Comparison of transthecal and subcutaneous single-injection digital block techniques in cadaver hands

Calmodulin-dependent protein kinase IV (CaM-kinase IV) is markedly activated on the phosphorylation of Thr196 by an upstream protein kinase, CaM-kinase kinase. The phosphorylation of CaM-kinase IV by CaM-kinase kinase is strongly suppressed on incubation with calmodulin in the presence of Ca2+, which results in a decrease in the enzyme activity, and completely restored on subsequent incubation with Mg2+ in the absence of Ca2+, which results in an increase in the activity. That a downstream protein kinase regulates its activity through reversible changes in its susceptibility to phosphorylation by an upstream protein kinase is a new regulatory mechanism.     

Distribution of Ca2+/calmodulin-dependent protein kinase kinase alpha in the rat central nervous system: an immunohistochemical study

Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) is activated by CaM-kinase IV kinase. We provided a rabbit antiserum against 20 amino acid residues at the carboxyl-terminal end of CaM-kinase IV kinase, and examined regional and intracellular distribution of CaM-kinase IV kinase immunohistochemically in the central nervous system of the rat by light and electron microscopy. The immunoreactivity was found in cellular nuclei of virtually all neurons. However, the immunoreactivity was weak in the nuclei of the granule cells in the cerebellar cortex, although the nuclei of the granule cells were reported to contain high CaM-kinase IV activity. Thus, it was suggested that other types of CaM-kinase IV kinase might exist in the cerebellum, and the present CaM-kinase IV kinase was named as CaM-kinase kinase alpha.     

Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2

The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit alpha (eIF2alpha) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2alpha kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.     

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1)

BACKGROUND: Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop. RESULTS: This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC betaII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC alpha and betaII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme. CONCLUSIONS: We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.     

Relationship between phosphorylation and activity of heme-regulated eukaryotic initiation factor 2 alpha kinase

In heme-deficient reticulocytes and their lysates, a heme-regulated inhibitor of protein synthesis is activated; this inhibitor is a cyclic AMP-independent protein kinase that specifically phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eIF-2 alpha). Heme regulates this kinase by inhibiting its activation and activity. The purified heme-regulated kinase (HRI) undergoes autophosphorylation; at least 3 mol of phosphate can be incorporated per HRI subunit (Mr 80,000). The phosphorylation of HRI, its eIF-2 alpha kinase activity, and its ability to inhibit protein synthesis are diminished by hemin (5 microM) and increased by N-ethylmaleimide (MalNEt). Treatment of MalNEt-activated HRI with hemin reduces its autophosphorylation and its ability to inhibit protein synthesis . These findings demonstrate a correlation of the phosphorylation of HRI, its eIF-2 alpha kinase activity, and its inhibition of protein synthesis. The mechanism of hemin regulation of HRI activity was studied by examining the binding of hemin to purified HRI. Significant binding was demonstrable by difference spectroscopy which revealed a pronounced shift in the absorption spectrum of hemin with the appearance of a peak at 418 nm, a shift similar to that observed with proteins known to bind hemin. These findings are consistent with a direct effect of hemin on HRI.     

Partial activation of the insulin receptor kinase domain by juxtamembrane autophosphorylation

Increased enzymatic activity of receptor tyrosine kinases occurs after trans-phosphorylation of one or two tyrosines in the activation loop, located near the catalytic cleft. Partial activation of the insulin receptor's kinase domain was observed at dilute concentrations of kinase, suggesting that cis-autophosphorylation was occurring. Autophosphorylation during partial activation mapped to the juxtamembrane (JM) tyrosines and not to activation loop tyrosines. Furthermore, a double JM Tyr-to-Phe mutant kinase (JMY2F) did not undergo partial activation but catalyzed substrate phosphorylation at a very low rate. Steady-state kinetics of peptide phosphorylation were determined with and without JM autophosphorylation. The JMY2F mutant was used to prevent concurrent cis-autophosphorylation and therefore to approximate the basal state apoenzyme in the kinetic analysis. Partial activation was dominated by a decreased Michaelis constant for peptide substrate, from KM,PEP >/= 2.5 mM in the basal state to 0.2 mM in the partially activated state; the KM,ATP remained virtually unchanged at approximately 1 mM, and kcat increased from 180 to 600 min-1. The high KM,PEP suggests weak binding of peptide substrates to the apoenzyme. This was confirmed by Ki > 1 mM for peptide substrates used as inhibitors of JM autophosphorylation. The absence of comparably large changes in kcat and KM,ATP suggests that the JM region is primarily a strong barrier to the peptide entry step of trans-phosphorylation reactions. The JM region therefore functions as an intrasteric inhibitor in the basal state of the insulin receptor's kinase domain.     

Acute cholecystitis: comparison of MR cholangiography and US

p70 S6 kinase plays an important role in growth factor-induced translational control and in cell cycle progression. Although the mechanism of p70 S6 kinase regulation is not fully understood, phosphorylation of serine and threonine residues of the enzyme is essential for its activation. The possible role of the serine-threonine kinase Akt in the activation of p70 S6 kinase induced by exposure of cells to heat has now been investigated. Overexpression of a mutant Akt1 (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine was shown to exert a dominant negative effect on Akt activation induced by platelet-derived growth factor (PDGF) or by heat treatment in CHO cells. Akt-AA also inhibited p70 S6 kinase activation induced by these stimuli. However, Akt-AA had no effect on the activation of p70 S6 kinase induced by 12-O-tetradecanoylphorbol 13-acetate, which did not stimulate Akt activity in these cells. These data suggest that Akt is required for heat treatment-induced activation of p70 S6 kinase.     

Identification of the major autophosphorylation sites of Nyk/Mer, an NCAM-related receptor tyrosine kinase

Nyk/Mer receptor tyrosine kinase is a new member of the Ufo/Axl tyrosine kinase family and is characterized by its neural cell adhesion molecule-like extracellular domain. By using a vaccinia virus expression system to express a constitutively activated form of Nyk, we identified the major sites of Nyk autophosphorylation in tryptic peptide IY749SGDY753Y754R. Tyr-749, Tyr-753, and Tyr-754 in this peptide lie in the activation loop of the kinase domain. We also studied a series of Nyk mutants in which the three tyrosine residues were replaced individually, in pairs, or all together by phenylalanine. Single mutations of Tyr-749 or Tyr-753 to phenylalanine reduced Nyk kinase activity toward exogenous substrate to 39 or 10% of that of the wild type Nyk, respectively, whereas the Tyr-754 mutant is completely inactive. All of the double and triple Tyr-Phe mutants reduced Nyk kinase activity to a level below the background. Similar results were obtained when Nyk autophosphorylation levels were examined. Our studies suggest that full activity of Nyk/Mer kinase requires phosphorylation of all three tyrosine residues in the kinase domain (Tyr-749, Tyr-753, and Tyr-754) and that Nyk kinase activity is modulated by the level of autophosphorylation in the kinase domain. Given the highly conserved nature of this region among the Ufo/Axl receptor family members, the information presented in this report may provide insight to the biochemical properties of other members of this family.     

Full activation of the platelet-derived growth factor beta-receptor kinase involves multiple events

Activation of receptor tyrosine kinases is thought to involve ligand-induced dimerization, which promotes receptor transphosphorylation and thereby increases the receptor's phosphotransferase activity. We used two platelet-derived growth factor beta-receptor (beta-PDGFR) mutants to identify events that are required for full engagement (autophosphorylation and activation of the kinase activity) of the beta-PDGFR kinase. The F79/81 receptor (Tyr to Phe substitution at 579 and 581 in the juxtamembrane domain of the receptor) was capable of only very modest ligand-dependent autophosphorylation and also failed to associate with numerous SH2 domain-containing proteins. Furthermore, stimulation with platelet-derived growth factor (PDGF) did not increase the kinase activity of the F79/81 mutant toward exogenous substrates. However, the F79/81 receptor had basal kinase activity and could be artificially stimulated by incubation with ATP. Because the low kinase activity of the F857 mutant (Tyr to Phe substitution at 857 in the putative activation loop) could not be increased by incubation with ATP, failure to phosphorylate Tyr-857 may be the reason why the F79/81 mutant has low kinase activity. Surprisingly, the F857 mutant underwent efficient PDGF-dependent autophosphorylation. Thus the PDGF-dependent increase in the kinase activity of the receptor is not required for autophosphorylation. We conclude that full activation of the beta-PDGFR kinase requires at least two, apparently distinct events.     

Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases

Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gbetagamma subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc*Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gbetagamma subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gbetagamma subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gbetagamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc.Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gbetagamma subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc.Grb2 complex formation, and MAP kinase activation. These data suggest that Gbetagamma subunit-mediated formation of Shc.c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.     

Ribonucleotide reductase R2 protein is phosphorylated at serine-20 by P34cdc2 kinase

Ribonucleotide reductase is a rate-limiting enzyme in DNA synthesis and is composed of two different proteins, R1 and R2. The R2 protein appears to be rate-limiting for enzyme activity in proliferating cells, and it is phosphorylated by p34cdc2 and CDK2, mediators of cell cycle transition events. A sequence in the R2 protein at serine-20 matches a consensus sequence for p34cdc2 and CDK2 kinases. We tested the hypothesis that the serine-20 residue was the major p34cdc2 kinase site of phosphorylation. Three peptides were synthesized (from Asp-13 to Ala-28) that contained either the wild type amino acid sequence (Asp-Gln-Gln-Gln-Leu-Gln-Leu-Ser-Pro-Leu-Lys-Arg-Leu-Thr-Leu-Ala, serine peptide) or a mutation, in which the serine residue was replaced with an alanine residue (alanine peptide) or a threonine residue (threonine peptide). Only the serine peptide and threonine peptide were phosphorylated by p34cdc2 kinase. In two-dimensional phosphopeptide mapping experiments of serine peptide and Asp-N endoproteinase digested R2 protein, peptide co-migration patterns suggested that the synthetic phosphopeptide containing serine-20 was identical to the major Asp-N digested R2 phosphopeptide. To further test the hypothesis that serine-20 is the primary phosphorylated residue on R2 protein, three recombinant R2 proteins (R2-Thr, R2-Asp and R2-Ala) were generated by site-directed mutagenesis, in which the serine-20 residue was replaced with threonine, aspartic acid or alanine residues. Wild type R2 and threonine-substituted R2 proteins (R2-Thr) were phosphorylated by p34cdc2 kinase, whereas under the same experimental conditions, R2-Asp and R2-Ala phosphorylation was not detected. Furthermore, the phosphorylated amino acid residue in the R2-Thr protein was determined to be phosphothreonine. Therefore, by replacing a serine-20 residue with a threonine, the phosphorylated amino acid in R2 protein was changed to a phosphothreonine. In total, these results firmly establish that a major p34cdc2 phosphorylation site on the ribonucleotide reductase R2 protein occurs near the N-terminal end at serine-20, which is found within the sequence Ser-Pro-Leu-Lys-Arg-Leu. Comparison of ribonucleotide reductase activities between wild type and mutated forms of the R2 proteins suggested that mutation at serine-20 did not significantly affect enzyme activity.     

Components of a calmodulin-dependent protein kinase cascade. Molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase beta

Ca2+/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV, respectively) require phosphorylation on an equivalent single Thr in the activation loop of subdomain VIII for maximal activity. Two distinct CaMKI/IV kinases, CaMKKalpha and CaMKKbeta, were purified from rat brain and partially sequenced (Edelman, A. M., Mitchelhill, K., Selbert, M. A., Anderson, K. A., Hook, S. S., Stapleton, D., Goldstein, E. G., Means, A. R., and Kemp, B. E. (1996) J. Biol. Chem. 271, 10806-10810). We report here the cloning and sequencing of cDNAs for human and rat CaMKKbeta, tissue and regional brain localization of CaMKKbeta protein, and mRNA and functional characterization of recombinant CaMKKbeta in vitro and in Jurkat T cells. The sequences of human and rat CaMKKbeta demonstrate 65% identity and 80% similarity with CaMKKalpha and 30-40% identity with CaMKI and CaMKIV themselves. CaMKKbeta is broadly distributed among rat tissues with highest levels in CaMKIV-expressing tissues such as brain, thymus, spleen, and testis. In brain, CaMKKbeta tracks more closely with CaMKIV than does CaMKKalpha. Bacterially expressed CaMKKbeta undergoes intramolecular autophosphorylation, is regulated by Ca2+/CaM, and phosphorylates CaMKI and CaMKIV on Thr177 and Thr200, respectively. CaMKKbeta activates both CaMKI and CaMKIV when coexpressed in Jurkat T cells as judged by phosphorylated cAMP response element-binding protein-dependent reporter gene expression. CaMKKbeta activity is enhanced by elevation of intracellular Ca2+, although substantial activity is observed at the resting Ca2+ concentration. The strict Ca2+ requirement of CaMKIV-dependent phosphorylation of cAMP response element-binding protein, is therefore controlled at the level of CaMKIV rather than CaMKK.     

MLCK-A, an unconventional myosin light chain kinase from Dictyostelium, is activated by a cGMP-dependent pathway

Dictyostelium myosin II is activated by phosphorylation of its regulatory light chain by myosin light chain kinase A (MLCK-A), an unconventional MLCK that is not regulated by Ca2+/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with Con A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A's activation mechanism. MLCK-A does not alter its cellular location upon treatment of cells with Con A, nor does it localize to the myosin-rich caps that form after treatment. However, MLCK-A activity rapidly increases 2- to 13-fold when Dictyostelium cells are exposed to Con A. This activation can occur in the absence of MLCK-A autophosphorylation. cGMP is a promising candidate for an intracellular messenger mediating Con A-triggered MLCK-A activation, as addition of cGMP to fresh Dictyostelium lysates increases MLCK-A activity 3- to 12-fold. The specific activity of MLCK-A in cGMP-treated lysates is 210-fold higher than that of recombinant MLCK-A, which is fully autophosphorylated, but lacks threonine-166 phosphorylation. Purified MLCK-A is not directly activated by cGMP, indicating that additional cellular factors, perhaps a kinase that phosphorylates threonine-166, are involved.     

Replacement of Ser657 of protein kinase C-alpha by alanine leads to premature down regulation after phorbol-ester-induced translocation to the membrane

Protein kinase C (PKC) is activated at the cell membrane by interacting with both the acidic lipid phosphatidylserine and the second messenger diacylglycerol. A direct activation of the kinase is also possible by substituting diacylglycerol with phorbol esters such as the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). Transphosphorylation of the activation loop followed by autophosphorylation at sites located on various domains of the protein have been suggested to be required as permissive activation of the alpha and beta isoforms of PKC [Cazaubon, S., Bornancin, F. & Parker, P. (1994) Biochem. J. 301, 443-448; Keranen, L. M., Dutil, E. M. & Newton, A. C. (1995) Curr. Biol. 5, 1393-1403]. Ser657, located near the C-terminus of PKC-alpha, represents a site which is very conserved among the members of the PKC protein family. Circumstantial evidence suggested that this residue represents a possible site of phosphorylation. The conversion of Ser657 to alanine caused a 70% loss of the catalytic activity as well as a drastically increased down regulation upon translocation of this isozyme to the membrane when induced by phorbol ester. The faster electrophoretic mobility of the mutant protein compared to that of the wild-type enzyme suggested that Ser657 represents a phosphorylation site.     

Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway

Kss1 protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae. In MATa haploids exposed to alpha-factor, Kss1 was rapidly phosphorylated on both Thr183 and Tyr185, and both sites were required for Kss1 function in vivo. De novo protein synthesis was required for sustained pheromone-induced phosphorylation of Kss1. Catalytically inactive Kss1 mutants displayed alpha-factor-induced phosphorylation on both residues, even in kss1 delta cells; hence, autophosphorylation is not obligatory for these modifications. In kss1 delta fus3 delta double mutants, Kss1 phosphorylation was elevated even in the absence of pheromone; thus, cross-phosphorylation by Fus3 is not responsible for Kss1 activation. In contrast, pheromone-induced Kss1 phosphorylation was eliminated in mutants deficient in two other protein kinases, Ste11 and Ste7. A dominant hyperactive allele of STE11 caused a dramatic increase in the phosphorylation of Kss1, even in the absence of pheromone stimulation, but required Ste7 for this effect, suggesting an order of function: Ste11-->Ste7-->Kss1. When overproduced, Kss1 stimulated recovery from pheromone-imposed G1 arrest. Catalytic activity was essential for Kss1 function in signal transmission, but not for its recovery-promoting activity. Kss1 was found almost exclusively in the particulate material and its subcellular fractionation was unaffected by pheromone treatment. Indirect immunofluorescence demonstrated that Kss1 is concentrated in the nucleus and that its distribution is not altered detectably during signaling.     

3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase

BACKGROUND: The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (Ptdlns(3,4,5)P3) or phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2). RESULTS: We have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB alpha only in the presence of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2. Overexpression of PDK1 in 293 cells activated PKB alpha and potentiated the IGF1-induced phosphorylation of PKB alpha at Thr308. Experiments in which the PH domains of either PDK1 or PKB alpha were deleted indicated that the binding of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2 to PKB alpha is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. CONCLUSIONS: PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of Ptdlns(3,4,5)P3/Ptdlns(3,4)P2 in the activation of PKB alpha is at least partly substrate directed.     

Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cells

A short model genome RNA and also the genome RNA of influenza A virus bearing both 5'- and 3'-terminal common sequences activated the interferon-induced double-stranded-RNA-dependent protein kinase, PKR, by stimulating autophosphorylation in vitro. The activated PKR catalyzed phosphorylation of the alpha subunit of eucaryotic translation initiation factor 2 (eIF2alpha). The NS1 protein efficiently eliminated the PKR-activating activity of these RNAs by binding to them. Two mutant NS1 proteins, each harboring a single amino acid substitution at different regions, exhibited temperature sensitivity in their RNA binding activity in the mutant virus-infected cell lysates as well as when they were prepared as fusion proteins expressed in bacteria. The virus strains carrying these mutant NS1 proteins exhibited temperature sensitivity in virus protein synthesis at the translational level, as reported previously, and could not repress the autophosphorylation of PKR developing during the virus growth, which is normally suppressed by a viral function(s). As a result, the level of eIF2alpha phosphorylation was elevated 2.5- to 3-fold. The defect in virus protein synthesis was well correlated with the level of phosphorylation of PKR and eIF2alpha.     

Identification of in vivo phosphorylation sites required for protein kinase D activation

Protein kinase D (PKD) is activated by phosphorylation in intact cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides, and growth factors, but the critical activating residues in PKD have not been identified. Here, we show that substitution of Ser744 and Ser748 with alanine (PKD-S744A/S748A) completely blocked PKD activation induced by phorbol-12,13-dibutyrate (PDB) treatment of intact cells as assessed by autophosphorylation and exogenous syntide-2 peptide substrate phosphorylation assays. Conversely, replacement of both serine residues with glutamic acid (PKD-S744E/S748E) markedly increased basal activity (7.5-fold increase compared with wild type PKD). PKD-S744E/S748E mutant was only slightly further stimulated by PDB treatment in vivo, suggesting that phosphorylation of these two sites induces maximal PKD activation. Two-dimensional tryptic phosphopeptide analysis obtained from PKD mutants immunoprecipitated from 32P-labeled transfected COS-7 cells showed that two major spots present in the PDB-stimulated wild type PKD or the kinase-dead PKD-D733A phosphopeptide maps completely disappeared in the kinase-deficient triple mutant PKD-D733A/S744E/S748E. Our results indicate that PKD is activated by phosphorylation of residues Ser744 and Ser748 and thus provide the first example of a non-RD kinase that is up-regulated by phosphorylation of serine/threonine residues within the activation loop.     

Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase

The AMP-activated protein kinase is a heterotrimeric enzyme, important in cellular adaptation to the stress of nutrient starvation, hypoxia, increased ATP utilization, or heat shock. This mammalian enzyme is composed of a catalytic alpha subunit and noncatalytic beta and gamma subunits and is a member of a larger protein kinase family that includes the SNF1 kinase of Saccharomyces cerevisiae. In the present study, we have identified by truncation and site-directed mutagenesis several functional domains of the alpha1 catalytic subunit, which modulate its activity, subunit association, and protein turnover. C-terminal truncation of the 548-amino acid (aa) wild-type alpha1 protein to aa 312 or 392 abolishes the binding of the beta/gamma subunits and dramatically increases protein expression. The full-length wild-type alpha1 subunit is only minimally active in the absence of co-expressed beta/gamma, and alpha1(1-392) likewise has little activity. Further truncation to aa 312, however, is associated with a large increase in enzyme specific activity, thus revealing an autoinhibitory sequence between aa 313 and 392. alpha-1(1-312) still requires the phosphorylation of the activation loop Thr-172 for enzyme activity, yet is now independent of the allosteric activator, AMP. The increased levels of protein expression on transient transfection of either truncated alpha subunit cDNA are because of a decrease in enzyme turnover by pulse-chase analysis. Taken together, these data indicate that the alpha1 subunit of AMP-activated protein kinase contains several features that determine enzyme activity and stability. A constitutively active form of the kinase that does not require participation by the noncatalytic subunits provides a unique reagent for exploring the functions of AMP-activated protein kinase.     

Phosphorylation of vitronectin by casein kinase II. Identification of the sites and their promotion of cell adhesion and spreading

The cell adhesion protein vitronectin (Vn) was previously shown to be the major target in human blood for an extracellular protein kinase A, which is released from platelets upon their physiological stimulation with thrombin and also prevails as an ectoenzyme in several other types of blood cells. Because plasma Vn was shown to have only one protein kinase A phosphorylation site (Ser378) but to contain approximately 3 mol of covalently bound phosphate, and because human serum and blood cells were shown to contain also a casein kinase II (CKII) on their surface, we studied the phosphorylation of Vn by CKII attempting to find out whether such phosphorylation modulates Vn function, an acid test for its having a physiological relevance. Here we show (i) that the CKII phosphorylation of Vn has a Km of 0.5-2 microM (lower than the Vn concentration in blood, 3-6 microM), (ii) that it is targeted to Thr50 and Thr57, which are vicinal to the RGD site of Vn, and (iii) that the phosphorylation of Thr57 facilitates the phosphorylation of Thr50. The maximal stoichiometry of the CKII phosphorylation of plasma Vn was found to be low, which, in principle, could be due to its partial prephosphorylation in vivo. However, for the detection of a functional modulation, we needed a comparison between a fully phosphorylated Vn (at Thr57 and Thr50) and a nonphosphorylated Vn. Therefore, we expressed Vn in a baculovirus system and show (i) that the CKII phosphorylation of wt-Vn enhances the adhesion of bovine aorta endothelial cells; (ii) that the double mutant T50E/T57E (in which the neutral Thr residues are replaced by the negatively charged Glu residues considered analogs of Thr-P) has a significantly enhanced capacity to promote cell adhesion and to accelerate cell spreading when compared with either wild-type Vn or to the neutral T50A/T57A mutant; and (iii) that, at least in the case of bovine aorta endothelial cells, the T50E/T57E mutant exhibits an enhanced adhesion, which seems to be due to an increased affinity toward the alphav beta3 Vn receptors.