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.     

Synthetic peptide substrates for the membrane tyrosine protein kinase stimulated by epidermal growth factor

The substrate specificity of the epidermal-growth-factor-stimulated tyrosine protein kinase of A431 cell membranes has been studied using a series of synthetic peptide analogs of the sequence around the phosphorylated tyrosine-419 of pp60src. The nine-residue peptide Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Thr-Ala was phosphorylated on tyrosine with an apparent Km of 0.4 mM and a V of 5.7 nmol X min-1 X mg-1. Synthetic peptide tyrosine phosphorylation was stimulated by epidermal growth factor but not by insulin or relaxin. Extension of the nine-residue peptide to include the basic residues, arginine-412, arginine-422 and lysine-423 led to an increased apparent Km. Substitution of glutamic-418 by leucine also increased the apparent Km. In the model peptide Ile-Xaa-Xaa-Ala-Ala-Tyr-Thr-Ala a lower apparent Km was obtained when Xaa was glutamic rather than aspartic acid. Poly(aspartic acid) and poly(glutamic acid) had only weak effects on peptide tyrosine phosphorylation. The results support the concept that acidic residues and not basic residues are important specificity determinants for the epidermal-growth-factor-stimulated tyrosine protein kinase.     

Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase

The specificities of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase were probed using synthetic peptides corresponding to the sequence around phosphorylation sites 1 and 2 on pyruvate dehydrogenase [Tyr-His-Gly-His-Ser(P1)-Met-Ser-Asp-Pro-Gly-Val-Ser(P2)-Tyr-Arg]. The dephosphotetradecapeptide containing aspartic acid at position 8 was a better substrate for the kinase than was the tetradecapeptide containing asparagine at position 8. The apparent Km and V values for the two peptides were 0.43 and 6.1 mM and 2.7 and 2.4 nmol of 32P incorporated/min/mg, respectively. Methylation of the aspartic acid residue also increased the apparent Km of the tetradecapeptide about 14-fold. These results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase. Phosphate was incorporated only into site 1 of the synthetic peptide by the kinase. The phosphatase exhibited an apparent Km of 0.28 mM and a V of 2.3 mumol of 32P released/min/mg for the phosphorylated tetradecapeptide containing aspartic acid. Methylation of the aspartic acid residue had no significant effect on dephosphorylation. The octapeptide and phosphooctapeptide produced by cleavage of the aspartyl-prolyl bond by formic acid were poorer substrates for the kinase and phosphatase than were the tetradecapeptide and phosphotetradecapeptide, respectively. Modification of the amino terminal by acetylation or lysine addition had only a slight effect on the kinase and phosphatase activities.     

Different specificities of spleen tyrosine protein kinases for synthetic peptide substrates

20 synthetic peptides, each of which includes a tyrosyl residue flanked by either neutral or acidic amino acids in different proportions and at variable positions, have been employed as model substrates for investigation of the site specificity of three tyrosine protein kinases previously isolated from spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] and conventionally termed TPK-I, TPK-IIB and TPK-III. Comparison of the phosphorylation efficiencies shows that each tyrosine protein kinase is considerably different from the others in both the stringency and the nature of its specificity determinants. By considering, in particular, the kinetic constants obtained with the pentapeptides AAYAA, EEYAA, AEYAA, EAYAA, with the tetrapeptides AYAA and EYAA and with the tripeptides AYA and EYA, it turns out that N-terminal acidic residue(s) are only essential with TPK-IIB for efficient phosphorylation with multiple residues displaying a synergistic effect. The very similar Km (130 microM) but 14-fold-different Vmax values with YEEEEE vs. EEEEEY indicate that an N-terminal rather than C-terminal location of acidic residues is required for a high phosphorylation rate with, though not for binding to TPK-IIB. Acidic residues decrease the phosphorylation rate with TPK-I, a kinase related to the src family which is immunologically indistinguishable from the lyn TPK; they are nearly ineffective, however, with TPK-III, the least specific of the tyrosine protein kinases, which exhibits appreciable activity toward tripeptides and dipeptides like GAY and AY which are not significantly affected by TPK-I and TPK-IIB. While the peptide substrate specificity of TPK-I is similar to that of TPK-IIA, a spleen tyrosine protein kinase previously considered [Brunati, A. M., Marchiori, F., Ruzza, P., Calderan, A., Borin, G. & Pinna, L. A. (1989) FEBS Lett. 254, 145-149], the remarkable requirement of TPK-IIB alone for acidic peptides may suggest the involvement of this enzyme, which is also unique in its failure to autophosphorylate, in the phosphorylation of the highly conserved and quite acidic phosphoacceptor sites of the src family protein kinases.     

Synthetic tyrosine polymers as substrates and inhibitors of tyrosine-specific protein kinases

Several synthetic random polymers of tyrosine containing glutamic acid, alanine, and lysine in various proportion served as substrates for tyrosine-specific protein kinases. The Km values for these substrates were much lower than for small polypeptides such as angiotensin. For the protein kinase coded by Fujinami virus, the best substrates (with the lowest Km) were polymers containing glutamic acid, alanine, and 8 to 10% tyrosine; for the insulin receptor protein kinase, the best substrate was a polymer containing 80% glutamic acid and 20% tyrosine. These polymers serve as inexpensive and tyrosine-specific substrates that can be used even with crude extracts and analyzed by the convenient filter paper assay. Several synthetic polymers with ordered sequences were found to be potent inhibitors of these tyrosine-specific protein kinases.     

Synthetic peptide substrates for casein kinase 2. Assessment of minimum structural requirements for phosphorylation

Unlike the peptides SAEAAA and SEEAAA which are not substrates for casein kinase 2 (CK-2) their analogs SAAEAE and SAAEAA are still significantly phosphorylated. Their K_m values, however, (13.3 and 18.9 mM, respectively) are almost two orders of magnitude higher than that of SEEEEE and their V_max values are 3- and 14-fold lower than that of SAAEEE. The peptide ESEEEEE, but not ASEEEEE, is a slightly better substrate than SEEEEE, while both RSEEEEE and SEEEKE are very poor substrates compared to ASEEEEE and SEEEAE, respectively. SAAEAE is much more responsive to polylysine stimulation and polyphosphate inhibition than is SEEEEE. Taken together these data show that a single acidic residue at the third position from the C-terminal side of the phosphorylatable amino acid represents not only a necessary, but also a sufficient condition for site recognition by CK-2. Optimal phosphorylation efficiency, however, requires an extended C-terminal cluster of several acidic residues, and can be compromised by the presence of only a basic residue either inside the acidic cluster or adjacent to the N-terminal side of the phosphoacceptor amino acid. The structure of the phosphoacceptor site can greatly influence the efficacy of substrate-directed effectors of CK-2.     

Structure-activity relationship of a novel peptide substrate for p60c-src protein tyrosine kinase

Summary We recently reported the identification of a peptide (YIYGSFK) as an efficient substrate for p60^c-src using a random combinatorial peptide library screening method. Over 70 analogues of YIYGSFK were designed and synthesized on beads and their phosphorylation on solid phase by p60^c-src was quantitated by the PhosphorImager. A hydrophobic l-amino acid in position 2 and a basic amino acid in position 7 proved crucial for activity as a substrate. In addition, the l-tyrosine residue at position 3 was critical as the phosphorylation site and was found to be stereospecific, as substitution with the d-enantiomer at this position rendered the peptide totally inactive.Abbreviations -alanine - -aminocaproic acid - Ac N-acetyl - BOP benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - Cha l-cyclohexylalanine - Chg l-cyclohexylglycine - Dab l-diaminobutyric acid - Dap l-diaminoproprionic acid - DIEA N,N-diisopropylethylamine - DMF dimethylformamide - Fmoc fluorenylmethyloxycarbonyl - HOBt N-hydroxybenzotriazole - MeF N-methyl-l-phenylalanine - MeG N-methylglycine - MeI N-methyl-l-isoleucine - MES 2-[N-morpholinolethanesulfonic acid - Nle l-norleucine - Orn l-ornithine - TFA trifluoroacetic acid - Z-Sar benzyloxycarbonyl-sarcosine - Z-Tyr benzyloxycarbonyl-l-tyrosine     

Discovery of cellular substrates for protein kinase A using a peptide array screening protocol

Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth.     

Characterization of synthetic peptide substrates for p34cdc2 protein kinase

Synthetic peptide substrates for the cell division cycle regulated protein kinase, p34cdc2, have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H-A-D-A-Q-H-A-T-P-P-K-K-K-R-K-V-E-D-P-K-D-F-OH (T antigen) and H-K-R-A-L-P-N-N-T-S-S-S-P-Q-P-K-K-K-P-L-D-G-E-Y-NH2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent Km of 74 and 120 microM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexes of p34cdc2. In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34cdc2 using either antibodies to p34cdc2 or by immobilized p13, a p34cdc2-binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34cdc2. These peptides should be useful in the investigation of p34cdc2 protein kinase and their regulation throughout the cell division cycle.     

A tandem chromatographic column method for assaying cAMP-dependent protein kinase and protein kinase C with synthetic peptide substrates

A method was devised for assaying protein kinases that phosphorylate either Kemptide, such as cAMP-dependent protein kinase, or a glycogen synthase peptide, which is an excellent substrate for protein kinase C. Upon sequential processing of reaction mixtures through tandem columns of cation and anion exchange resins, radioactivity in background samples is nearly nil and the yield of phosphorylated peptides is high. This method reduces labor, radioactivity, enzyme requirements, and costs of assaying protein kinases.     

A peptide library approach identifies a specific inhibitor for the ZAP-70 protein tyrosine kinase

We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase involved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates (K(I) of 2 microM). The related protein Tyr kinases, Lck and Syk, were not significantly inhibited by the peptide. When introduced into intact T cells, the peptide blocked signaling downstream of ZAP-70, including ZAP-70-dependent gene induction, without affecting upstream Tyr phosphorylation. Thus, screening Tyr-oriented peptide libraries can identify selective peptide inhibitors of protein Tyr kinases.     

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.     

Development of a selective pseudosubstrate-based peptide inhibitor of pp60c-src protein tyrosine kinase

Summary Using a combinatorial peptide library method, we identified YIYGSFK as an efficient and specific peptide substrate for pp60^c-src protein tyrosine kinase (PTK) [Lam et al., Int. J. Pept. Protein Res., 45 (1995) 587]. Employing YIYGSFK as a template, we synthesized and evaluated a series of pseudosubstrate-based inhibitors for pp60^c-src. We found that the efficiency of a given inhibitor was highly dependent on the specific tyrosine analog used at the phosphorylation site of the substrate. One of these pseudosubstrate inhibitors, YI(2-Nal)GSFK, selectively inhibited the kinase activity of pp60^c-src, with a K_i of 24 M. This peptide inhibitor exhibited selectivity for pp60^c-src as compared to other PTKs tested, such as c-Abl and Bcr-Abl. Our results suggest that selective inhibitors for a specific PTK can be developed when the structure of a specific and efficient small peptide substrate for this PTK can be used as a template for structure modification.Abbreviations 1-Nal l-1-naphthylalanine - 2-Nal l-2-naphthylalanine - BOP benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - cAPK cyclic AMP-dependent protein kinase - DIEA diisopropylethylamine - EGFR epidermal growth factor receptor - Fmoc fluorenylmethoxycarbonyl - HOBt 1-hydroxybenzotriazole - MES 2-[N-morpholino]ethanesulfonic acid - PBS phosphate-buffered salts - pCl l-p-chlorophenylalanine - pF l-p-fluorophenylalanine - PTK protein tyrosine kinase - TLC thin-layer chromatography     

Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein

We have applied intein-mediated peptide ligation (IPL) to the use of peptide substrates for kinase assays and subsequent Western blot analysis. IPL allows for the efficient ligation of a synthetic peptide with an N-terminal cysteine residue to an intein-generated carrier protein containing a cysteine reactive C-terminal thioester through a native peptide bond. A distinct advantage of this procedure is that each carrier protein molecule ligates only one peptide, ensuring that the ligation product forms a sharp band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrate the effectiveness of this approach by mutational analysis of peptide substrates derived from human cyclin-dependent kinase, Cdc2, which contains a phosphorylation site of human c-Src protein tyrosine kinase.     

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 oncogenelyndisplays a particularly high affinity (K_m20 μm ) 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 aK_mvalue similar to those obtained for the dimeric linear and cyclic unmodified analogues, theK_mvalue 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. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine phosphatase receptor type z

Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective PTPs. Several years ago, substrate-trap mutants were developed to identify the substrates of PTPs, but only a limited number of PTP substrates have been identified using typical biochemical techniques in vitro. The application of this strategy to all the PTPs seems difficult, because the substrates identified to date were restricted to relatively abundant and highly tyrosine phosphorylated cellular proteins. Therefore, the development of a standard method applicable to all PTPs has long been awaited. We report here a genetic method to screen for PTP substrates which we have named the "yeast substrate-trapping system." This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait. This method is probably applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs. Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.     

Synthetic myelin basic protein peptide analogs are specific inhibitors of phospholipid/calcium-dependent protein kinase (protein kinase C)

Synthetic peptide analogs of the bovine myelin basic protein (MBP) corresponding to residues 104-118 were found to specifically inhibit phospholipid/ Ca2+-dependent protein kinase (protein kinase C). The peptides [Ala107]MBP (104-118) and [Ala113]MBP (104-118) inhibited protein phosphorylation of intact MBP, histone H1 and peptide phosphorylation with MBP(104-123), MBP(104-118) or [Ala105]MBP (104-118) as substrates. The inhibitor peptides [Ala107]MBP(104-118) and [Ala113]MBP (104-118), containing alanine in place of the arginine recognition sites, apparently inhibited the enzyme noncompetitively with respect to substrates, with IC50 values ranging from 46-145 and 28-62 microM, respectively. These peptide analogs did not inhibit cyclic AMP-dependent protein kinase or myosin light chain kinase but inhibited phospholipid/Ca2+-dependent phosphorylation of endogenous proteins in the total, solubilized fraction of rat brain.