Mg X ATP2-dependent interaction of the inhibitor protein of the cAMP-dependent protein kinase with the catalytic subunit

The interaction between the inhibitor protein and the catalytic subunit of the cAMP-dependent protein kinase has been investigated by steady state kinetics and by an assessment of the requirement of this interaction for ATP. By analysis for tightly bound inhibitors, inhibition by the inhibitor protein was shown to be competitive versus peptide substrate and uncompetitive versus Mg X ATP2-. This, together with the observations of Gronot et al. (Gronot, J., Mildvan, A.S., Bramson, H. N., Thomas, N., and Kaiser, E.T. (1981) Biochemistry 20, 602-610) and those given in the accompanying paper (Whitehouse, S., Feramisco, J.R., Casnellie, J.E., Krebs, E.G., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3693-3701), would indicate that the probable reaction mechanism of the protein kinase is ordered with the nucleotide binding first and that the inhibitor protein blocks catalysis by interaction with the catalytic subunit-Mg X ATP complex. The Ki for this interaction at saturating Mg X ATP and zero peptide substrate is 0.49 nM. Multiple inhibition analysis in the presence of 5'-adenylimidodiphosphate (AMP X PNP) indicates that the inhibitor protein does not interact with a catalytic subunit-AMP X PNP complex. The requirement for ATP for the inhibitor protein-catalytic subunit interaction has also been demonstrated by direct binding measurements and by the observation that the efficiency of the inhibitor protein is increased by preincubation of the inhibitor protein, catalytic subunit, and ATP in the absence of peptide substrate. By either measurement, the catalytic subunit in the presence of the inhibitor protein, was shown to exhibit an apparent Kd of 20 approximately 60 nM for ATP; this value is two orders of magnitude higher than the affinity for ATP by the catalytic subunit alone. This high apparent affinity of the catalytic subunit for ATP (in the presence of the inhibitor) does not require that there be a specific binding site on the inhibitor protein for some moiety of the ATP but may simply be a reflection of the formation of a catalytic subunit-Mg X ATP X inhibitor protein complex with resultant displacement of the equilibrium of ATP binding to the protein kinase.     

Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992) were tested as inhibitors of cGMP-dependent protein kinase. The peptides themselves were not substrates. cGMP-dependent protein kinase activity was assayed using histone H2B and two synthetic peptide substrates. Consistent with previous observations of other peptide inhibitors of this enzyme (Glass, D. B. (1983) Biochem. J. 213, 159-164), the inhibitory peptides had no effect on the phosphorylation of histone H2B, but they competitively inhibited cGMP-dependent phosphorylation of the two peptide substrates. The parent inhibitor peptide, PKI(5-24)amide, and a series of analogs had Ki (or IC50) values for cGMP-dependent protein kinase in the range of 15-190 microM. In contrast to their effects on the cAMP-dependent protein kinase, the inhibitory peptides were substantially less potent with cGMP-dependent protein kinase, and potency was reduced by the presence of the NH2-terminal residues (residues 5-13). We conclude that the two protein kinases share a recognition of the basic amino acid cluster within the pseudosubstrate region of the peptide, but that the cGMP-dependent protein kinase does not recognize additional NH2-terminal determinants that make the inhibitor protein extremely potent toward the cAMP-dependent enzyme. Even- when tested at high concentrations and with peptide substrates, the native inhibitor protein did not inhibit cGMP-dependent protein kinase under assay conditions in which the peptides derived from it were inhibitory. Thus, the native inhibitor protein appears to have structural features which block interaction with the cGMP-dependent enzyme and enhance its selectivity for cAMP-dependent protein kinase.     

Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases

The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.     

Genistein inhibits protein histidine kinase.

Protein histidine kinase was prepared from whole cell extracts of the yeast, Saccharomyces cerevisiae. The enzyme was assayed using either histone H4 or a synthetic peptide corresponding to residues 70 to 102 of histone H4 as an in vitro substrate. With either substrate, both genistein and its solvent, dimethyl sulfoxide (Me2SO), inhibited protein histidine kinase. Me2SO alone gave a cooperative dose-response curve, with inhibition changing from almost zero below 10% Me2SO to 80% at 20% Me2SO with either substrate. Genistein gave a simple dose-response curve with 50% inhibition of protein histidine kinase at 110 microM genistein. In experiments with protein histidine kinase, genistein was a noncompetitive inhibitor with respect to ATP, histone H4 or the synthetic peptide, although, in the case of the synthetic peptide, the data were also consistent with competitive inhibition. These data gave Km values for both ATP and histone H4 of 15 microM, in satisfactory agreement with previously reported values (Huang, J., Wei, Y., Kim, Y., Osterberg, L., and Matthews, H. R. (1991) J. Biol. Chem. 266, 9023-9031). The Km for the synthetic peptide was 80 microM. The KI values were 270 or 310 microM measured with histone H4 or the synthetic peptide as substrate, respectively. While these KI values are relatively high, relative to published KI values for genistein inhibition of protein tyrosine kinases, many reported experiments use genistein at concentrations where inhibition of protein histidine kinase occurs. It is possible that some of the observed effects of genistein in vivo may be due to inhibition of protein histidine kinase.     

A phenylalanine in peptide substrates provides for selectivity between cGMP- and cAMP-dependent protein kinases.

Bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) is a potent and relatively specific substrate for cGMP-dependent protein kinase (cGK) as compared to cAMP-dependent protein kinase (cAK) (Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14971-14978). A synthetic peptide, RKISASEFDRPLR (BPDEtide), was synthesized corresponding to the sequence surrounding the phosphorylation site in cG-BPDE. BPDEtide retained the cGK/cAK kinase specificity demonstrated by native cG-BPDE: the apparent Km of BPDEtide for cGK was 5-fold lower than that for cAK (Km = 68 and 320 microM, respectively). Vmax values were 11 mumol/min/mg for cGK and 3.2 mumol/min/mg for cAK. The peptide was not phosphorylated to a measurable extent by protein kinase C or by calcium/calmodulin-dependent protein kinase II. Thus, the primary amino acid sequence of the peptide substrate was sufficient to confer kinase specificity. Studies in crude tissue extracts indicated that BPDEtide was the most selective peptide substrate documented for measuring cGK activity. Peptide analogs of BPDEtide were synthesized to determine the contribution of specific residues to cGK or cAK substrate specificity. Substitution of a Lys for the amino-terminal Arg did not reduce cGK/cAK specificity; neither did the exchange of an Ala for the non-phosphorylated Ser nor the removal of the 3 carboxyl-terminal residues. A truncated BPDEtide (RKISASE) served equally well as substrate (Km approximately 90 microM) for both kinases. However, restoration of the Phe, to yield RKISASEF, reproduced the original cGK/cAK specificity for BPDEtide (Km = 120 and 480 microM, respectively), primarily by decreasing the affinity of cAK. Addition of a carboxyl-terminal Phe to the peptide RKRSRAE (derived from the sequence of the cGK phosphorylation site in histone H2B) or to the peptide LRRASLG (derived from the sequence of the cAK phosphorylation site in pyruvate kinase) also improved the cGK/cAK specificity by decreasing the affinity of cAK. These data suggested that the Phe in each substrate tested is a negative determinant for cAK.     

Protein kinase C substrate and inhibitor characteristics of peptides derived from the myristoylated alanine-rich C kinase substrate (MARCKS) protein phosphorylation site domain.

The phosphorylation sites in the myristoylated alanine-rich C kinase substrate or MARCKS protein consist of four serines contained within a conserved, basic region of 25 amino acids, termed the phosphorylation site domain. A synthetic peptide comprising this domain was phosphorylated by both protein kinase C and its catalytic fragment with high affinity and apparent positive cooperativity. Tryptic phosphopeptides derived from the peptide appeared similar to phosphopeptides derived from the phosphorylated intact protein. The peptide was phosphorylated by cAMP- and cGMP-dependent protein kinases with markedly lower affinities. In peptides containing only one of the four serines, with the other three serines replaced by alanine, the affinities for protein kinase C ranged from 25 to 60 nM with Hill constants between 1.8 and 3.0. The potential pseudosubstrate peptide, in which all four serines were replaced by alanines, inhibited protein kinase C phosphorylation of histone or a peptide substrate with an IC50 of 100-200 nM with apparently non-competitive kinetics; it also inhibited the catalytic fragment of protein kinase C with a Ki of 20 nM, with kinetics of the mixed type. The peptide did not significantly inhibit the cAMP- and cGMP-dependent protein kinases. It inhibited Ca2+/calmodulin-dependent protein kinases I, II, and III by competing with the kinases for calmodulin. In addition, the peptide inhibited the Ca2+/calmodulin-independent activity of a proteolytic fragment of Ca2+/calmodulin protein kinase II, with an IC50 approximately 5 microM. Thus, the phosphorylation site domain peptide of the MARCKS protein is a high affinity substrate for protein kinase C in vitro; the cognate peptide containing no serines is a potent but not completely specific inhibitor of both protein kinase C and its catalytic fragment.     

Architecture of the Outer Membrane of Escherichia coli III. Protein-Lipopolysaccharide Complexes in Intramembraneous Particles

In a previous paper (A. Verkleij, L. van Alphen, J. Bijvelt, and B. Lugtenberg, Biochim. Biophys. Acta 466:269-282, 1977) we have hypothesized that particles on the outer fracture face of the outer membrane ([Formula: see text]), with corresponding pits on the inner fracture face of the outer membrane ([Formula: see text]), consist of lipopolysaccharide (LPS) aggregates stabilized by divalent cations and that they might contain protein and/or phospholipid. In the present paper the roles of LPS, cations, and proteins in these [Formula: see text] particles are described more extensively, using a strain that lacks the major outer membrane proteins, b, c, and d (b(-) c(-) d(-)), and has a reduction in the number of [Formula: see text] particles of 75%. To study the role of divalent cations in the formation of [Formula: see text] particles, these b(-) c(-) d(-) cells were grown or incubated with Ca(2+), Mg(2+), or putrescine. The presence of Ca(2+) resulted in the appearance of many [Formula: see text] particles and [Formula: see text] pits. Mg(2+) and putrescine were less effective than Ca(2+). Introduction of these particles was not accompanied by alterations in the relative amounts of LPS and cell envelope proteins. Ca(2+) treatment of a heptoseless derivative of a b(-) c(-) d(-) strain did not result in morphological changes. Incubation of Ca(2+)-treated cells with ethylenediaminetetraacetate caused the disappearance of the introduced particles as well as the release of more than 60% of the cellular LPS. These results strongly support the hypothesis that LPS is involved in the formation of [Formula: see text] particles and [Formula: see text] pits. The roles of various outer membrane proteins in the formation of [Formula: see text] particles were studied by comparing the freeze-fracture morphology of b(-) c(-) d(-) cells with that of cells which contain one of the outer membrane proteins b, c, d, and e or the receptor protein for bacteriophage lambda. The results showed that the presence of any of these five proteins in a b(-) c(-) d(-) background resulted in a large increase in the number of [Formula: see text] particles and [Formula: see text] pits, indicating that these proteins are, independent of each other, involved in the formation of [Formula: see text] particles and [Formula: see text] pits. The simplest explanation for the results is that in wild-type cells each particle consists of LPS complexed with some molecules of a single protein species, stabilized by either divalent cations or polyamines. It is hypothesized that the outer membrane of the wild-type cell contains a heterogeneous population of particles, of which 75% consists of protein b-LPS, protein c-LPS, and protein d-LPS particles. A function of these particles as aqueous pores is proposed.     

Application of DFT methods to the study of the coordination environment of the VO2+ ion in V?proteins

Density functional theory (DFT) methods were used to simulate the environment of vanadium in several V proteins, such as vanadyl-substituted carboxypeptidase (sites A and B), vanadyl-substituted chloroplast F(1)-ATPase (CF(1); site 3), the reduced inactive form of vanadium bromoperoxidase (VBrPO; low- and high-pH sites), and vanadyl-substituted imidazole glycerol phosphate dehydratase (IGPD; sites α, β, and γ). Structural, electron paramagnetic resonance, and electron spin echo envelope modulation parameters were calculated and compared with the experimental values. All the simulations were performed in water within the framework of the polarizable continuum model. The angular dependence of [Formula: see text] and [Formula: see text] on the dihedral angle θ between the V=O and N-C bonds and on the angle φ between the V=O and V-N bonds, where N is the coordinated aromatic nitrogen atom, was also found. From the results it emerges that it is possible to model the active site of a vanadium protein through DFT methods and determine its structure through the comparison between the calculated and experimental spectroscopic parameters. The calculations confirm that the donor sets of sites B and A of vanadyl-substituted carboxypeptidase are [[Formula: see text], H(2)O, H(2)O, H(2)O] and [N(His)(||), N(His)(⊥), [Formula: see text], H(2)O], and that the donor set of site 3 of CF(1)-ATPase is [[Formula: see text], OH(Thr), H(2)O, H(2)O, [Formula: see text]]. For VBrPO, the coordination modes [N(His)(||), N(His)(∠), OH(Ser), H(2)O, H(2)O(ax)] for the low-pH site and [N(His)(||), N(His)(∠), OH(Ser), OH(-), H(2)O(ax)] or [N(His)(||), N(His)(∠), [Formula: see text], H(2)O] for the high-pH site, with an imidazole ring of histidine strongly displaced from the equatorial plane, can be proposed. Finally, for sites α, β, and γ of IGPD, the subsequent deprotonation of one, two, and three imidazole rings of histidine and the participation of a carboxylate group of a glutamate residue ([N(His)(||), [Formula: see text], H(2)O, H(2)O], [N(His)(||), N(His)(||), [Formula: see text], H(2)O], and [N(His)(||), N(His)(||), [Formula: see text], OH(-), [Formula: see text]], respectively) seems to be the most plausible hypothesis.     

(13)C NMR Reveals No Evidence of n-π* Interactions in Proteins

An [Formula: see text] interaction between neighboring carbonyl groups has been postulated to stabilize protein structures. Such an interaction would affect the [Formula: see text]C chemical shielding of the carbonyl groups, whose paramagnetic component is dominated by [Formula: see text] and [Formula: see text] excitations. Model compound calculations indicate that both the interaction energetics and the chemical shielding of the carbonyl group are instead dominated by a classical dipole-dipole interaction. A set of high-resolution protein structures with associated carbonyl [Formula: see text]C chemical shift assignments verifies this correlation and provides no evidence for an inter-carbonyl [Formula: see text] interaction.     

Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. Effect on tyrosine kinase activity and ligand binding affinity

The effect of autophosphorylation and protein kinase C-catalyzed phosphorylation on the tyrosine-protein kinase activity and ligand binding affinity of the epidermal growth factor (EGF) receptor has been studied. Kinetic parameters for the phosphorylation by the receptor kinase of synthetic peptide substrates having sequences related to the 3 in vitro receptor autophosphorylation sites (tyrosine residues 1173 (P1), 1148 (P2), and 1068 (P3)) were measured. The Km of peptide P1 (residues 1164-1176) was significantly lower than that for peptides P2 (residues 1141-1151) or P3 (residues 1059-1072). The tyrosine residue 1173 was also the most rapidly autophosphorylated in purified receptor preparations, consistent with previous observations for the receptor in intact cells (Downward, J., Parker, P., and Waterfield, M. D. (1984) Nature 311, 483-485). Variation in the extent of receptor autophosphorylation from 0.1 to 2.8 mol of phosphate/mol of receptor did not influence kinase activity or EGF binding affinity either for purified receptor or receptor in membrane preparations. Phosphorylation of the EGF receptor by protein kinase C was shown to cause a 3-fold decrease in the affinity of purified EGF receptor for EGF and to reduce the receptor kinase activity. In membrane preparations, phosphorylation of the EGF receptor by protein kinase C resulted in conversion of high affinity EGF binding sites to a low affinity state. This suggests that activation of protein kinase C by certain growth promoting agents and tumor promoters is directly responsible for modulation of the affinity of the EGF receptor for its ligand EGF. The regulation of the EGF receptor function by protein kinase C is discussed.     

Prevention of lethal murine candidiasis using HP (2-20), an antimicrobial peptide derived from the N-terminus of Helicobacter pylori ribosomal protein L1

Peptide HP (2-20), A(2)KKVFKRLEKLFSKIQNDK(20), is a cationic antimicrobial peptide derived from the N-terminus of Helicobacter pylori ribosomal protein 1, HpRpL1. Native peptide HP (2-20) and its synthetic derivatives have been shown in vitro to exhibit potent killing activity against Gram-positive, Gram-negative and yeast cells, thus, making them promising candidates for treatment of polymicrobial infections. However, the therapeutic potential of peptide HP (2-20) or its synthetic derivatives in any animal model of either bacterial or fungal diseases has not yet been investigated. In this study, we demonstrate that synthetic peptide amide HP (2-20), administered in six doses (300microg each; one intraperitoneal dose at the time of the infection, followed by five intravenous doses at 12h intervals) to CBA/J male mice experimentally infected with a lethal inoculum ( [Formula: see text] CFU) of Candida albicans, delayed the onset of disease, suppressed disease progression, and greatly increased survival rate and time (16.6% by day 14), as compared with the untreated infected control mice (100% mortality by day 5). Further, using isotonic buffer systems differing in ionic strength, peptide HP (2-20) was shown in vitro to exhibit an ionic strength-dependent hemolytic activity, previously not detected. Repeated intravenous administration of uninfected control CBA/J male mice with peptide HP (2-20), however, caused neither morbidity nor mortality. These findings strongly evidence the therapeutic efficacy and safety values of peptide HP (2-20) as a lead drug for the treatment of acquired candidiasis.     

Identification of the protein kinase C phosphorylation site in neuromodulin

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin [Alexander, K. A., Cimler, B. M., Meier, K. E., & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113], and we have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar Km values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [gamma-32P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32P-labeled tryptic peptide was generated from phosphorylated neuromodulin. The sequence of this peptide was IQASFR. The serine in this peptide corresponds to position 41 of the entire protein, which is adjacent to or contained within the calmodulin binding domain of neuromodulin. A synthetic peptide, QASFRGHITRKKLKGEK, corresponding to the calmodulin binding domain with a few flanking residues, including serine-41, was also phosphorylated by protein kinase C. We conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmodulin to neuromodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural basis for the low affinities of yeast cAMP-dependent and mammalian cGMP-dependent protein kinases for protein kinase inhibitor peptides.

Affinities of the catalytic subunit (C1) of Saccharomyces cerevisiae cAMP-dependent protein kinase and of mammalian cGMP-dependent protein kinase were determined for the protein kinase inhibitor (PKI) peptide PKI(6-22)amide and seven analogues. These analogues contained structural alterations in the N-terminal alpha-helix, the C-terminal pseudosubstrate portion, or the central connecting region of the PKI peptide. In all cases, the PKI peptides were appreciably less active as inhibitors of yeast C1 than of mammalian C alpha subunit. Ki values ranged from 5- to 290-fold higher for the yeast enzyme than for its mammalian counterpart. Consistent with these results, yeast C1 exhibited a higher Km for the peptide substrate Kemptide. All of the PKI peptides were even less active against the mammalian cGMP-dependent protein kinase than toward yeast cAMP-dependent protein kinase, and Kemptide was a poorer substrate for the former enzyme. Alignment of amino acid sequences of these homologous protein kinases around residues in the active site of mammalian C alpha subunit known to interact with determinants in the PKI peptide [Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N-h, Taylor, S. S., & Sowadski, J. M. (1991) Science 253, 414-420] provides a structural basis for the inherently lower affinities of yeast C1 and cGMP-dependent protein kinase for binding peptide inhibitors and substrates. Both yeast cAMP-dependent and mammalian cGMP-dependent protein kinases are missing two of the three acidic residues that interact with arginine-18 in the pseudosubstrate portion of PKI. Further, the cGMP-dependent protein kinase appears to completely lack the hydrophobic/aromatic pocket that recognizes the important phenylalanine-10 residue in the N-terminus of the PKI peptide, and binding of the inhibitor by the yeast protein kinase at this site appears to be partially compromised.     

Isolation and characterization of a dimeric cAMP-dependent protein kinase from the fungus Saccobolus platensis

A cAMP-dependent protein kinase from mycelia of Saccobolus platensis was characterized. The holoenzyme seems to be a dimer (i.e., regulatory subunit--catalytic subunit) of 78,000 Da, slightly activated by cAMP but susceptible to dissociation into its subunits by cAMP, or by kemptide and protamine, the best substrates for Saccobolus protein kinase. The regulatory subunit was purified to homogeneity by affinity chromatography. It is highly specific for cAMP and has two types of binding sites but failed to inhibit the phosphotransferase activity of the homologous or the heterologous (bovine heart) catalytic components. The activity of the catalytic subunit was completely abolished by the regulatory component of the bovine heart protein kinase as well as by a synthetic peptide corresponding to the active site of the mammalian protein kinase inhibitor. The data suggest that interaction between the subunits of the S. platensis protein kinase is different than that found in cAMP-dependent protein kinases from other sources. Similarities and differences between the Saccobolus protein kinase and enzymes from low eucaryotes and mammalian tissues are discussed.     

Characterization of the interaction of 5'-p-fluorosulfonylbenzoyl adenosine with the epidermal growth factor receptor/protein kinase in A431 cell membranes

Treatment of membrane vesicles from A431 cells, a human epidermoid carcinoma line, with the affinity label 5'-p-fluorosulfonylbenzoyl [8-14C]adenosine (5'-p-FSO2Bz[14C]Ado) results in an inhibition of the epidermal growth factor (EGF)-stimulable protein kinase and in the modification of proteins having the same molecular weight (Mr = 170,000 and 150,000) as the receptor for EGF (Buhrow, S. A., Cohen, S., and Staros, J. V. (1982) J. Biol. Chem. 257, 4019-4022). Modification of the vesicles with 5'-p-FSO2BzAdo inhibits not only the EGF-stimulated phosphorylation of endogenous membrane proteins but also the EGF-stimulated phosphorylation of an exogenous synthetic tyrosine-containing peptide substrate. This indicates that the EGF-stimulable protein kinase is modified by 5'-p-FSO2BzAdo at a site affecting catalytic activity. Membrane vesicles were treated with 5'-p-FSO2Bz-[14C]Ado to affinity label the kinase, then the EGF receptor was purified by affinity chromatography on immobilized EGF. The EGF receptor thus purified contains the 5'-p-SO2Bz[14C]Ado moiety. These data strongly support our hypothesis that the EGF receptor and EGF-stimulable kinase are two parts of the same polypeptide chain.     

Phosphorylation of membrane-bound acetylcholine receptor by protein kinase C: characterization and subunit specificity

Acetylcholine receptor (AChR) from Torpedo electric organ in its membrane-bound or solubilized form is phosphorylated by the Ca2+/phospholipid-dependent protein kinase (PKC). The subunit specificity for PKC is different from that observed for cAMP-dependent protein kinase (PKA). Whereas PKC phosphorylates predominantly the delta subunit and the phosphorylation of the gamma subunit by this enzyme is very low, PKA phosphorylates both subunits to a similar high extent. We have extended our phosphorylation studies to a synthetic peptide from the gamma subunit, corresponding to residues 346-359, which contains a consensus PKA phosphorylation site. This synthetic peptide is phosphorylated by both PKA and PKC, suggesting that in the intact receptor both kinases may phosphorylate the gamma subunit at a similar site, as has been previously demonstrated by us for the delta subunit [Safran, A., et al. (1987) J. Biol. Chem. 262, 10506-10510]. The diverse pattern of phosphorylation of AChR by PKA and PKC may play a role in the regulation of its function.     

NMR studies of the backbone protons and secondary structure of pentapeptide and heptapeptide substrates bound to bovine heart protein kinase

The conformations of enzyme-bound pentapeptide (Arg-Arg-Ala-Ser-Leu) and heptapeptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) substrates of protein kinase have been studied by NMR in quaternary complexes of the type (Formula: see text). Paramagnetic effects of Mn2+ bound at the inhibitory site of the catalytic subunit on the longitudinal relaxation rates of backbone Ca protons, as well as on side-chain protons of the bound pentapeptide and heptapeptide substrates, have been used to determine Mn2+ to proton distances which range from 8.2 to 12.4 A. A combination of the paramagnetic probe-T1 method with the Redfield 2-1-4-1-2 pulse sequence for suppression of the water signal has been used to measure distances from Mn2+ to all of the backbone amide (NH) protons of the bound pentapeptide and heptapeptide substrates, which range from 6.8 to 11.1 A. Paramagnetic effects on the transverse relaxation rates yield rate constants for peptide exchange, indicating that the complexes studied by NMR dissociate rapidly enough to participate in catalysis. Model-building studies based on the Mn2+-proton distances, as well as on previously determined distances from Cr3+-AMPPCP to side-chain protons [Granot, J., Mildvan, A.S., Bramson, H. N., & Kaiser, E. T. (1981) Biochemistry 20, 602], rule out alpha-helical, beta-sheet, beta-bulge, and all possible beta-turn conformations within the bound pentapeptide and heptapeptide substrates. The distances are fit only by extended coil conformations for the bound peptide substrates with a minor difference between the pentapeptides and heptapeptides in the phi torsional angle at Arg3C alpha and in psi at Arg2C alpha. An extended coil conformation, which minimizes the number of interactions within the substrate, would facilitate enzyme-substrate interaction and could thereby contribute to the specificity of protein kinase.     

Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val -Lys-Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an alpha-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RII alpha or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RII alpha and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 microM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RII alpha at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type II alpha PKA holoenzyme may be specifically targeted to different locations in each type of cell.