Insulin treatment of Drosophila melanogaster Kc 167 cells induces the multiple phosphorylation of a Drosophila ribosomal protein, as judged by its decreased electrophoretic mobility on two-dimensional polyacrylamide gels. The extent to which insulin induces this response is potentiated by cycloheximide and blocked by pretreatment with rapamycin. Isolation and mass spectrometric analysis revealed that the multiply phosphorylated protein was the larger of two Drosophila melanogaster orthologues of mammalian 40S ribosomal protein S6, termed here DS6A. Proteolytic cleavage of DS6A derived from stimulated Kc 167 cells with the endoproteinase Lys-C released a number of peptides, one of which contained all the putative phosphorylation sites. Conversion of phosphoserines to dehydroalanines with Ba(OH)(2) showed that the sites of phosphorylation reside at the carboxy terminus of DS6A. The sites of phosphorylation were identified by Edman degradation after conversion of the phosphoserine residues to S-ethylcysteine as Ser(233), Ser(235), Ser(239), Ser(242), and Ser(245) Finally, phosphopeptide mapping of individual phosphoderivatives, isolated from two-dimensional polyacrylamide gels, indicated that DS6A phosphorylation, in analogy to mammalian S6 phosphorylation, appears to proceed in an ordered fashion. The importance of these observations in cell growth and development is discussed.

Autophosphorylation is a key event in the activation of protein kinases. In this study, we demonstrate that autophosphorylation of the recombinant Src family kinase Hck leads to a 20-fold increase in its specific enzymatic activity. Hck was found to autophosphorylate readily to a stoichiometry of 1.3 mol of phosphate per mol of enzyme, indicating that the kinase autophosphorylated at more than one site. Solid phase sequencing and two-dimensional mapping of the phosphopeptide fragments derived from the autophosphorylated enzyme revealed that the kinase can undergo autophosphorylation at the following two sites: (i) Tyr-388, which is located to the consensus autophosphorylation site commonly found in the activation loop of many protein kinases, and (ii) Tyr-29, which is located in the unique domain of Hck. Hck purified from mouse bone marrow-derived macrophages could also autophosphorylate in vitro at both Tyr-388 and Tyr-29, indicating that naturally occurring Hck can also autophosphorylate at Tyr-29. Furthermore, Hck transiently expressed in human embryonic kidney 293T cells was found to be phosphorylated at Tyr-29 and Tyr-388, proving that Hck can also undergo autophosphorylation at both sites in vivo. The recombinant enzyme carrying the mutation of Tyr-388 to Phe was also able to autophosphorylate at Tyr-29, albeit at a significantly slower rate. A 2-fold increase in the specific enzymatic activity was seen with this mutant despite the stoichiometry of autophosphorylation only approaching 0.2 mol of phosphate per mol of enzyme. This indicates that autophosphorylation of Tyr-29 contributes significantly to the activation of Hck. Regulation of the catalytic activity by phosphorylation of Tyr-29 in the unique domain may represent a new mechanism of regulation of Src family tyrosine kinases.

The complete amino acid sequences of rat and yeast (Saccharomyces cerevisiae) ribosomal proteins derived from precursors containing an N-terminal ubiquitin or ubiquitin-like sequence (C-terminal extension proteins or CEPs) were determined and investigated for any post-translational modifications by reverse-phase HPLC purification, direct amino acid sequence and mass spectrometric analyses. Covalent modifications were detected in the rat liver proteins RS27a (CEP-80), RL29, RL37 and RL40 (CEP-52), while RS30 (CEP), RL36a, RL39 and RL41 were unmodified. Heterogeneity of RS27a was due to C-terminal truncations, with Lys80 missing from about 20% of the liver RS27a population; C-terminal processing was also detected with RL29 and RL37. No other covalent modifications of liver, brain or thymus RS27a were detected. The rat RL40 structure was identical to the cDNA-predicted sequence except for complete stoichiometric N epsilon-trimethylation of Lys22 within its zinc-finger motif; this modification occurred in the ribosomes of all three rat tissues investigated but not in yeast ribosomes. The methylation characteristics of RL40 were distinct from those of ribosomal protein RL29 in the rat, which was differentially monomethylated at Lys4 in the liver, brain and thymus (27%, > 99% and 95% methylation, respectively). In the case of liver, there was no appreciable difference in the RL29 methylation status of free and membrane-bound ribosomes. The possibilities of an essential role for RL40 methylation in the formation of rat ribosomes, and a distinct regulatory role for RL29 methylation in the rat, are discussed.

A homologue of human protein kinase C (PKC)-related kinase-2, PRK2, which had previously escaped identification in normal mammalian tissues, was isolated from rat liver as the protease-activated kinase (PAK) originally named PAK-2. The 130-kDa cytosolic enzyme was purified to homogeneity and shown by tryptic peptide and reverse transcriptase- polymerase chain reaction (RT-PCR)-amplified rat cDNA sequence analyses to be structurally related to the 116-kDa rat hepatic PAK-1/protein kinase N (PKN) and, even more closely (95% sequence identity) to the 130-kDa human PKC-related kinase, PRK2. Rat myeloma RNA was used as the RT-PCR template because of its relative abundance in PAR-2/PRK2 mRNA compared with liver and other rat tissues. The catalytic properties of PAK-2/PRK2 in many respects resembled those of hepatic PAK-1/PKN, but were distinguished by more favorable kinetics with several peptide substrates, and greater sensitivity to PKC pseudosubstrate and polybasic amino acid inhibitors. PAK-2/PRK2 was also activated by lipids, particularly cardiolipin and to a lesser extent by other acidic phospholipids and unsaturated fatty acids. Cardiolipin activation was most evident with autophosphorylation and histone H2B phosphorylation, but only marginally evident with the favored ribosomal S6-(229-239) peptide substrate for the protease-activated kinase activity. It was concluded that PAK-2 is the rat homologue of human PRK2, with biochemical properties distinct from although overlapping those of the PAK-1/PKN/PRK1 isoform.

The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.

In mammals, the 59-residue ribosomal protein S30 (rpS30)is synthesized as a fusion to a 74-residue ubiquitin-like protein, which is cleaved to yield mature rpS30. An artificial fusion of this ubiquitin-like protein to E. coli beta-galactosidase was not cleaved when expressed in yeast (Saccharomyces cerevisiae), indicating that yeast lack this cleaving activity, The yeast rpS30 homolog (yrpS30) was purified and sequenced to reveal a 63-residue protein with 61% sequence identity to mammalian rpS30, Degenerate oligonucleotides based on the yrpS30 sequence were used to isolate full-length yrpS30 cDNAs, Sequence analysis of five cDNA clones revealed that yrpS30 is not synthesized as a fusion to a ubiquitin-like protein but is extended at its N terminus by a single methionine residue, The corresponding gene was identified in the GenBank(TM) data base by sequence alignment and termed RPS30A, The gene consists of two exons separated by a 430-base pair intron, which contains consensus splicing elements, Exon 1 encodes the initiator methionine residue and is preceded by canonical yeast ribosomal protein gene promoter elements, Exon 2 encodes the 62-residue mature yrpS30, Genomic hybridization reveals that the RPS30A gene is duplicated. Disruption of the RPS30A gene is not lethal but confers a slow growth phenotype. Ribosomes in the mutant strains contain an authentic yrpS30 protein, indicating that a functional yrpS30 is expressed from the duplicated gene but that the reduced capacity for yrpS30 synthesis restricted the growth rate. Analysis of avail able DNA sequence data bases reveals that rpS30 is synthesized as a fusion to a ubiquitin-like protein in nematodes and mammals but unfused in yeast, plants, and protazoa.

The middle ear cleft connects by membranes to the inner ear and the subarachnoid space particularly in infants. In order to gain more insight about the permeability between the two compartments we quantified the concentration of beta-trace protein – a highly specific marker for CSF and perilymph but not for serum and mucosal effusion – in middle ear secretions from children with otitis media with effusion.One-hundred and three patients were included and 93 samples from secretory otitis media were collected during myringotomy or explorative tympanotomy. Thirty-eight patients of 103 had to be excluded (36.9%). Of the 93 collected samples from 65 subjects, 82 viscous samples were pre-diluted 1:1 with tyloxapol. In spite of the attempt to pre-dilute the viscous samples, 30 glue-like samples of 93 were not applicable for nephelometry. The final analysis was made on 63 samples of 52 subjects (median age 3 years) which were quantified for beta-trace protein using immunonephelometry.In 3/63 samples the beta-trace protein values were below the detection range (<0.2 mg/L) and in 1/63 it was beyond with 18.3 mg/L. The median beta-trace protein value for 59 samples within the measuring range was 2.4 mg/L, range 0.2–14.2.In pediatric middle ear effusions, the beta-trace protein concentration was found to have a high range compared to other body fluids from other studies. In other studies, the values for serum (0.59 mg/L) or mucosal secretion (0.003–0.12 mg/L) were lower and the values in CSF (18.4 mg/L) or perilymphatic fluid (23.5 mg/L) were highest. This finding might indicate a weak barrier between the cerebrospinal fluid space or inner ear fluid compartments on the one side and the tympanic cavity on the other side given the condition of otitis media with effusion. The detection of beta-trace protein might be important to assess the risk of impending complications.

The occurrence of the thyroid hormone-binding plasma protein transthyretin in the bloodstream was investigated for four American marsupial species. Serum samples were analyzed by incubation with radioactive T4, followed by electrophoresis, then autoradiography, and Western blotting. Transthyretin was found in serum from Monodelphis domestica, Didelphis virginiana, Caluromys lanatus, and Dromiciops australis. For unambiguous identification, transthyretin from D, virginiana was purified from serum and its N-terminal amino acid sequence was determined. The obtained results suggest that the initiation of transthyretin gene expression in the liver of marsupials occurred independently in several lineages of American marsupials, all of which are at the ends of phylogenetic branches. The expression of the transthyretin gene in the liver of the American polyprotodont marsupials contrasts with the lack of transthyretin gene expression in the liver of all 22 previously investigated Australian Polyprotodonta.

Cardiolipin- or protease-activated protein kinase, isolated from rat liver cytosol and originally named liver PAK-1, was found to be the natural form of protein kinase N (PKN) by comparing the sequences of 43 tryptic peptides of the purified liver enzyme and determining the corresponding liver cDNA sequence. These analyses also identified (i) Arg-546 as the major site of proteolytic activation, (ii) the protease resistance of the C-terminal extension beyond the catalytic domain, and (iii) in vivo stoichiometric phosphorylation of Thr-778 in the mature enzyme. Homology modeling of the catalytic domain indicated that phosphothreonine 778 functions as an anchoring site similar to Thr-197 in cAMP-dependent protein kinase, which stabilizes an active site compatible with preferred substrate sequences of PAK-1/PKN. Sigmoidal autophosphorylation kinetics and increased S6-(229-239) peptide kinase activity following preincubation with ATP suggested phosphorylation-dependent activation of PAK-1/PKN. The onset of activation corresponded with phosphorylation of the regulatory domain site Ser-377 (located within a spectrin homology region), followed by Thr-504 (within a limited protein kinase C homology region), and, to a lesser extent, Thr-64 (in the RhoA-binding region). Several additional sites in the hinge region adjacent to a PEST protein degradation signal were selectively autophosphorylated following cardiolipin activation. Overall, these observations suggest that the regulation of this class of protein kinase involves complex interactions among phosphorylation-, lipid-, and other ligand-dependent activation events.

The evolution of the expression and the structure of the gene for transthyretin, a thyroxine-binding plasma protein formerly called prealbumin, was studied in three marsupial species: the South American polyprotodont Monodelphis domestica, the Australian polyprotodont Sminthopsis macroura and the Australian diprotodont Petaurus breviceps. The transthyretin gene was found to be expressed in the choroid plexus of all three species. In liver it was expressed in P. breviceps and in M. domestica, but not in S. macroura. This, together with previous studies [Richardson, S. J., Bradley, A. J., Duan, W., Wettenhall, R. E. H., Harms, P. J., Babon, J. J., Southwell, B. R., Nicol, S., Donnellan, S. C. & Schreiber, G. (1994) Am. J. Physiol. 266, R1359-R1370], suggests the independent evolution of transthyretin synthesis in the liver of the American Polyprotodonta and the Australian Diprotodonta. The results obtained from cloning and sequencing of the cDNA for transthyretin from the three species suggested that, in the evolution of the structure of transthyretin in vertebrates, marsupial transthyretin structures are intermediate between bird/reptile and eutherian transthyretin structures. In marsupials, as in birds and reptiles, a hydrophobic tripeptide beginning with valine and ending with histidine was found in transthyretin at a position which has been identified in eutherians as the border between exon 1 and intron 1. In humans, rats and mice, the nine nucleotides, coding for this tripeptide in marsupials/reptiles/birds, are found at the 5' end of intron 1. They are no longer present in mature transthyretin mRNA. This results in a change in character of the N-termini of the subunits of transthyretin from hydrophobic to hydrophilic. This change might affect the accessibility of the thyroxine-binding site in the central channel of transthyretin, since, at least in humans, the N-termini of the subunits of transthyretin are located in the vicinity of the channel entrance [Hamilton, J. A., Steinrauf, L. K., Braden, B. C., Liepnieks, J., Benson, M. D., Holmgren, G., Sandgren, O. & Steen, L. (1993) J. Biol. Chem. 268, 2416-2424].

The retention behavior of a series of amphipathic peptide multimers based on the amino acid sequence [KSEEQLA]n has been investigated using reversed-phase high performance liquid chromatography (RP-HPLC). Structure-retention parameters which are related to the hydrophobic contact area and affinity of these peptides for the immobilized hydrocarbonaceous ligands were determined over a range of operating temperatures between 5 degrees and 85 degrees C. The influence of ligand hydrophobicity was assessed by comparison of peptide retention behavior using an n-octadecyl (C18)- and an n-butyl (C4)-silica of similar ligand density. The results demonstrated that ligand-mediated conformational effects can stabilize peptide structure depending on the chromatographic residence time and peptide length. In particular, more highly stabilized secondary structures were evident for the longer peptides. In addition, the amphipathic secondary structure of the peptides were more effectively stabilized by the more hydrophobic C18 ligands relative to the shorter C4 ligands. Additional information on the interactive dynamics of these peptide multimers was obtained from analysis of bandwidth dependencies under the different chromatographic conditions. These studies provide further insight into the role which hydrophobic forces can play in the stabilization of peptide structures.

The immunosuppressive agent rapamycin induces inactivation of p70s6k with no effect on other mitogen-activated kinases. Here we have employed a combination of techniques, including mass spectrometry, to demonstrate that this effect is associated with selective dephosphorylation of three previously unidentified p70s6k phosphorylation sites: T229, T389 and S404. T229 resides at a conserved position in the catalytic domain, whose phosphorylation is essential for the activation of other mitogen-induced kinases. However, the principal target of rapamycin-induced p70s6k inactivation is T389, which is located in an unusual hydrophobic sequence outside the catalytic domain. Mutation of T389 to alanine ablates kinase activity, whereas mutation to glutamic acid confers constitutive kinase activity and rapamycin resistance. The importance of this site and its surrounding motif to kinase function is emphasized by its presence in a large number of protein kinases of the second messenger family and its conservation in putative p70s6k homologues from as distantly related organisms as yeast and plants.

Binding of radioactive thyroxine to proteins in the plasma of vertebrates was studied by electrophoresis followed by autoradiography. Albumin was found to be a thyroxine carrier in the blood of all studied fish, amphibians, reptiles, monotremes, marsupials, eutherians (placental mammals), and birds. Thyroxine binding to transthyretin was detected in the blood of eutherians, diprotodont marsupials, and birds, but not in blood from fish, toads, reptiles, monotremes, and Australian polyprotodont marsupials. Globulins binding thyroxine were only observed in the plasma of some mammals. Apparently, albumin is the phylogenetically oldest thyroxine carrier in vertebrate blood. Transthyretin gene expression in the liver developed in parallel, and independently, in the evolutionary lineages leading to eutherians, to diprotodont marsupials, and to birds. In contrast, high transthyretin mRNA levels, strong synthesis, and secretion of transthyretin in choroid plexus from reptiles and birds indicate that transthyretin gene expression in the choroid plexus evolved much earlier than in the liver, probably at the stage of the stem reptiles. NH2-terminal sequence analysis suggests a change of transthyretin pre-mRNA splicing during evolution.

A cardiolipin- and protease-activated protein kinase (PAK) has been isolated from cytoplasmic extracts of rat liver. The enzyme (PAK-1) phosphorylates the ribosomal protein S6-(229-239) peptide analogue and can be activated by limited proteolysis. Partial amino acid sequences of tryptic peptides derived from both the purified 116-kDa PAK-1 holoenzyme and its active catalytic fragment reveal that the catalytic domain is most related (50-58% identity) to the protein kinase C family. PAK-1 has protein and peptide substrate specificities distinct from those of known protein kinase C isoforms and is insensitive to inhibition by the protein kinase C-alpha-(19-31) pseudosubstrate peptide. Phosphatidylserine, diacylglycerol, and phorbol ester do not activate PAK-1 toward the S6 peptide substrate. However, other acidic phospholipids, the most effective being cardiolipin, activate PAK-1 to a similar extent as trypsin. The PAK-1 catalytic activities generated through activation by cardiolipin or limited proteolysis were kinetically similar, with Km values of 3.6 and 3.4 microM, respectively, for the S6-(229-239) peptide substrate. However, differences were observed in the catalytic activities with protamine sulfate and the glycogen synthase-(1-12) peptide analogue as substrates. It was concluded that PAK-1 is a phospholipid-regulated protein kinase with a primary structure, substrate specificity, and mechanism of regulation in vitro distinct from those of any known member of the protein kinase C superfamily.

The lipid responsiveness of the structurally unique protein kinase, referred to as PAK‐1, recently isolated from rat liver [(1994) J. Biol. Chem. 269, in press], is characterised by the high sensitivity (low micromolar) of its ribosomal S6(229–239) peptide kinase activity to both cardiolipin and the cis ‐unsaturated fatty acids and insensitivity to phosphatidylserine. Autophosphorylation of PAK‐1 exhibited even greater sensitivity (submicromolar) to cardiolipin, but was relatively less affected by phosphatidylserine. Oleate, the most potent activator of PAK‐1's peptide kinase activity was relatively ineffectual with autophosphorylation. These and other unusual characteristics, including high levels of basal catalytic activities, suggest a novel mechanism of regulation distinct from that of the protein kinase Cs

The presence of transthyretin in mammals and birds, but not amphibia, suggested that transthyretin expression first appeared in stem reptiles. Therefore, transthyretin synthesis was studied in a lizard. Transthyretin synthesis in choroid plexus pieces from Tiliqua rugosa was demonstrated by incorporation of radiactive amino acids. Oligonucleotides corresponding to conserved regions of transthyretin were used as primers in polymerase chain reaction with lizard choroid plexus cDNA. Amplified DNA was used to screen a lizard choroid plexus cDNA library. A full-length transthyretin cDNA clone was isolated and sequenced. A three-dimensional model of lizard transthyretin was obtained by homology modeling. The central channel of transthyretin, containing the thyroxine-binding site, was found to be completely conserved between reptiles and mammals. Transthyretin expression was not detected in lizard liver. These data suggest that transthyretin first evolved in the choroid plexus of the brain. Due to a change in tissue distribution of gene expression, occurring much later during evolution, transthyretin also became a plasma protein, synthesized in the liver.

1. Transthyretin was found to be synthesized and secreted by choroid plexus from rats, echidnas, and lizards, but not toads. 2. Transthyretin was observed in blood from placental mammals, birds, and marsupials, but not reptiles and monotremes. 3. The obtained data suggest that transthyretin synthesis by the liver evolved independently in the lineage leading to the placental mammals and marsupials and in that leading to the birds. 4. It is proposed that transthyretin gene expression in mammalian liver appeared about 200 million years later than its first occurrence in the choroid plexus of the stem reptiles.

Phosphorylated ribosomal proteins were isolated from Xenopus 40 S ribosomal subunits by reversed-phase high performance liquid chromatography (HPLC) to enable direct analysis of the phosphorylation sites in ribosomal protein S6. Xenopus S6 closely resembled mammalian S6 with respect to the following properties: (i) reversed-phase HPLC elution behavior, (ii) amino-terminal sequence (96% identity in the first 37 residues), and (iii) an identical sequence within the region of its phosphorylation sites. Whereas S6 was the only ribosomal protein phosphorylated in vitro by Xenopus S6 kinase II, ribosomes phosphorylated in vivo were found to be associated with an additional phosphoprotein having an amino-terminal sequence identical to that of the ubiquitin carboxyl-terminal extension protein CEP 80. S6 kinase II phosphorylated at least four sites (serines 1-3 and 5) in the sequence Arg-Arg-Leu-Ser(1)-Ser(2)-Leu-Arg-Ala-Ser(3)-Thr-Ser(4)-Lys-Ser(5)-, which correspond to the residues known to be phosphorylated in the carboxyl-terminal region of mammalian S6. The in vivo S6 phosphorylation sites in maturing Xenopus oocytes were shown to be located within the same cluster of serine residues, although individual sites were not identified. Kinetic analysis of S6 kinase II-catalyzed phosphorylation events indicated a simple sequential mechanism of multisite phosphorylation initiating at either serine 2 (preferred) or serine 1, with the rates of phosphorylation of individual sites occurring in the order serine 2 greater than serine 1 greater than serine 3 greater than serine 5.