Volker Kinzel

Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5-24).

The crystal structure of the porcine heart catalytic subunit of cAMP‐dependent protein kinase in a ternary complex with the MgATP analogue MnAMP‐PNP and a pseudosubstrate inhibitor peptide, PKI(5‐24), has been solved at 2.0 A resolution from monoclinic crystals of the catalytic subunit isoform CA. The refinement is presently at an R factor of 0.194 and the active site of the molecule is well defined. The glycine‐rich phosphate anchor of the nucleotide binding fold motif of the protein kinase is a beta ribbon acting as a flap with conformational flexibility over the triphosphate group. The glycines seem to be conserved to avoid steric clash with ATP. The known synergistic effects of substrate binding can be explained by hydrogen bonds present only in the ternary complex. Implications for the kinetic scheme of binding order are discussed. The structure is assumed to represent a phosphotransfer competent conformation. The invariant conserved residue Asp166 is proposed to be the catalytic base and Lys168 to stabilize the transition state. In some tyrosine kinases Lys168 is functionally replaced by an Arg displaced by two residues in the primary sequence, suggesting invariance in three‐dimensional space. The structure supports an in‐line transfer with a pentacoordinate transition state at the phosphorus with very few nuclear movements.

Staurosporine-induced conformational changes of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential.

BACKGROUND Staurosporine inhibits most protein kinases at low nanomolar concentrations. As most tyrosine kinases, along with many serine/threonine kinases, are either proto oncoproteins or are involved in oncogenic signaling, the development of protein kinase inhibitors is a primary goal of cancer research. Staurosporine and many of its derivatives have significant biological effects, and are being tested as anticancer drugs. To understand in atomic detail the mode of inhibition and the parameters of high-affinity binding of staurosporine to protein kinases, the molecule was cocrystallized with the catalytic subunit of cAMP-dependent protein kinase. RESULTS The crystal structure of the protein kinase catalytic subunit with staurosporine bound to the adenosine pocket shows considerable induced-fit rearrangement of the enzyme and a unique open conformation. The inhibitor mimics several aspects of adenosine binding, including both polar and nonpolar interactions with enzyme residues, and induces conformational changes of neighboring enzyme residues. CONCLUSIONS The results explain the high inhibitory potency of staurosporine, and also illustrate the flexibility of the protein kinase active site. The structure, therefore, is not only useful for the design of improved anticancer therapeutics and signaling drugs, but also provides a deeper understanding of the conformational flexibility of the protein kinase.

Direct Effects of Phosphorylation on the Preferred Backbone Conformation of Peptides: A Nuclear Magnetic Resonance Study

Control of protein activity by phosphorylation appears to work principally by inducing conformational change, but the mechanisms so far reported are dependent on the structural context in which phosphorylation occurs. As the activity of many small peptides is also regulated by phosphorylation, we decided to investigate possible direct consequences of this on the preferred backbone conformation. We have performed 1H nuclear magnetic resonance (NMR) experiments with short model peptides of the pattern Gly-Ser-Xaa-Ser, where Xaa represents Ser, Thr, or Tyr in either phosphorylated or unphosphorylated form and with either free or blocked amino and carboxy termini. The chemical shifts of amide protons and the 3JNH-Halpha coupling constants were estimated from one-dimensional and two-dimensional scalar correlated spectroscopy (COSY) spectra at different pH values. The results clearly indicate a direct structural effect of serine and threonine phosphorylation on the preferred backbone dihedrals independent of the presence of charged groups in the surrounding sequence. Tyrosine phosphorylation does not induce such a charge-independent effect. Additionally, experiments with p-fluoro- and p-nitro-phenylalanine-containing peptides showed that the mere presence of an electronegative group on the aromatic ring of tyrosine does not produce direct structural effects. In the case of serine and threonine phosphorylation a strong dependence of the conformational shift on the protonation level of the phosphoryl group could be observed, showing that phosphorylation induces the strongest effect in its dianionic, i.e., physiological, form. The data reveal a hitherto unknown mechanism that may be added to the repertoire of conformational control of peptides and proteins by phosphorylation.

The human papillomavirus type 16 E5 protein modulates phospholipase C-γ-1 activity and phosphatidyl inositol turnover in mouse fibroblasts

The human papillomavirus type 16 E5 (HPV16-E5) protein is a membrane protein that has been associated with malignant growth. The protein affects growth factor-mediated signal transduction in a ligand-dependent manner. We show now that E5 expression in A31 fibroblasts results in an increased level of diacylglycerol (DAG) and inositol phosphates. Immunoprecipitation of phospholipase C-γ-1 (PLC-γ-1) with specific antibodies and immunoblotting with anti-phosphotyrosine antibodies reveal a large increase in tyrosine phosphorylation of the enzyme in E5-expressing cells compared to control vector-transfected cells. This activation of tyrosine phosphorylation is growth factor independent. In addition, an enhanced formation of phosphatidic acid (PA) was observed in E5 cells. This increase did not result from activation of phospholipase D (PLD), although the enzyme was activatable by treatment with phorbol ester. Thus, a phosphohydrolase-mediated DAG synthesis from PLD-produced PA can be excluded. The observed effects were not further enhanced by EGF showing that the presence of the growth factor is not necessary for maintaining permanent activation of PLC-γ-1 in E5-expressing cells. The DAG- and inositol phosphate-mediated signal cascade within the cells is thus effectively uncoupled from external control via EGF and its receptor in the presence of E5 protein.

Chromatographic separation of two heterogeneous forms of the catalytic subunit of cyclic AMP-dependent protein kinase holoenzyme type I and type II from striated muscle of different mammalian species☆

Electrophoretically homogeneous preparations of catalytic subunit (C) of cAMP-dependent protein kinase isolated according to two different procedures from holoenzyme type I and type II from rabbit and from holoenzyme type II from rat skeletal muscle and from bovine cardiac muscle can be separated on carboxymethyl cellulose or on a Mono S column (Pharmacia) by salt gradient elution into two enzymatically active peaks called A and B, which do not interconvert on rechromatography. Cochromatography of peak A fractions or of peak B fractions derived from both holoenzymes respectively yields single enzyme peaks in each case, thus indicating that both represent different entities, which were named CA and CB. The separate character of both enzyme forms is supported by the fact that CB under all conditions is degraded faster by the C-specific protease (E. Alhanaty et al. (1981) Proc. Natl. Acad. Sci. USA 78, 3492-3495) than CA, a phenomenon which is enhanced in both enzyme forms by substrate (Kemptide). The separation of both subtypes from each other is probably based on differences in isoelectric values (delta pH less than or equal to 0.5 units). The reason for the charge difference is not presently known. CA and CB do not differ significantly in their phosphate content. No differences between CA and CB have been detectable so far with respect to their migration in SDS gels, kinetic behavior regarding both substrates and cosubstrate, pH dependence, inhibition by regulatory subunits of holoenzyme type I (rabbit skeletal muscle) and of type II (bovine cardiac muscle), and inhibition by specific-heat and acid-stable inhibitor-modulator. The peptide pattern of both forms after limited proteolysis exhibits small differences.

Phorbol ester stimulates choline incorporation.

Im Zusammenhang mit der Verwendung yon MetalI(III)-telluraten als Modifikatoren bei der hydrothermalen Herstellung von CrO~ aus CrO~ unter Sauerstoffdruck nntersuchte man neben den bekann ten Telluraten(VI) yon Tri rut i l -Struktur [1] der allgemeinen Formel A~TeO~ (A ---A1, Ga, Cr und Fe) auch das Mn~TeOg. Man erhielt die bisher nicht bekannte Verbindung auf folgendem Wege: 2 Liter ether ~ m MnSOcL6sung wurden mi t 2 Liter einer 0,5 m H~TeO~-L6sung vereinigt, bet 25 ~ unter Riihren in einem GuB mit 2 L i t e r einer 2rn NHaOH-L6sung gef~llt (pH = 7,0--7,5) und die Suspension noch 30 rain unter Einleiten yon 250 Liter Luft]h oxydiert. Der Niederschlag wurde abfiltriert, sulfatfrei gewaschen und bet 120 ~ an der Luf t getrocknet. Das Produkt wurde pulverisiert und 16h bet 600 ~ an der Luf t getempert. ] )ann wurden das ents tandene Mn~TeO~ analysiert, die pyknometr ische Dichte und nach dem Debye-Scherrer-Verfahren aus Diffraktometer-Aufnahmen die tetragonalen Gi t terkonstanten best immt. Gefunden: 33 ,3%Mn, 37 ,9%Te; berechnet: 32,95%Mn, 38,26% Te @pyk20 ---5,69 :k 0,01 g/cm~; ~rSnt = 5 , 7 2 g/cm a a o -4,6079 • 0,0002; c o = 9A 144 • 0,0004 A; c/a = t,978; Z = 1,99 Molekein Mn~TeO~]Zelle. Die Git terkonstanten und Elementarvolumina der im TrirutiIGitter kristallisierenden Tellurate(VI) der allgemeinen Formel A~TeOs (A = A1, Ga, Cr, Fe, Mn) sind linear abhXngig yon den Goldschmidtschen Metall(III)-Ionenradien.

Phosphorylation by cell surface protein kinase of bovine and human fibrinogen and fibrin.

Human and bovine fibrinogen as well as fibrin are shown to be phosphorylated by Co631 (monolayer, hamster) and RPL12 (suspension, chicken) cells by their surface protein kinase of the casein kinase II type. The phosphate label is introduced into the alpha-peptide. The kinase system phosphorylates serine residues and utilizes GTP equally well as ATP. The participation of intact cell surfaces indicates the possibility of phosphorylation of extracellular fibrinogen independently of the site of its biosynthesis.

Role of cdc25-C phosphatase in the immediate G2 delay induced by the exogenous factors epidermal growth factor and phorbolester.

Studies on the link between cellular signalling and cell cycle control at the G2 checkpoint have shown that, in HeLa cells, epidermal growth factor (EGF) and the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) rapidly inhibit the G2‐M transition by preventing the key component of mitosis‐promoting factor (MPF), p34cdc2, from expressing protein kinase activity. The kinase activity of active MPF is not inhibited; rather, the conversion of pre‐MPF to MPF, i.e., the activating dephosphorylation of p34cdc2, at tyrosine is rapidly blocked (Barth and Kinzel, 1994, Exp. Cell Res. 212:383–388; 1995, J. Cell. Physiol., 162:44–51). The phosphatase responsible, cdc25‐C, is activated by phosphorylation in mitotic cells starting at the G2‐M transition in an autocatalytic loop with MPF (Hoffmann et al., 1993, EMBO J. 12:53–63). We now show that, concomitant with the prevention of MPF activation, EGF and TPA induced a reduction of the activity of cdc25‐C in synchronized cultures. Furthermore, treatment of mitotic HeLa cells with TPA did not influence the kinase activity of MPF but caused a rapid decrease of the specific enzyme activity of cdc25‐C, probably due to dephosphorylation of the enzyme, as indicated by reduced binding of monoclonal MPM‐2 antibody specific for phosphoepitopes in M phase. Because of its inability to induce signalling during division, EGF failed to influence the activity of cdc25‐C in mitotic cells. The scenario in cells late in G2 that are committed to enter mitosis may be as follows: In those cells where the signalling pathways responding to EGF as well as those responding to TPA are still open, cdc25‐C is prevented by dephosphorylation from exceeding the threshold level of activity required to initiate the activation of and the autocatalytic feedback loop with p34cdc2 and to enter mitosis. Therefore, cdc25‐C appears to represent part of an interface between cellular signalling and cell cycle control in G2 phase.

In vivo activation of recombinant cAPK catalytic subunit active site mutants by coexpression of the wild-type enzyme, evidence for intermolecular cotranslational phosphorylation

The catalytic subunit of cAMP dependent protein kinase (cAPK) carries two stable autophosphorylated residues. One of them, Thr197, residues in the so‐called protein kinase activation segment, and needs to be phosphorylated for full activity and protein kinase inhibitor binding of the enzyme. While wild‐type recombinant mammalian C‐subunit, expressed in E. coli, can fully autoactivate itself by phosphorylation at Thr197, many active site mutants lack this autophosphorylation activity, so that the primary effects of the mutations become obscured. Two active site mutants of bovine C‐subunit, defective in protein kinase inhibitor peptide binding, were activated by wild‐type enzyme in vivo, but could not be activated in vitro, demonstrating intermolecular and presumably cotranslational autophosphorylation. The results may delineate strategies for the expression and mutagenesis of other protein kinases with requirements for activation segment phosphorylation.

Rat Cα catalytic subunit of the cAMP-dependent protein kinase : cDNA sequence and evidence that it is the only isoform expressed in myoblasts

A full length cDNA clone encoding the C alpha type catalytic subunit of cAMP-dependent protein kinase was isolated from a cDNA library of differentiated rat myoblast L6 cell line. The 2137 bp clone codes for a protein of 351 amino acid residues having more than 90% sequence identity to C alpha subunits of other mammalians. The C alpha isoform was found to be the only isoform of catalytic subunits expressed in myoblast cells as was determined in Northern blot analysis.