A "SYDE" effect of hierarchical phosphorylation: possible relevance to the cystic fibrosis basic defect.

The motif "SYDE", incorporating the protein kinase CK2 consensus sequence (S-x-x-E) has been found to be phosphorylated at both its serine and tyrosine residues in several proteins. Of special interest is the case of cystic fibrosis Transmembrane-conductance Regulator (CFTR), where this motif is close to the residue (F508), whose deletion is the by far commonest cause of cystic fibrosis. Intriguingly, however, CFTR S511 cannot be phosphorylated by CK2 to any appreciable extent. Using a number of peptide substrates encompassing the CFTR "SYDE" site we have recently shown that: (1) failure of CK2 to phosphorylate the S(511)YDE motif is due to the presence of Y512; (2) CK2 readily phosphorylates S511 if Y512 is replaced by a phospho-tyrosine; (3) the Src family protein tyrosine kinase Lyn phosphorylates Y512 in a manner that is enhanced by the deletion of F508. These data, in conjunction with the recent observation that by inhibiting CK2 the degradation of F508delCFTR is reduced, lead us to hypothesize that the hierarchical phosphorylation of the motif SYDE by the concerted action of protein tyrosine kinases and CK2 is one of the mechanisms that cooperate to the premature degradation of F508delCFTR.

Protein kinase CK2: a newcomer in the 'druggable kinome'.

The acronym CK2 (derived from the misnomer 'casein kinase' 2) denotes one of the most pleiotropic members of the eukaryotic protein kinase superfamily, characterized by an acidic consensus sequence in which a carboxylic acid (or pre-phosphorylated) side chain at position n+3 relative to the target serine/threonine residue plays a crucial role. The latest repertoire of CK2 substrates includes approx. 300 proteins, but the analysis of available phosphopeptide databases from different sources suggests that CK2 alone may be responsible for the generation of a much larger proportion (10-20%) of the eukaryotic phosphoproteome. Although for the time being CK2 is not included among protein kinases whose inhibitors are in clinical practice or in advanced clinical trials, evidence is accumulating that elevated CK2 constitutive activity co-operates to induce a number of pathological conditions, including cancer, infectious diseases, neurodegeneration and cardiovascular pathologies. The development and usage of cell-permeant, selective inhibitors discloses a scenario whereby CK2 plays a global anti-apoptotic role, which under special circumstances may lead to untimely and pathogenic cell survival.

Autocatalytic tyrosine-phosphorylation of protein kinase CK2 alpha and alpha' subunits: implication of Tyr182.

CK2 is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits, whose mechanism of modulation is still obscure. Here we show that CK2 alpha/alpha' subunits undergo intermolecular (trans) tyrosine-autophosphorylation, which is dependent on intrinsic catalytic activity and is suppressed by the individual mutation of Tyr182, a crucial residue of the activation loop, to phenylalanine. At variance with serine-autophosphorylation, tyrosine-autophosphorylation of CK2alpha is reversed by ADP and GDP and is counteracted by the beta-subunit and by a peptide reproducing the activation loop of CK2alpha/alpha' (amino acids 175-201). These results disclose new perspectives about the mode of regulation of CK2 catalytic subunits.

Distribution of protein disulphide isomerase in rat liver mitochondria.

Here we report the localization of protein disulphide isomerase (PDI) in the mitochondrial compartments, comparing it with that of thioredoxin reductase. The latter enzyme is present mostly in the matrix, whereas PDI is located at the level of the outer membrane. We characterize the different submitochondrial fractions with specific marker enzymes. PDI, whether isolated from whole mitochondria or from purified outer membranes, exhibits the same electrophoretic mobility, indicating identical molecular masses. Moreover, immunoblot analysis with monoclonal anti-PDI antibody shows immunoreactivity only with the microsomal PDI, indicating the specificity of the mitochondrial isoform. The significance of these findings is discussed with reference to the potential role of PDI and thioredoxin reductase in regulating the mitochondrial functions dependent on the thiol-disulphide transition.

Generation of mutants of CK2alpha which are dependent on the beta-subunit for catalytic activity.

To shed light on the structural features underlying high constitutive activity of protein kinase CK2 a number of mutants of the human CK2alpha-subunit altered in the interactions between the N-terminal segment and the activation loop have been generated and shown to be defective in catalytic activity. In particular the truncated mutant delta2-12 displays under standard conditions an almost complete loss of catalytic activity accounted for by a dramatic rise in its Km forATP (from 10 to 206 microM) and a reduced Kcat. Such a drop in efficiency is paralleled by conformational disorganization, as judged from Superdex 75 gel filtration profile. Both catalytic properties and gel filtration behaviour similar to those of wild type CK2alpha were restored upon association with the regulatory beta-subunit, suggesting that constitutive activity is conferred to CK2alpha and to CK2 holoenzyme through different molecular mechanisms. In the holoenzyme an assumable release of tension at the backbone of Ala-193 (as seems to be indicated by a comparison of the crystal structures of maize CK2alpha alone vs. a CK2alpha-beta peptide complex) may result in the ability of the activation loop to adopt its proper conformation independently of interactions with the N-terminal segment.

Isolation, purification, and characterization of a rat liver mitochondrial protein disulfide isomerase.

The isolation and purification to electrophoretical homogeneity and characterization of a protein disulfide isomerase from rat liver mitochondria is reported. The purified enzyme exhibits a single band on sodium dodecylsulfatepolyacrylamide gel electrophoresis with an apparent molecular weight of approximately 54 kDa. Comparatively, the microsomal form shows an apparent molecular weight of 57 kDa indicating that the two forms are slightly different. The antibody raised against the microsomal isoform does not recognize the mitochondrial enzyme. To characterize the enzyme, different classical methodologies utilized for protein disulfide isomerase estimation have been adopted. The isolated enzyme is active with all of them, indicating that it comprises all the features of a typical protein disulfide isomerase. At the mitochondrial level the enzyme appears mostly localized at the membrane level. Its potential involvement in mitochondrial membrane permeability control is also discussed.

Tyrosine versus serine/threonine phosphorylation by protein kinase casein kinase-2. A study with peptide substrates derived from immunophilin Fpr3.

Protein kinase casein kinase-2 (CK2) is a spontaneously active, ubiquitous, and pleiotropic enzyme that phosphorylates seryl/threonyl residues specified by multiple negatively charged side chains, the one at position n + 3 being of crucial importance (minimum consensus S/T-x-x-E/D/S(P)/T(P). Recently CK2 has been reported to catalyze phosphorylation of the yeast nucleolar immunophilin Fpr3 at a tyrosyl residue (Tyr(184)) fulfilling the consensus sequence of Ser/Thr substrates (Wilson, L.K., Dhillon, N., Thorner, J., and Martin, G.S. (1997) J. Biol. Chem. 272, 12961-12967). Here we show that, by contrast to other tyrosyl peptides fulfilling the consensus sequence for CK2, a peptide reproducing the sequence around Fpr3 Tyr(184) (DEDADIY(184)DEEDYDL) is phosphorylated by CK2, albeit with much higher K(m) (384 versus 4. 3 microM) and lower V(max) (8.4 versus 1,132 nmol.min(-1).mg(-1)) than its derivative with Tyr(184) replaced by serine. The replacement of Asp at position n + 1 with alanine and, to a lesser extent, of Ile at n - 1 with Asp are especially detrimental to tyrosine phosphorylation as compared with serine phosphorylation, which is actually stimulated by the Ile to Asp modification. In contrast the replacement of Glu at n + 3 with alanine almost suppresses serine phosphorylation but not tyrosine phosphorylation. It can be concluded that CK2 is capable to phosphorylate, under special circumstances, tyrosyl residues, which are specified by structural features partially different from those that optimize Ser/Thr phosphorylation.

CK2: a protein kinase in need of control.

Protein kinase CK2 is a heterotetrameric alpha2beta2 Ser/Thr protein kinase with some features unusual among the eukaryotic protein kinases: (1) CK2 recognizes phosphoacceptor sites specified by several acidic determinants; (2) CK2 can use both ATP and GTP as phosphoryl donors; and (3) the regulatory properties of CK2 are poorly understood; it is insensitive to any known second messenger and displays high basal activity. To gain insight into CK2 regulation and to understand its unusual properties, site-directed mutagenesis experiments on both subunits and X-ray crystallographic studies of the catalytic alpha-subunit were performed. The noncatalytic beta-subunit has at least three functions: (1) it protects the alpha-subunit against denaturing agents or conditions; (2) it alters the substrate specificity of the alpha-subunit; and (3) it modulates the activity of the enzyme, i.e., depending on the substrate, it increases or decreases the activity of the alpha-subunit. Mutagenesis experiments revealed that an acidic stretch between amino acids 55 and 64 has a down-regulatory and autoinhibitory function. Mutational analysis of the alpha-subunit has revealed a network of unique basic residues that are responsible for the recognition of phosphoacceptor substrates and for down-regulation by the beta-subunit and by polyanionic inhibitors. The resolution of the crystal structure of Zea mays CK2 alpha-subunit has disclosed the structural features that are responsible for high basal activity and for unusual response to nucleotide analogs. The increasing knowledge of CK2 structure-function relationships will allow the design of highly selective inhibitors of this pleiotropic kinase with oncogenic potential.

A multifunctional network of basic residues confers unique properties to protein kinase CK2.

Protein kinase CK2 is characterized by a number of features, including substrate specificity, inhibition by polyanionic compounds and intrasteric down-regulation by its beta-subunit, which denote a special aptitude to interact with negatively charged ligands. This situation may reflect the presence in CK2 catalytic subunits of several basic residues that are not conserved in the majority of other protein kinases. Some of these residues, notably K49 in the 'Gly rich loop', K74, K75, K76, K77, K79, R80, K83 in the 'Lys rich segment' and R191, R195, K198 in the 'p+1 loop', have been shown by mutational studies to be implicated to various extents and with distinct roles in substrate recognition, inhibition by heparin and by pseudosubstrate and instrasteric regulation. Molecular modelization based on crystallographic data provide a rationale for the biochemical observations, showing that several of these basic residues are clustered around the active site where they make contact with individual acidic residues of the peptide substrate. They can also mediate the effect of polyanionic inhibitors (e.g. heparin) and of regulatory elements present in the beta-subunit, in the N terminal segment of the catalytic subunit and possibly in other proteins interacting with CK2. Our data also disclose a unique mode of binding of the phosphoacceptor substrate which bridges across the catalytic cleft making contacts with both the lower and upper lobes of CK2.

Linear and cyclic peptides as substrates for Lyn tyrosine kinase.

Two Tyr residues are supposed to play a crucial role in the regulation of protein tyrosine kinases of the Src family. Autophosphorylation of Src Tyr416 correlates with enzyme activation, while phosphorylation of C-terminal Tyr527 by Csk gives rise to inactive forms of Src kinases. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a particularly high affinity (Km 20 microm) toward the dimeric linear and cyclic derivatives of the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH which reproduces the main autophosphorylation site of most of the Src enzymes. Under the experimental conditions used only one Tyr residue of the dimeric sequence can be phosphorylated [P. Ruzza, A. Calderan, B.Filippi, B. Biondi, A. Donella Deana, L. Cesaro, L. A. Pinna & G. Borin (1995) Int. J. Peptide Protein Res. 45, 529-539]. The present study addresses the problem of the efficiency displayed by Lyn towards the two Tyr residues located at positions 5 and 12 of the dimeric peptide. To this purpose, two tetradecapeptides were synthesized by the classical solution method, each containing one of the two Tyr residues alternatively replaced by Phe, and the corresponding univocal cyclic form. A possible correlation between the different structural properties induced by the modifications of the native sequence and the ability of the peptides to act as Lyn substrates was noted. The kinetic data obtained indicate that Lyn phosphorylates the residues located at different positions in the two linear analogues differently. In particular, while the Tyr5, Phe12 derivative presents a Km value similar to those obtained for the dimeric linear and cyclic unmodified analogues, the Km value of the Phe5, Tyr12 derivative is two-fold higher than those found for the above-mentioned peptides. Moreover, as previously reported for the linear and cyclic dimeric forms of the native sequence, in the mono-tyrosine containing series of dimers the still conformationally flexible cyclic derivative shows a phosphorylation efficiency two-fold higher than those found for the linear derivatives.

Spontaneous autophosphorylation of Lyn tyrosine kinase at both its activation segment and C-terminal tail confers altered substrate specificity.

Two tyrosyl residues have been reported to play a crucial role in the regulation of protein tyrosine kinases of the Src family: autophosphorylation of Tyr416 (c-Src numbering) located in the catalytic domain correlates with enzyme activation, while Csk-mediated phosphorylation of the C-terminal tyrosine Tyr527 (c-Src numbering) gives rise to inactive forms of Src kinases. Here we show that the Src-related Lyn kinase undergoes spontaneous and stoichiometric autophosphorylation at both Tyr396 (homologous to c-Src Tyr416) and Tyr507 (homologous to c-Src Tyr527). Such a doubly autophosphorylated form of Lyn is hyperactive toward peptide substrates and insensitive to Csk-induced downregulation. In contrast, doubly autophosphorylated Lyn exhibits reduced activity toward protein substrates such as phospho-p50/HS1 (hematopoietic-lineage cell-specific protein) and p57/PDI (protein disulfide isomerase related protein), whose multiple sequential/processive phosphorylation relies on the accessibility of the SH2 domain of the kinase. These data disclose a novel conformation of Lyn that is catalytically active despite the presence of an intramolecular interaction between the phosphorylated tail and the SH2 domain. This enzyme conformation is expected to display a reduced oncogenic potential resulting from its defective recognition of a subset of protein substrates whose targeting is mediated by the Lyn SH2 domain.

Correlation between cytosolic Ca2+ concentration, protein phosphorylation and platelet secretion.

Addition of the calcium-ionophore ionomycin to acetylsalicylate-treated platelets suspended in a low Ca2+ concentration-containing medium (about 0.1 microM), induced a dose-dependent (range 0.25-3 microM) and transient increase in the cytosolic Ca2+ concentration ([Ca2+]c). Less than 10% of the maximal releasable amount of serotonin was secreted at [Ca2+]c lower than 1 microM, whereas secretion was almost maximal at [Ca2+]c higher than 2 microM. In all cases the secretion stopped after about 1 min even if the [Ca2+]c was kept constant by repeated small additions of CaCl2 (25-40 microM). A rapid phosphorylation of pleckstrin (47 kDa) and myosin light chain (20 kDa) was found in all cases, whereas a weak phosphorylation of a 27 kDa protein occurred at [Ca2+]c lower than 1.5 microM. Addition of 0.2 mM CaCl2 to platelets pretreated for 4 min with 0.5-1 microM ionomycin brought about a serotonin secretion remarkably lower than obtained by the simultaneous addition of CaCl2 and ionophore. Platelets suspended in a low calcium-containing medium and exposed to ionomycin showed a major increase in tyrosine phosphorylation of 60 and 72 kDa proteins and a slight increment in tyrosine phosphorylation of 115 and 130 kDa proteins. Subsequent addition of 0.2 mM CaCl2 induced a widespread phosphotyrosine dephosphorylation, particularly evident in the 60 kDa protein identified as p60c-src kinase. The protein kinase inhibitor genistein caused, together with a marked prevention of the protein tyrosine phosphorylation, a remarkable increase in the ionomycin-elicited secretory activity of platelets All together these results indicate that protein kinase C-dependent pleckstrin phosphorylation is a prerequisite of platelet secretion, but that the latter process is apparently regulated by a network of phosphoproteins, in particular the serine/threonine phosphorylation of 27 and 68 kDa proteins and the tyrosine phosphorylation of the p60c-src were found to be associated with a decrease in the secretory activity.

An exploration of the effects of constraints on the phosphorylation of synthetic protein tyrosine kinase peptide substrates.

We synthesized by classical solution methods three conformational constrained analogues of EDNEYTA, a heptapeptide sequence that represents the common major autophosphorylation site of the protein tyrosine kinases (PTKs) of the Src family. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as PTK substrates was examined. The kinetic data obtained indicate that the introduction of the tyrosine-analogue constraints Tic(OH) and MeTyr, which block the ring flexibility, completely prevents the phosphorylation catalysed by the kinases Lyn and Fgr. On the other hand PTKIIB/p38syk can phosphorylate the two derivatives albeit with an efficiency lower than that found with the native sequence. A third derivative contained side chain to side chain cyclization. This analogue, in which the freedom of the phenolic moiety is not altered, can be phosphorylated by all the PTKs tested with kinetic constants comparable to the parent peptide.

A comparative study of the phosphotyrosyl phosphatase specificity of protein phosphatase type 2A and phosphotyrosyl phosphatase type 1B using phosphopeptides and the phosphoproteins p50/HS1, c-Fgr and Lyn.

The phosphotyrosyl phosphatase (PTPase) specificity of phosphotyrosyl-phosphatase-activator-(PTPA)-stimulated protein phosphatase (PP)2A(D) (rabbit muscle) and a bona fide PTP-1B (Xenopus laevis oocytes) were examined in vitro using phosphotyrosine-containing peptides, derived from the phosphorylation sites of p34cdc2, p50/HS1 protein, Abl, c-Src and c-Fgr, as well as the intact phosphoprotein p50/HS1 and the Src-related tyrosine kinases, Lyn and c-Fgr. The local specificity determinants were found to be different for both PTPases. The length of the phosphopeptides is more important for PP2A(D) than for PTP-1B, C-terminal acidic residues adjacent to the phosphotyrosine are detrimental for the PTPase activity of PP2A(D), but they do not affect the PTP-1B activity. Acidic residues at the --2 and --3 position relative to Tyr(P) primarily dictate dephosphorylation by PTP-1B. The higher-order structure of the protein substrates also differentially influences both enzymes: the phospho-octapeptide KDDEYpNPA, which reproduces the autophosphorylation site in c-Fgr (Tyr400), is only dephosphorylated by PP2A(D) if embedded in the intact protein, whereas the opposite is true for PTP-1B. Both the intact p50/HS1 phosphoprotein and the derived phosphopeptide are substrates only for PTP-1B and not for PP2A(D). Lyn and c-Fgr phosphorylated by C-terminal Src kinase (CSK) at their down-regulatory site are resistant to the action of both PTPases while the [Phe6]Src-(514-533) phosphopeptide, representing the highly similar site affected by CSK in c-Src, is readily dephosphorylated by both PTPases, although to a different extent. In vitro dephosphorylation of the c-Fgr Tyr400 site by PP2A(D) is correlated with a decreased tyrosine kinase activity towards exogenous substrates. Under experimental conditions in which both Tyr400 (autophosphorylation site) and Tyr511 (down-regulatory site) of c-Fgr are phosphorylated, PP2A(D) can reverse both phosphorylations.

Isolation from spleen of a 57-kDa protein substrate of the tyrosine kinase Lyn. Identification as a protein related to protein disulfide-isomerase and localisation of the phosphorylation sites.

A 57-kDa protein (p57) has been purified to homogeneity from a microsomal fraction of rat spleen. It is specifically and efficiently phosphorylated by the Src-like tyrosine kinase Lyn purified from the same source with a Km of 0.34 microM. The tyrosine kinases c-Fgr, Fyn, C-terminal Src kinase and p72syk, as well as the Ser/Thr-specific cAMP-dependent protein kinase and protein kinases CK1 and CK2 do not phosphorylate p57. C-terminal Src kinase, which acts to down-regulate the Src-like protein-tyrosine kinases, almost completely prevents the protein phosphorylation catalysed by Lyn. Protein mass fingerprinting with tryptic fragments identified p57 as a protein related to protein disulfide-isomerase which belongs to the superfamily of Cys-Gly-His-Cys-containing sequences. Lyn phosphorylates tyrosine residues Y444, Y453 and Y466 which are located in a highly acidic region of the protein at the C-terminus. Upon phosphorylation, p57 forms a complex with Lyn which can be immunoprecipitated with anti-Lyn IgG. The association which occurs between the phosphorylated substrate and the SH2 domain of the kinase is consistent with the suggested 'processive phosphorylation' model, which implies that a primary phosphorylation site of the substrate binds to the SH2 domain of the enzyme and triggers the phosphorylation at secondary site(s).

Synthetic Tyr-phospho and non-hydrolyzable phosphonopeptides as PTKs and TC-PTP inhibitors.

Tyrosine-specific protein kinases and phosphatases are important signal transducing enzymes in normal cellular growth and differentiation and have been implicated in the etiology of a number of human neoplastic processes. In order to develop agents which inhibits the function of these two classes of enzymes by interfering with the binding of their substrates, we synthesized analogs derived from the peptide EDNEYTA. This sequence reproduces the main autophosphorylation site of Src tyrosine kinases. In this work we report the synthesis, by classical solution methods, of the phosphotyrosyl peptide EDNEYpTA as well as of three analogs in which the phosphotyrosine is replaced by a phosphinotyrosine and by two unnatural, non-hydrolyzable amino acids 4-phosphonomethyl-L-phenylalanine and 4-phosphono-L-phenylalanine. The Src peptide and its derivatives were tested as inhibitors of three non-receptor tyrosine kinases (Lyn, belonging to the Src family, CSK and PTK-IIB) and a non-receptor protein tyrosine phosphatase obtained from human T-cell (TC-PTP). The biomimetic analogues, which do not significantly affect the activity of CSK, PTK-IIB and TC-PTP, act as efficient inhibitors on Lyn, influencing both the exogenous phosphorylation and, especially, its autophosphorylation. In particular, the Pphe derivative may provide a basis for the design of a class of inhibitors specific for Lyn and possibly Src tyrosine kinases, capable of being used in vivo and in vitro conditions.

Different susceptibility of protein kinases to staurosporine inhibition. Kinetic studies and molecular bases for the resistance of protein kinase CK2.

A systematic analysis reveals that out of 20 protein kinases examined, specific for either Ser/Thr or Tyr, the majority are extremely sensitive to staurosporine, with IC50 values in the low nanomolar range. A few of them however, notably protein kinases CK1 and CK2, mitogen-activated protein (MAP) kinase and protein-tyrosine kinase CSK, are relatively refractory to staurosporine inhibition, exhibiting IC50 values in the micromolar range. With all protein kinases tested, namely PKA, CK1, CK2, MAP kinase (ERK-1), c-Fgr, Lyn, CSK and TPK-IIB/p38Syk, staurosporine inhibition was competitive with respect to ATP, regardless of its inhibitory power. In contrast, either uncompetitive or noncompetitive kinetics of inhibition with respect to the phosphoacceptor substrate were exhibited by Ser/Thr and Tyr-specific protein kinases, respectively, consistent with a different mechanism of catalysis by these two sub-families of kinases. Computer modeling based on PKA crystal structure in conjunction with sequence analysis suggest that the low sensitivity to staurosporine of CK2 may be accounted for by the bulky nature of three residues, Val66, Phe113 and Ile174 which are homologous to PKA Ala70, Met120 and Thr183, respectively. In contrast these PKA residues are either conserved or replaced by smaller ones in protein kinases highly sensitive to staurosporine inhibition. On the other hand, His160 which is homologous to PKA Glu170, appears to be responsible for the unique behaviour of CK2 with respect to a staurosporine derivative (CGP44171A) bearing a negatively charged benzoyl substituent: while CGP44171A is 10- 100-fold less effective than staurosporine against PKA and most of the other protein kinases tested, it is actually more effective than staurosporine for CK2 inhibition, but it looses part of its efficacy if it is tested on a CK2 mutant (H160D) in which His160 has been replaced by Asp. It can be concluded from these data that the catalytic sites of protein kinases are divergent enough as to allow a competitive inhibitor like staurosporine to be fairly selective, a feature that can be enhanced by suitable modifications designed based on the structure of the catalytic site of the kinase.

Linear and cyclic synthetic peptides related to the main autophosphorylation site of the Src tyrosine kinases as substrates and inhibitors of Lyn.

Tyrosine protein kinases (TPKs) of the src family contain two major phosphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60c-src. The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased kinase activity. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a high affinity toward the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH, which reproduces the main autophosphorylation site of the Src family enzymes [Donella-Deana, A., Marin, O., Brunati, A.M. & Pinna, L.A. (1992) Eur. J. Biochem. 204, 1159-1163]. Our study was addressed to the synthesis of some derivatives of this sequence in order to obtain both peptide substrates suitable for the detection of the Src-like tyrosine kinase activity and active site-directed inhibitors specific for this class of enzymes. For this purpose we synthesized by classical solution methods the heptapeptide and its dimeric form. Moreover, in order to improve the proteolytic resistance of these peptides we also synthesized their cyclic derivatives and their N-terminal acetylated and C-terminal amidated analogs. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as Lyn substrates was examined. The kinetic data obtained indicate that the extent of the peptide phosphorylation varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C-terminal amidation of the heptapeptide are detrimental for the Lyn activity, dimeric derivatives display very favourable kinetic constants. In particular the cyclic dimer is an especially suitable substrate for the tyrosine kinase and a powerful inhibitor of both the phosphorylation activity of Lyn and the enzyme autophosphorylation.

Post-traumatic cerebral air embolism. Case report.

A case of post-traumatic cerebral air embolism is presented. In the chest-X-radiography fractures of many ribs and hemo-pneumothorax were visible on the right side associated with intra-alveolar, interstitial hemorrhages and mediastinal emphysema. At the end of a CT scan of the head appeared features of arterial air embolism. The patient was diagnosed as brain dead and cardiac arrest occurred one hour after.

Phosphorylated residues as specificity determinants for an acidophilic protein tyrosine kinase. A study with src and cdc2 derived phosphopeptides.

Spleen TPK-IIB is an acidophilic protein tyrosine kinase, devoid of autophosphorylation activity, whose phosphorylation of the src-peptide NEYTA is crucially specified by Glu-2[(1991) J. Biol. Chem. 266, 17798-17803]. We show that phosphothreonine, phosphotyrosine and phosphoserine are, in this order, specificity determinants even more effective than glutamic acid if they are replacing Glu-2, to give the phosphopeptides NTpYTA, NYpYTA, NSpYTA, respectively. Non-phosphorylated threonine, tyrosine and serine are conversely ineffective. Consequently also the heptapeptide GEGTYGV reproducing the phosphoacceptor and inhibitory site of p34cdc2 is not appreciably affected by TPK-IIB, unless its threonyl residue is previously phosphorylated, the phosphoderivative GEGTpYGV being readily phosphorylated at its tyrosyl residue. Such a behaviour is unique for TPK-IIB among the protein tyrosine kinases tested (lyn-TPK, fgr-TPK and EGF-receptor, besides TPK-IIB). These data provide the first evidence that, in some instances, the targeting by protein tyrosine kinases can be specifically determined by the previous phosphorylation of the peptide substrate, thus extending the concept of 'hierarchal phosphorylation' [(1991) J. Biol. Chem. 266, 14139-14142] to tyrosyl residues as well.