Effect of the DEF motif on phosphorylation of peptide substrates by ERK

MAP kinase ERK maintains specificity by binding to docking sites such as the DEF domain or D domain. It was previously shown that appending peptides derived from D domains to a substrate peptide increased apparent efficiency of peptide phosphorylation while preserving its apparent specificity for ERK. Here we determine the effect of the DEF motif on efficiency and specificity of peptide phosphorylation by ERK. The DEF motif modulated the apparent affinity of the peptide for ERK while the substrate motif dominated the apparent catalytic rate. Attachment of the DEF sequence improved apparent phosphorylation efficiency by 60-fold. Addition of peptides possessing both the DEF and D motif to a substrate sequence did not yield additive effects on the K_M of the substrate for ERK. Further, the DEF motif diminished the apparent specificity for ERK and increased the apparent efficiencies of phosphorylation of the substrate peptide by p38α kinase and JNK1.     

Specificity determinants of substrate recognition by the protein kinase DYRK1A

DYRK1A is a dual-specificity protein kinase that is thought to be involved in brain development. We identified a single phosphorylated amino acid residue in the DYRK substrate histone H3 (threonine 45) by mass spectrometry, phosphoamino acid analysis, and protein sequencing. Exchange of threonine 45 for alanine abolished phosphorylation of histone H3 by DYRK1A and by the related kinases DYRK1B, DYRK2, and DYRK3 but not by CLK3. In order to define the consensus sequence for the substrate specificity of DYRK1A, a library of 300 peptides was designed in variation of the H3 phosphorylation site. Evaluation of the phosphate incorporation into these peptides identified DYRK1A as a proline-directed kinase with a phosphorylation consensus sequence (RPX(S/T)P) similar to that of ERK2 (PX(S/T)P). A peptide designed after the optimal substrate sequence (DYRKtide) was efficiently phosphorylated by DYRK1A (K(m) = 35 microM) but not by ERK2. Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A, phosphorylated the mitogen-activated protein kinase substrate ELK-1. This marked difference in substrate specificity between DYRK1A and ERK2 can be explained by the requirement for an arginine at the P -3 site of DYRK substrates and its presumed interaction with aspartate 247 conserved in all DYRKs.     

A kinetic approach for the study of protein phosphatase-catalyzed regulation of protein kinase activity

The activities of many protein kinases are regulated by phosphorylation. The phosphorylated protein kinases thus represent an important class of substrates for protein phosphatases. However, our ability to study the phosphatase-catalyzed substrate dephosphorylation has been limited in many cases by the difficulty in preparing sufficient amount of stoichiometrically phosphorylated kinases. We have applied the kinetic theory of substrate reaction during irreversible modification of enzyme activity to the study of phosphatase-catalyzed regulation of kinase activity. As an example, we measured the effect of the hematopoietic protein-tyrosine phosphatase (HePTP) on the reaction catalyzed by the fully activated, bisphosphorylated extracellular signal-regulated protein kinase 2 (ERK2/pTpY). Because only a catalytic amount of ERK2/pTpY is required, this method alleviates the need for large quantities of phospho-ERK2. Kinetic analysis of the ERK2/pTpY-catalyzed substrate reaction in the presence of HePTP leads to the determination of the rate constants for the HePTP-catalyzed dephosphorylation of free ERK2/pTpY and ERK2/pTpY*substrate(s) complexes. The data indicate that ERK2/pTpY is a highly efficient substrate for HePTP (k(cat)/K(m) = 3.05 x 10(6) M(-1) s(-1)). The data also show that binding of ATP to ERK2/pTpY has no effect on ERK2/pTpY dephosphorylation by HePTP. In contrast, binding of an Elk-1 peptide substrate to ERK2/pTpY completely blocks the HePTP action. This result indicates that phosphorylation of Tyr185 is important for ERK2 substrate recognition and that binding of the Elk-1 peptide substrate to ERK2/pTpY blocks the accessibility of pTyr185 to HePTP for dephosphorylation. Collectively, the results establish that the kinetic theory of irreversible enzyme modification can be applied to study the phosphatase catalyzed regulation of kinase activity.     

Examining docking interactions on ERK2 with modular peptide substrates

ERK2 primarily recognizes substrates through two recruitment sites, which lie outside the active site cleft of the kinase. These recruitment sites bind modular-docking sequences called docking sites and are potentially attractive sites for the development of non-ATP competitive inhibitors. The D-recruitment site (DRS) and the F-recruitment site (FRS) bind D-sites and F-sites, respectively. For example, peptides that target the FRS have been proposed to inhibit all ERK2 activity (Galanis, A., Yang, S. H., and Sharrocks, A. D. (2001) J. Biol. Chem. 276, 965-973); however, it has not been established whether this inhibition is steric or allosteric in origin. To facilitate inhibitor design and to examine potential coupling of recruitment sites to other ligand recognition sites within ERK2, energetic coupling within ERK2 was investigated using two new modular peptide substrates for ERK2. Modeling shows that one peptide (Sub-D) recognizes the DRS, while the other peptide (Sub-F) binds the FRS. A steady-state kinetic analysis reveals little evidence of thermodynamic linkage between the peptide substrate and ATP. Both peptides are phosphorylated through a random-order sequential mechanism with a k(cat)/K(m) comparable to Ets-1, a bona fide ERK2 substrate. Occupancy of the FRS with a peptide containing a modular docking sequence has no effect on the intrinsic ability of ERK2 to phosphorylate Sub-D. Occupancy of the DRS with a peptide containing a modular docking sequence has a slight effect (1.3 ± 0.1-fold increase in k(cat)) on the intrinsic ability of ERK2 to phosphorylate Sub-F. These data suggest that while docking interactions at the DRS and the FRS are energetically uncoupled, the DRS can exhibit weak communication to the active site. In addition, they suggest that peptides bound to the FRS inhibit the phosphorylation of protein substrates through a steric mechanism. The modeling and kinetic data suggest that the recruitment of ERK2 to cellular locations via its DRS may facilitate the formation of F-site selective ERK2 signaling complexes, while recruitment via the FRS will likely inhibit ERK2 through a steric mechanism of inhibition. Such recruitment may serve as an additional level of ERK2 regulation.     

Substrate recognition determinants for rhodopsin kinase: studies with synthetic peptides, polyanions, and polycations

Rhodopsin kinase phosphorylates serine- and threonine-containing peptides from bovine rhodopsin's carboxyl-terminal sequence. Km's for the peptides decrease as the length of the peptide is increased over the range 12-31 amino acids, reaching 1.7 mM for peptide 318-348 from the rhodopsin sequence. The Km for phosphorylation of rhodopsin is about 10(3) lower than that for the peptides, which suggests that binding of rhodopsin kinase to its substrate, photolyzed rhodopsin, involves more than just binding to the carboxyl-terminal peptide region that is to be phosphorylated. A synthetic peptide from the rhodopsin sequence that contains both serines and threonines is improved as a substrate by substitution of serines for the threonines, suggesting that serine residues are preferred as substrates. Analogous 25 amino acid peptides from the human red or green cone visual pigment, a beta-adrenergic receptor, or M1 muscarinic acetylcholine receptors are better substrates for bovine rhodopsin kinase than is the peptide from bovine rhodopsin. An acidic serine-containing peptide from a non-receptor protein, alpha s1B-casein, is also a good substrate for rhodopsin kinase. However, many basic peptides that are substrates for other protein kinases--histone IIA, histone IIS, clupeine, salmine, and a neurofilament peptide--are not phosphorylated by rhodopsin kinase. Polycations such as spermine or spermidine are nonessential activators of phosphorylation of rhodopsin or its synthetic peptide 324-348. Polyanions such as poly(aspartic acid), dextran sulfate, or poly(adenylic acid) inhibit the kinase. Poly(L-aspartic acid) is a competitive inhibitor with respect to rhodopsin (KI = 300 microM) and shows mixed type inhibition with respect to ATP.     

Phosphorylation of synthetic peptide analogs of rabbit cardiac troponin inhibitory subunit by the cyclic AMP-dependent protein kinase.

A series of synthetic peptide analogs of the cardiac troponin inhibitory subunit (TN-1) phosphorylation site sequence, Arg12-Pro-Ala-Pro-Ala-Val-Arg18-Arg19-Ser20-Asp21-Arg22-Ala, have been tested as substrates for the catalytic subunit of the cyclic AMP-dependent protein kinase (EC 2.7.1.37, ATP:protein phosphotransferase). As substrates, these peptides were generally inferior to the pyruvate kinase analog peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly or its COOH-terminal amide analog. Replacing Arg-19 with alanine had only a minor effect on the kinetics of phosphorylation of the TN-1 peptide analog. In contrast, replacement of Arg-22 and Arg-18 with alanine resulted in marked enhancement and reduction of the Vmax, respectively. The results of this study have demonstrated that synthetic peptide analogs of the local phosphorylation site sequences of natural substrates may differ widely in their capacity to act as substrates for this protein kinase. In the case of the TN-1 peptide analogs, the contribution of the 4 arginine residues can be distinguished in terms of their influence on the kinetics of phosphorylation.     

Studies of the mechanism of action and regulation of cAMP-dependent protein kinase

Magnetic resonance and kinetic studies of the catalytic subunit of a Type II cAMP-dependent protein kinase from bovine heart have established the active complex to be an enzyme-ATP-metal bridge. The metal ion is β,γ coordinated with Δ chirality at the β-phosphorous atom. The binding of a second metal ion at the active site which bridges the enzyme to the three phosphoryl groups of ATP, partially inhibits the reaction. Binding of the metal-ATP substrate to the enzyme occurs in a diffusion-controlled reaction followed by a 40 ° change in the glycosidic torsional angle. This conformational change results from strong interaction of the nucleotide base with the enzyme. NMR studies of four ATP-utilizing enzymes show a correlation between such conformational changes and high nucleotide base specificity. Heptapeptide substrates and substrate analogs bind to the active site of the catalytic subunit at a rate significantly lower than collision frequency indicating conformational selection by the enzyme or a subsequent slow conformational change. NMR studies of the conformation of the enzyme-bound peptide substrates have ruled out α-helical and β-pleated sheet structures. The results of kinetic studies of peptide substrates in which the amino acid sequence was systematically varied were used to rule out the obligatory requirement for all possible β-turn conformations within the heptapeptide although an enzymatic preference for a β_2–5 or β_3–6 turn could not be excluded. Hence if protein kinase has an absolute requirement for a specific secondary structure, then this structure must be a coil. In the enzyme-substrate complex the distance along the reaction coordinate between the γ-P of ATP and the serine oxygen of the peptide substrate (5.3 ± 0.7 Å) allows room for a metaphosphate intermediate. This finding together with kinetic observations as well as the location of the inhibitory metal suggest a dissociative mechanism for protein kinase, although a mechanism with some associative character remains possible. Regulation of protein kinase is accomplished by competition between the regulatory subunit and peptide or protein substrates at the active site of the catalytic subunit. Thus, the regulatory subunit is found by NMR to block the binding of the peptide substrate to the active site of protein kinase but allows the binding of the nucleotide substrate and divalent cations. The dissociation constant of the regulatory subunit from the active site (10^?10m) is increased ~10-fold by phosphorylation and ~10⁴-fold by the binding of cAMP, to a value (10^?5m) which exceeds the intracellular concentration of the R₂C₂ holoenzyme complex (10^?6m). The resulting dissociation of the holoenzyme releases the catalytic subunit, permitting the active site binding of peptide or protein substrates.     

Ligand regulates epidermal growth factor receptor kinase specificity: activation increases preference for GAB1 and SHC versus autophosphorylation sites

The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, K(m) and k(cat), revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFR-mediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3-7-fold). Conversely, EGF stimulation increased k(cat) approximately 5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of approximately 5-fold in the specificity constants (k(cat)/K(m)) for EGFR peptides, whereas approximately 15-40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins approximately 4-6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.     

Phosphorylation of atrial natriuretic peptides by cyclic AMP-dependent protein kinase

Atrial natriuretic peptides refer to a family of related peptides secreted by atria that appear to have an important role in the control of blood pressure. The structure of these peptides shows the amino acid sequence Arg101-Arg102-Ser103-Ser104, which is a typical recognition sequence (Arg-Arg-X-Ser) for phosphorylation by cyclic AMP-dependent protein kinase. With this background, we tested two synthetic atrial natriuretic peptides (Arg101-Tyr126 and Gly96-Tyr126) as substrates for in vitro phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. The tested atrial natriuretic peptides were found to be substrates for the reaction. Sequence studies demonstrated that the site of phosphorylation was located, as expected, at Ser104. Kinetic studies demonstrate that both atrial natriuretic peptides are excellent substrates for cyclic AMP-dependent protein kinase. In particular, the longer peptide Gly96-Tyr126 exhibited an apparent Km value of about 0.5 microM, to our knowledge the lowest reported Km for a cyclic AMP-dependent protein kinase substrate. Preliminary studies to measure the biological activity of the in vitro phosphorylated atrial peptides indicate that these compounds are more effective than the corresponding dephospho forms in stimulating Na/K/Cl cotransport in cultured vascular smooth muscle cells.     

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.     

Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase.

The beta-adrenergic receptor kinase (beta-ARK) phosphorylates G protein coupled receptors in an agonist-dependent manner. Since the exact sites of receptor phosphorylation by beta-ARK are poorly defined, the identification of substrate amino acids that are critical to phosphorylation by the kinase are also unknown. In this study, a peptide whose sequence is present in a portion of the third intracellular loop region of the human platelet alpha 2-adrenergic receptor is shown to serve as a substrate for beta-ARK. Removal of the negatively charged amino acids surrounding a cluster of serines in this alpha 2-peptide resulted in a complete loss of phosphorylation by the kinase. A family of peptides was synthesized to further study the role of acidic amino acids in peptide substrates of beta-ARK. By kinetic analyses of the phosphorylation reactions, beta-ARK exhibited a marked preference for negatively charged amino acids localized to the NH2-terminal side of a serine or threonine residue. While there were no significant differences between glutamic and aspartic acid residues, serine-containing peptides were 4-fold better substrates than threonine. Comparing a variety of kinases, only rhodopsin kinase and casein kinase II exhibited significant phosphorylation of the acidic peptides. Unlike beta-ARK, RK preferred acid residues localized to the carboxyl-terminal side of the serine. A feature common to beta-ARK and RK was a much greater Km for peptide substrates as compared to that for intact receptor substrates.     

Phosphorylation of rat hepatic fructose-1,6-bisphosphatase and pyruvate kinase

Fructose-1,6-bisphosphatase from rat liver was phosphorylated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Brief exposure of the 32P-labeled enzyme to trypsin removed all radioactivity from the enzyme core and produced a single-labeled peptide. The partial sequence of the 17-amino acid peptide was found to be Ser-Arg-Pro-Ser(P)-Leu-Pro-Leu-Pro-(Ser2, Glx2, Pro2, Leu, Arg2). The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of native fructose bisphosphatase were compared with those of rat liver type L pyruvate kinase where the sequence around the phosphoserine is known (Arg-Arg-Ala-Ser(P)-Val; Hjelmquist, G., Anderson, J., Edlund, B., and Engstrom, L. (1974) Biochem. Biophys. Res. Commun. 61, 559-563). The Km for pyruvate kinase (17 microM) was less than that for fructose bisphosphatase (58 microM); the Vmax was about 3-fold greater with pyruvate kinase as substrate. The relationship between the rates of phosphorylation of these native substrates and the amino acid sequences surrounding the phosphorylated sites is discussed.     

Phosphorylation of the 165-kDa dihydropyridine/phenylalkylamine receptor from skeletal muscle by protein kinase C

Dihydropyridine-sensitive Ca2+ channels exist in many different types of cells and are believed to be regulated by various protein phosphorylation and dephosphorylation reactions. The present study concerns the phosphorylation of a putative component of dihydropyridine-sensitive Ca2+ channels by the calcium and phospholipid-dependent protein kinase, protein kinase C. A skeletal muscle peptide of 165 kDa, which is known to contain receptors for dihydropyridines, phenylalkylamines, and other Ca2+ channel effectors, was found to be an efficient substrate for protein kinase C when the peptide was phosphorylated in its membrane-bound state. Protein kinase C incorporated 1.5-2.0 mol of phosphate/mol of peptide within 2 min into the 165-kDa peptide in incubations carried out at 37 degrees C. In contrast to the membrane-bound peptide, the purified 165-kDa peptide in detergent solution was phosphorylated to a markedly less extent than its membrane-bound counterpart; less than 0.1 mol of phosphate/mol of peptide was incorporated. Preincubation of the membranes with several types of drugs known to be Ca2+ channel activators or inhibitors had no specific effects on the rate and/or extent of phosphorylation of the 165-kDa peptide by protein kinase C. The phosphorylation of the membrane-bound 165-kDa peptide by protein kinase C was compared to that catalyzed by cAMP-dependent protein kinase and was found to be not additive. Prior phosphorylation of the 165-kDa peptide by cAMP-dependent protein kinase prevented subsequent phosphorylation of the peptide by protein kinase C. Phosphoamino acid analysis indicated that protein kinase C phosphorylated the 165-kDa peptide at both serine and threonine residues. Phosphopeptide mapping experiments showed that protein kinase C phosphorylated one unique site in the 165-kDa peptide, and, in addition, other sites that were phosphorylated by either cAMP-dependent protein kinase or a multifunctional Ca2+/calmodulin-dependent protein kinase. The results suggest that the 165-kDa dihydropyridine/phenylalkylamine receptor could serve as a physiological substrate of protein kinase C in intact cells. It is therefore possible that the regulation of dihydropyridine-sensitive Ca2+ channels by activators of protein kinase C may occur at the level of this peptide.     

Protein kinase C phosphorylates the synthetic peptide Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val in the presence of phospholipid plus either Ca2+ or a phorbol ester tumor promoter

The synthetic nonapeptide Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val is a substrate for in vitro phosphorylation by a partially purified preparation of rat brain protein kinase C, with Kmapp of about 130 microM. The closely related peptide kemptide was a much weaker substrate, bovine serum albumin was not a substrate and the peptide Arg-Arg-Lys-Ala-Ala-Gly-Pro-Pro-Val was a weak inhibitor of the enzyme. Protein kinase C-catalyzed phosphorylation of histone III-S and the nonapeptide are regulated by identical mechanisms since with both substrates the reaction required added phospholipid and either Ca2+ (1mM) or TPA (200 nM TPA). Our findings show that polypeptides containing multiple basic residues followed by the sequence Ala-Ser can be substrates for TPA-stimulated phosphorylation by protein kinase C.     

The intrinsic ATPase activity of protein kinase C is catalyzed at the active site of the enzyme.

We recently reported that autophosphorylated protein kinase C (PKC) has an intrinsic Ca(2+)- and phospholipid-dependent ATPase activity and that the ATPase and histone kinase activities of PKC have similar metal-ion cofactor requirements and Km,app(ATP) values. We hypothesized that the intrinsic ATPase activity of PKC may represent the bond-breaking step of its protein kinase activity. The rate of the ATPase reaction is several times slower than the histone kinase reaction rate. At subsaturating concentrations, various peptide and protein substrates stimulate the ATPase reaction by as much as 1.5-fold. In contrast, non-phosphorylatable substrate analogs are not stimulatory. These observations support a mechanism of PKC catalysis in which the productive binding of phosphoacceptor substrates enhances the rate of phosphodonor substrate (ATP) hydrolysis at the active site of PKC. However, this mechanism contains an assumption that the ATPase activity of PKC is catalyzed at the active site. In fact, sequence analysis indicates that PKC contains a potential second nucleotide binding site outside of its active site. In this report, we provide a detailed analysis of the relationship between the active site of PKC and the intrinsic ATPase activity of the enzyme. We show that the regulatory and catalytic properties of the ATPase reactions of three PKC isozymes are similar, despite critical differences among the isozymes in their consensus sequences for the potential non-active-site nucleotide binding site in their catalytic domains. We also show that the ATPase and histone kinase reactions of each isozyme have similar Km,app(ATP) values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the insulin receptor kinase purified from human placental membranes

The insulin receptor purified from human placenta by sequential affinity chromatography on wheat germ agglutinin- and insulin-Sepharose to near homogeneity retained tyrosine-specific protein kinase activity. This purified insulin receptor kinase specifically catalyzed the incorporation of 32P from [gamma-32P]ATP into not only the beta-subunit of the insulin receptor but also histone H2B, a synthetic peptide which is sequentially similar to the site of tyrosine phosphorylation in pp60src (a gene product of the Rous sarcoma virus) and antibodies to pp60src present in the sera obtained from three rabbits bearing tumors induced by the Rous sarcoma virus. In each case, phosphorylation occurred exclusively on tyrosine residues. Insulin stimulated phosphorylation of these substrates 3- to 5-fold. Kinetic analysis using the synthetic peptide indicated that insulin acted by increasing the Vmax of peptide phosphorylation from about 3.1 to 9.5 nmol X mg-1 of protein X min-1, whereas the value of the Km for the peptide, about 1.5 mM, was not significantly changed. This kinase acted weakly on casein, alpha-S-casein, actin, and a tyrosine-containing peptide analogue of a serine-containing peptide used commonly as a substrate for the cyclic AMP-dependent protein kinases. These data show that the insulin receptor kinase displays specificity toward exogenous substrates similar to the substrate specificity observed for pp60src and the protein kinase activity associated with the receptor for epidermal growth factor. The data suggest that the catalytic sites of these three tyrosine kinases are similar and that insulin activates its receptor kinase by increasing the Vmax.     

Synthetic segments of the mammalian beta AR are preferentially recognized by cAMP-dependent protein kinase and protein kinase C

Desensitization of the beta-adrenergic receptor has been correlated in some cell systems with receptor phosphorylation. Various kinases have been implicated in these phosphorylation processes, including both cAMP-dependent protein kinase and protein kinase C. In the present study, we have utilized the protein sequence information obtained from the cloning of the mammalian beta-adrenergic receptor to prepare synthetic peptides corresponding to regions of the receptor which would be predicted to act as possible substrates for these kinases in vivo. Two of these receptor-derived peptides were found to serve as substrates for these protein kinases. A peptide corresponding to amino acids 257-264 of the beta-receptor is the preferred substrate for the cAMP-dependent protein kinase, while protein kinase C showed a marked preference for phosphorylation of a peptide corresponding to residues 341-351 of the beta-adrenergic receptor.     

A protein kinase associated with apoptosis and tumor suppression: structure, activity, and discovery of peptide substrates

Death-associated protein kinase (DAPK) has been implicated in apoptosis and tumor suppression, depending on cellular conditions, and associated with mechanisms of disease. However, DAPK has not been characterized as an enzyme due to the lack of protein or peptide substrates. Therefore, we determined the structure of DAPK catalytic domain, used a homology model of docked peptide substrate, and synthesized positional scanning substrate libraries in order to discover peptide substrates with K(m) values in the desired 10 microm range and to obtain knowledge about the preferences of DAPK for phosphorylation site sequences. Mutagenesis of DAPK catalytic domain at amino acids conserved among protein kinases or unique to DAPK provided a link between structure and activity. An enzyme assay for DAPK was developed and used to measure activity in adult brain and monitor protein purification based on the physical and chemical properties of the open reading frame of the DAPK cDNA. The results allow insight into substrate preferences and regulation of DAPK, provide a foundation for proteomic investigations and inhibitor discovery, and demonstrate the utility of the experimental approach, which can be extended potentially to kinase open reading frames identified by genome sequencing projects or functional genetics screens and lacking a known substrate.     

A rapid method for determining protein kinase phosphorylation specificity

Selection of target substrates by protein kinases is strongly influenced by the amino acid sequence surrounding the phosphoacceptor site. Identification of the preferred peptide phosphorylation motif for a given kinase permits the production of efficient peptide substrates and greatly simplifies the mapping of phosphorylation sites in protein substrates. Here we describe a combinatorial peptide library method that allows rapid generation of phosphorylation motifs for serine/threonine kinases.     

Docking sites on substrate proteins direct extracellular signal-regulated kinase to phosphorylate specific residues

Mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase (ERK) are important signaling proteins that phosphorylate (S/T)P sites in many different protein substrates. ERK binding to substrate proteins is mediated by docking sites including the FXFP motif and the D-domain. We characterized the sequence of amino acids that can constitute the FXFP motif using peptide and protein substrates. Substitutions of the phenylalanines at positions 1 and 3 had significant effects, indicating that these phenylalanines provide substantial binding affinity, whereas substitutions of the residues at positions 2 and 4 had less effect. The FXFP and D-domain docking sites were analyzed in a variety of positions and arrangements in the proteins ELK-1 and KSR-1. Our results indicate that the FXFP and D-domain docking sites form a flexible, modular system that has two functions. First, the affinity of a substrate for ERK can be regulated by the number, type, position, and arrangement of docking sites. Second, in substrates with multiple potential phosphorylation sites, docking sites can direct phosphorylation of specific (S/T)P residues. In particular, the FQFP motif of ELK-1 is necessary and sufficient to direct phosphorylation of serine 383, whereas the D-domain directs phosphorylation of other (S/T)P sites in ELK-1.