Synthesis and biological activities of cyclic lactam peptides as substrates for non-receptors PTKs

The heptapeptide EDNEYTA, which reproduces the main autophosphorylation site of Src, has been previously shown to be a good substrate for both Src and Syk tyrosine kinases [Ruzza, P., et al., J. Pept. Sci., 2 (1996) 325]. Four lactam bridge conformationally constrained analogues of this peptide were synthesized by classical solution methods and screened for their suitability as c-Fgr and Syk tyrosine kinase substrates. The kinetic data obtained indicate that the different rings of the lactam peptides influence the capability of the peptides to act as PTK substrates. In general cyclization decreases the peptide phosphorylability, however the sequence containing the greatest lactam ring, ED(EEYTK), resulted in an especially suitable and selective substrate for Syk tyrosine kinase.     

Catalytic domains of tyrosine kinases determine the phosphorylation sites within c-Cbl

Catalytic (SH1) domains of protein tyrosine kinases (PTKs) demonstrate specificity for peptide substrates. Whether SH1 domains differentiate between tyrosines in a physiological substrate has not been confirmed. Using purified proteins, we studied the ability of Syk, Fyn, and Abl to differentiate between tyrosines in a common PTK substrate, c-Cbl. We found that each kinase produced a distinct pattern of c-Cbl phosphorylation, which altered the phosphotyrosine-dependent interactions between c-Cbl and CrkL or phosphatidylinositol 3'-kinase (PI3-K). Our data support the concept that SH1 domains determine the final sites of phosphorylation once PTKs reach their target proteins.     

Structural basis of specific and efficient phosphorylation of peptides derived from p34cdc2 by a pp60src-related protein tyrosine kinase.

Cys-cdc2(8-20), a synthetic peptide derived from p34cdc2, was previously reported to be a specific and efficient substrate of a pp60c-src-related tyrosine kinase isolated from bovine spleen (the spleen tyrosine kinase) (Litwin, C.M.E., Cheng, H.-C., and Wang, J.H. (1991) J. Biol. Chem. 266, 2557-2566). The longer peptide, cdc2(1-24), was found to be phosphorylated by the kinase with similar efficiency, and Tyr15 was the only amino acid residue phosphorylated. This indicated that the amino acid sequence of cdc2(8-20) peptide, EKI-GEGTYGVVYK, contained the structural features important for protein tyrosine kinase substrate activity. A stepwise procedure using synthetic peptides was employed to investigate such structural features. First, a computer search of protein sequences homologous to cdc2(8-20) uncovered five protein kinases containing homologous sequence with tyrosine at a position corresponding to Tyr15 of p34cdc2. Second, a peptide derived from ribosomal S6 protein kinase (rsk(436-456] was synthesized. The rsk(436-456) peptide contained a segment, ETIGVGSYSVCKR, which is highly homologous to that of cdc2(8-20). It was found to be a very poor substrate of the spleen tyrosine kinase. Third, peptide analogs of cdc2(6-20) with single substitutions of amino acid residues Lys9, Glu12, Thr14, Gly16, Val18, and Tyr19 by amino acid residues at corresponding positions of rsk were synthesized and tested as spleen tyrosine kinase substrates. Only Glu12 and Thr14 substituted peptide analogs showed decreased substrate activities. (The substrate activity of a peptide is the ability of the peptide to serve as the substrate of the spleen tyrosine kinase. It was determined of the spleen tyrosine kinase. It was determined either by the kinetic parameters (Km and Vmax) of phosphorylation of the peptide or by the initial phosphorylation rate of the peptide by the spleen tyrosine kinase.) An analog with double substitution at Glu12 An analog with double substitution at Glu12 and Thr14 was found to be almost as poor a substrate as the rsk peptide. In addition, peptide analogs with Tyr15 substituted by Phe or D-Tyr had poor substrate activities as well as weak inhibitory activities. Thus, Glu12, Thr14, and Tyr15 residues of p34cdc2 contained structural components essential for the efficient phosphorylation of the peptides derived from p34cdc2 by the pp60c-src-related spleen tyrosine kinase.     

Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors

Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS.     

Prediction and verification of novel peptide targets of protein tyrosine phosphatase 1B

Phosphotyrosine peptides are useful starting points for inhibitor design and for the search for protein tyrosine phosphatase (PTP) phosphoprotein substrates. To identify novel phosphopeptide substrates of PTP1B, we developed a computational prediction protocol based on a virtual library of protein sequences with known phosphotyrosine sites. To these we applied sequence-based methods, biologically meaningful filters and molecular docking. Five peptides were selected for biochemical testing of their potential as PTP1B substrates. All five peptides were equally good substrates for PTP1B compared to a known peptide substrate whereas appropriate control peptides were not recognized, showing that our protocol can be used to identify novel peptide substrates of PTP1B.     

Synthetic beta-turn peptides as substrates for a tyrosine protein kinase

An attempt has been made at defining the secondary structural requirement for phosphorylation of substrates of a protein tyrosine kinase from the leukemia virus-transformed LSTRA cell line. An examination of the sites of phosphorylation of substrates of protein tyrosine kinases indicated a relatively high probability of the beta-turn as the secondary structural feature at these sites. We have, therefore, synthesized three tyrosine peptides: Ala-Pro-Tyr-Gly-NHCH3, Leu-Pro-Tyr-Ala-NHCH3, and Pro-Gly-Ala-Tyr-NH2, of which the first two peptides, but not the third, would be expected to contain the tyrosine residue in a beta-turn. Circular dichroism and infrared spectral data on the peptides confirmed this expectation. Phosphorylation data on the peptides by the tyrosine kinase showed that the two beta-turn peptides were phosphorylated with Vmax and Km values comparable to those of the 13-residue-long arginine-containing synthetic peptide substrate having a sequence homologous to the autophosphorylation site of the LSTRA kinase. The peptides used here contain the shortest sequence length among the reported synthetic peptide substrates for protein tyrosine kinases. Their preference for the beta-turn indicated that this conformation may serve as the recognition site for tyrosine phosphorylation.     

Phosphorylation on Syk Y342 is important for both ITAM and hemITAM signaling in platelets

Immune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl₃ injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal.     

The kinase Syk as an adaptor controlling sustained calcium signalling and B-cell development

Upon B-cell antigen receptor (BCR) activation, the protein tyrosine kinase Syk phosphorylates the adaptor protein SH2 domain-containing leukocyte protein of 65 kDa (SLP-65), thus coupling the BCR to diverse signalling pathways. Here, we report that SLP-65 is not only a downstream target and substrate of Syk but also a direct binding-partner and activator of this kinase. This positive feedback is mediated by the binding of the SH2 domain of SLP-65 to an autophosphorylated tyrosine of Syk. The mutant B cells that cannot form the Syk/SLP-65 complex are defective in BCR-induced extracellular signal-regulated kinase, nuclear factor kappa B and nuclear factor of activated T cells, but not Akt activation, and are blocked in B-cell development. Furthermore, we show that formation of the Syk/SLP-65 complex is required for sustained Ca(2+) responses in activated B cells. We suggest that after activation and internalization of the BCR, Syk remains active as part of a membrane-bound Syk/SLP-65 complex controlling sustained signalling and calcium influx.     

Splicing Pattern of Gsα mRNA in Human and Rat Brain

The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromocytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 microM and 7.1 mumol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36-46), for example, exhibited a Km of 108 microM and a Vmax of 6.93 mumol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme.     

Purification and characterization of human Syk produced using a baculovirus expression system

The cytoplasmic tyrosine kinase p72syk (Syk) plays an essential role in signaling via a variety of immune and nonimmune cell receptors. Syk is activated in response to the engagement of the appropriate cell surface receptors and can phosphorylate downstream targets and recruit additional SH2-domain-containing proteins. In order to study the characteristics of Syk in vitro, we have overexpressed untagged, full-length human Syk in a recombinant baculovirus expression system. The enzyme was purified to 95% purity using a novel two-step affinity chromatography process using reactive yellow and phosphotyrosine columns. Yields of 3-10 mg purified Syk were obtained from 1 liter of infected insect cells. Western blotting, internal protein sequencing, and the specific tyrosine phosphorylation of a Syk peptide substrate indicated authenticity of the purified protein. The enzymatic properties of Syk were in good agreement with published data for the human enzyme, as the apparent K(m) of Syk for ATP was 10 microM and the peptide substrate was 3 microM. The recombinant protein also showed similar biochemical characteristics to the native protein isolated from B-cells such as autophosphorylation. Proteolytic cleavage of purified recombinant Syk was used to generate the kinase domain by micro-calpain. We therefore describe an efficient expression system and purification methodology to produce biologically active human Syk.     

Conformational constraints of tyrosine in protein tyrosine kinase substrates: Information about preferred bioactive side-chain orientation

The side-chain orientation of a tyrosine residue located in a peptide, which is an excellent substrate of Syk tyrosine kinase (A. M. Brunati, A. Donella-Deana, M. Ruzzene, O. Marin, L. A. Pinna, FEBS Letters, 1995, Vol. 367, pp. 149-152), was fixed in the gauche (+) or gauche (-) conformation by using the 7-hydroxy-1,2,3,4-tetrahydro isoquinoline-3-carboxylic (Htc) structure. The tyrosine trans conformation was blocked by using an aminobenzazepine-type (Hba) structure. The proposed side-chain orientations were confirmed by the analysis of the (1)H-NMR parameters: chemical shifts, coupling constants, and nuclear Overhauser effects to the tyrosine constraints in the different analogs. This "rotamer scan" of the phosphorylatable residue allowed us to generate optimal substrates in terms of both phosphorylation efficiency and selectivity for Syk tyrosine kinase. In contrast, these conformationally restricted tyrosine analogs were not tolerated by the Src-related tyrosine kinases Lyn and c-Fgr.     

New approach for analysis of the phosphotyrosine proteome and its application to the chicken B cell line, DT40

In this study, we have begun to analyze phosphotyrosyl and associated proteins present in a DT40 chicken B cell line overexpressing the nonreceptor protein-tyrosine kinase, Syk. An anti-phosphotyrosine antibody was used to select tyrosine-phosphorylated proteins. After tryptic digestion, peptides were subjected to a beta-elimination reaction and phosphotyrosine-containing peptides were enriched via immobilized metal affinity chromatography. Several known substrates and candidate substrates for Syk and the location of 22 tyrosine phosphorylation sites were identified.     

Purification of recombinant pp60v-src protein tyrosine kinase and phosphorylation of peptides with different secondary structure preference

The expression of the transforming gene product of Rous sarcoma virus (pp60v-src) in Saccharomyces cerevisiae has recently been reported (Kornbluth et al., 1987; Brugge et al., 1987). To carry out biochemical and structural studies of this enzyme, a facile purification was developed. The purification was accomplished in four chromatographic steps: Q-Sepharose, Affi-Gel Blue, phosphoagarose, and hydroxylapatite chromatography. The tyrosine kinase was isolated in milligram quantities as two highly active proteolytic fragments (52 and 54 kDa). Three model tyrosine kinase substrates with propensities to adopt helical or omega-loop conformations were synthesized and characterized. The peptides were based on the sites of phosphorylation of pp60v-src, lipocortin I, and lipocortin II. Circular dichroism spectroscopy was used to study the conformation of the helix-forming peptides in 50 mM Tris and in 50% trifluoroethanol/Tris. Peptide 1, which was designed to form an amphiphilic alpha-helix, displayed 24.2% helicity in buffer and 40.2% helicity in 50% TFE/buffer. Similar experiments for peptide 3, the other helix former, showed a lower helicity (8.1% helical and 26.0% helical in buffer and in 50% TFE/buffer, respectively). All three peptides were shown to be substrates for the recombinant tyrosine kinase. Kinetic measurements using high-voltage paper electrophoresis indicated that the helix-forming peptides exhibited low KM values (approximately 450 microM) for the purified src gene product, consistent with the notion that elements of secondary structure may be important in substrate recognition by tyrosine kinases.     

Role of acidic residues as substrate determinants for casein kinase I

Sites phosphorylated by casein kinase I have been characterized by the presence of acidic amino acids NH2-terminal to the modified residue. Recently, phosphoserine was shown to be a particularly effective determinant for casein kinase I action when present in the motif -S(P)-X-X-S- (Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W., and Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269). Nonetheless, nonphosphorylated substrates for casein kinase I are well documented. In this study, we examined the efficacy of Asp and Glu residues as determinants of casein kinase I action using synthetic peptide substrates. Peptides with runs of Asp residues in the motif Dn-X-X-S- were substrates for casein kinase I. Peptides with n = 3 or 4 were the most effective substrates, much better than n = 2. The peptide with n = 1, a single Asp residue, was a very poor substrate. A block of 4 Glu residues was a little less effective as a substrate determinant than 4 Asp residues in an otherwise identical peptide. The most effective substrate, with the motif -D-D-D-D-X-X-S-, was specific for casein kinase I and was not detectably phosphorylated by cyclic AMP-dependent protein kinase, casein kinase II, glycogen synthase kinase 3, or phosphorylase kinase and thus will be useful for the specific assay of casein kinase I. This peptide was nonetheless significantly worse as a substrate than peptides in which casein kinase I action was determined by phosphoserine in the -3 position. Still, the fact that Asp or Glu residues can specify a casein kinase I substrate suggests that acidic character has a role in substrate selection by this protein kinase.     

Molecular mechanism of the Syk activation switch

Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340-356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.     

Substrate specificity and kinetic mechanism of human placental insulin receptor/kinase

The insulin receptor has been shown to be a protein kinase which phosphorylates its substrates on tyrosine residues. To examine the acceptor specificity of affinity-purified insulin receptor/kinase, hydroxyamino acid containing analogues of the synthetic peptide substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly were prepared. Substitution of serine, threonine, or D-tyrosine for L-tyrosine completely ablated the acceptor activity of the synthetic peptides. These peptides, along with a phenylalanine-containing analogue, did serve as competitive inhibitors of the insulin receptor/kinase with apparent Ki values in the range of 2-4 mM. These data suggest that the insulin receptor/kinase is specific for tyrosine residues in its acceptor substrate and imply that serine phosphate or threonine phosphate present in receptor is due to phosphorylation by other protein kinases. The kinetics of the phosphorylation of the L-tyrosine-containing peptide were examined by using prephosphorylated insulin receptor/kinase. Prephosphorylation of the receptor was necessary to maximally activate the kinase and to linearize the initial velocity of the peptide phosphorylation reaction. The data obtained rule out a ping-pong mechanism and are consistent with a random-order rapid-equilibrium mechanism for the phosphorylation of this peptide substrate. Additional experiments demonstrated that the autophosphorylated insulin receptor was not able to transfer the preincorporated phosphate to the synthetic peptide substrate. Thus, the insulin receptor/kinase catalyzes the reaction via a mechanism that does not involve transfer of phosphate from a phosphotyrosine-containing enzyme intermediate.     

The v-Src and c-Src tyrosine kinases immunoprecipitated from Rous sarcoma virus-transformed cells display different peptide substrate specificities

In the cells transformed by Rous sarcoma virus (RSV), two Src proteins are expressed: the ubiquitous tyrosine kinase c-Src and the v-Src, the product of the transforming gene of the virus. Using three synthetic peptide substrates widely used for testing Src kinase activity, we show that they are phosphorylated with different efficiencies by the v-Src and c-Src tyrosine kinases immunoprecipitated from the tumor cell line H19. The v-Src displays higher efficiency (Vmax/Km ratio) toward all three peptides used, but the Vmax of v-Src is much lower than Vmax of c-Src with two peptides out of three. This difference in substrate specificity, if ignored, may cause misestimation of the amounts of active c-Src and v-Src in RSV-transformed cells. On the other hand, the different peptide substrate specificities may also reflect different protein substrate specificities of the v-Src and c-Src kinases in vivo.     

汉滩病毒G1蛋白胞质区ITAM样基序功能的研究

汉滩病毒(HTNV)的G1蛋白胞质区尾段包含保守的免疫受体酪氨酸活化基序(ITAM)样基序,该基序与许多重要的免疫受体胞质区ITAM基序同源性较高。为了研究HTNV的G1 ITAM样基序的免疫信号转导功能,首先人工合成了一段保守的酪氨酸残基磷酸化的G1 ITAM样基序多肽,应用体外蛋白激酶共沉淀实验,分别从Jur-kat细胞和Raji细胞裂解物中初筛到5~9种与该基序相互作用的磷酸化蛋白或激酶;然后通过突变体分析、体外磷酸化实验和体外激酶共沉淀-免疫印迹分析,进一步确证了G1 ITAM样基序在体外可以与Src家族蛋白酪氨酸激酶(PTK)Lyn、Fyn及其下游Syk家族激酶Syk、ZAP-70相互作用,而这种相互作用依赖于该基序中两个高度保守的酪氨酸残基的存在。上述研究表明,HTNV G1蛋白胞质区包含一个高度保守的功能性ITAM样基序,该基序在体外可以与TCR和BCR信号转导中关键的PTK相互作用,为进一步探讨HTNV G1蛋白ITAM样基序在肾综合征出血热(HFRS)免疫信号传递中的作用奠定了基础。