-1070-1093. Peptide beta-770-794 was of higher calmodulin affinity than either gamma-302-326 or gamma-342-366; it was of higher affinity than the model synthetic peptide IV defined by O' Neil, K. T., and DeGrado, W. F. ((1990) Trends Biochem. Sci. 15, 59-64); and it is currently the most potent calmodulin-binding peptide so far described. Correlated with their affinity for calmodulin, all six phosphorylase kinase-derived peptides and several other established calmodulin-binding peptides inhibited phosphorylase kinase previously activated by cAMP-dependent phosphorylation, reducing its activity to the level of the nonactivated enzyme. However, these peptides did not inhibit (and some peptides slightly activated) the nonphosphorylated enzyme. Even in the presence of these peptides both activated and nonactivated enzyme remained fully Ca2+-dependent. The beta-770-794 peptide has at least a 5-fold greater calmodulin binding affinity than the holo-phosphorylase kinase. This, and its higher affinity for calmodulin than either of the sites on the gamma-subunit, raises the possibility that in the native enzyme it may be involved in binding the intrinsic delta-subunit. Further, inhibition of activated but not nonactivated enzyme by calmodulin-binding peptides would suggest that the phosphorylation-dependent activation of phosphorylase kinase may be mediated by changes in the binding interactions of the intrinsic calmodulin delta-subunit.