The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB.

PKB/Akt, S6K1 and SGK are related protein kinases activated in a PI 3-kinase-dependent manner in response to insulin/growth factors signalling. Activation entails phosphorylation of these kinases at two residues, the T-loop and the hydrophobic motif. PDK1 activates S6K, SGK and PKB isoforms by phosphorylating these kinases at their T-loop. We demonstrate that a pocket in the kinase domain of PDK1, termed the 'PIF-binding pocket', plays a key role in mediating the interaction and phosphorylation of S6K1 and SGK1 at their T-loop motif by PDK1. Our data indicate that prior phosphorylation of S6K1 and SGK1 at their hydrophobic motif promotes their interaction with the PIF-binding pocket of PDK1 and their T-loop phosphorylation. Thus, the hydrophobic motif phosphorylation of S6K and SGK converts them into substrates that can be activated by PDK1. In contrast, the PIF-binding pocket of PDK1 is not required for the phosphorylation of PKBalpha by PDK1. The PIF-binding pocket represents a substrate recognition site on a protein kinase that is only required for the phosphorylation of a subset of its physiological substrates.

A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation.

The inhibition of GSK3 is required for the stimulation of glycogen and protein synthesis by insulin and the specification of cell fate during development. Here, we demonstrate that the insulin-induced inhibition of GSK3 and its unique substrate specificity are explained by the existence of a phosphate binding site in which Arg-96 is critical. Thus, mutation of Arg-96 abolishes the phosphorylation of "primed" glycogen synthase as well as inhibition by PKB-mediated phosphorylation of Ser-9. Hence, the phosphorylated N terminus acts as a pseudosubstrate, occupying the same phosphate binding site used by primed substrates. Significantly, this mutation does not affect phosphorylation of "nonprimed" substrates in the Wnt-signaling pathway (Axin and beta-catenin), suggesting new approaches to design more selective GSK3 inhibitors for the treatment of diabetes.

A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta ) and PKC-related kinase 2 by PDK1.

Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKCzeta, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKCzeta and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKCzeta and PKCiota, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKCzeta, full-length PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in vivo. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKCzeta. These findings indicate that the hydrophobic motif of PRK2 and PKCzeta acts as a "docking site" enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.

Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA.

The 3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates a number of protein kinases of the AGC subfamily. The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF), through a hydrophobic motif. Here we identify a hydrophobic pocket in the small lobe of the PDK1 kinase domain, separate from the ATP- and substrate-binding sites, that interacts with PIF. Mutation of residues predicted to form part of this hydrophobic pocket either abolished or significantly diminished the affinity of PDK1 for PIF. PIF increased the rate at which PDK1 phosphorylated a synthetic dodecapeptide (T308tide), corresponding to the sequences surrounding the PDK1 phosphorylation site of PKB. This peptide is a poor substrate for PDK1, but a peptide comprising T308tide fused to the PDK1-binding motif of PIF was a vastly superior substrate for PDK1. Our results suggest that the PIF-binding pocket on the kinase domain of PDK1 acts as a 'docking site', enabling it to interact with and enhance the phosphorylation of its substrates.

Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.

Role of Mg2+ in nucleoside diphosphate kinase autophosphorylation.

Nucleoside diphosphate (NDP) kinase is a ubiquitous enzyme that has been described to have regulatory functions. In addition to its classical enzymatic activity, NDP kinases have been characterized as inhibitors of metastasis, as a factor stimulating gene transcription, and as a protein kinase. In this report we show some characteristics of the autophosphorylation of homogeneous NDP kinase and make a comparison with that of other proteins in crude extracts. By using labeled substrates and fluorescence quenching analysis, we prove that Mg2+ is indeed necessary for the two steps of the ping-pong reaction to take place and present evidence that NTPs or NDPs, when uncomplexed to divalent cations, may not bind the active site in a comparable way to NTP . Mg2+ and NDP . Mg2+. However, even extremely small concentrations of Mg2+ suffice for maximal autophosphorylation which is obtained with Mg2+ in the nanomolar range and 100 microM ATP using homogeneous enzyme. Moreover, lower autophosphorylation levels were observed with increasing concentrations of Mg2+. The autophosphorylation equilibrium varied from 0.19 to 1.6 upon the inclusion of 10 mM EDTA to produce low Mg2+ concentrations. Under optimal conditions (low Mg2+ concentrations and short incubation times) NDP kinase was the only protein phosphorylated in crude extracts from Candida albicans, indicating that the autophosphorylation properties of the enzyme are very singular.

Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture.

Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated on histidine residues, however, only the B isoform appeared to be serine phosphorylated.

Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase.

We have critically analyzed current methodologies for distinguishing histidine and serine phosphorylated residues in proteins and report a simple technique that assures a reliable discrimination. Electro-transfer of a phosphorylated enzyme to Immobilon membranes and its treatment at pH 1 and 14 in buffers containing 5% methanol allows unambiguous distinction between serine/threonine and histidine phosphorylation (O-phosphomonoesters and phosphoramide, respectively) since under these conditions only one type of residue is dephosphorylated. The addition of 5% methanol to all buffers was indispensable to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP kinase cellular function, signaling the suppression of metastasis in the case of human NDP kinase A. Using this improved method, we show that human, Escherichia coli and Candida albicans NDP kinases are only autophosphorylated on histidine residues. In addition, we present evidence that the presence of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe that the technique will be generally useful in histidine phosphorylation screenings.

Tilarin in combination with astemizole.

This multicentre double-blind, placebo controlled study had a practical objective, based on the expectation that many patients with seasonal allergic rhinitis will be prescribed oral antihistamine monotherapy by their primary care physician, whereas allergy specialists are more likely to prescribe combination therapy including antiinflammatories. The specific question was, "Will the addition of nedocromil sodium 1% nasal spray to astemizole tablets improve control of symptoms of seasonal allergic rhinitis induced by ragweed pollen, as compared to astemizole therapy alone?'. Following a one-week baseline, planned to coincide with the start of the local ragweed pollen season, patients (aged 12-64) were randomly assigned to four weeks' double-blind test treatment with either nedocromil sodium 1% nasal spray four times daily (QID) + astemizole (n = 146) or placebo nasal spray + astemizole (n = 148) or double-dummy (nasal spray + capsules) placebo (n = 71). Patient diary cards were kept throughout the five weeks, and clinic visits were made before and after baseline and after one and four weeks' treatment. During the 10-day peak pollen period, the diary card rhinitis symptom summary score (0-4 severity scale) was significantly reduced in patients receiving either astemizole alone (p < 0.001) or the combination therapy (p < 0.001) as compared with placebo. Direct comparison of the active treatments further showed that symptoms were significantly less severe (p < 0.01) with the combined therapy than with astemizole alone, and this despite significantly greater reliance on permitted rescue medications (p < 0.05 for pseudoephedrine usage) in the astemizole group. Clinical assessments of rhinitis made during the peak pollen visit, after the first week of test treatment, were also significantly (p < 0.05 - p < 0.01) in favour of combined therapy with nedocromil sodium 1% nasal spray + astemizole rather than astemizole alone, and at the same time this preference was confirmed by physician (p = 0.011) and patient (p = 0.003) opinions of symptom control. In conclusion, this antiinflammatory + antihistamine treatment proved superior to antihistamine alone for effective management of allergic rhinitis. The combined therapy worked quickly and was well-tolerated, with no serious adverse events or untoward effects on blood or urine variables.

Candida albicans nucleoside-diphosphate kinase: purification and characterization.

Soluble nucleoside-diphosphate kinase (NDP kinase) from Candida albicans was purified to electrophoretic homogeneity and a partial sequence was determined. The enzyme was kinetically and physically characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single polypeptide of 17 kDa upon staining, by immunodetection with heterologous and homologous antibodies, and by autoradiography of the phosphorylated enzyme. Furthermore, isoelectric focusing of the purified native enzyme showed a single acidic band (pI 4.5). These results together with a native molecular mass of 98 kDa suggest a hexameric native enzyme composed of identical subunits. Like NDP kinases from other sources, the catalysis involves a phosphoenzyme intermediate that is rapidly formed upon incubation of the enzyme with ATP. The transfer of phosphate from phosphoprotein intermediate to nucleoside diphosphates is equally fast. Kinetic experiments indicated that GTP and ATP had the lowest Km compared to UTP, dTTP, and CTP. GDP acted as a preferred acceptor as assessed by kinetic measurements as well as by competition experiments. Experimental data are presented indicating the existence of a membrane-associated NDP kinase. Preliminary characterization of this enzyme suggests that cytosolic and membrane-associated NDP kinases are similar proteins.