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PubMed1998

Solution structure of the C-terminal SH2 domain of the p85 alpha regulatory subunit of phosphoinositide 3-kinase.

Heterodimeric class IA phosphoinositide 3-kinase (PI 3-kinase) plays a crucial role in a variety of cellular signalling events downstream of a number of cell-surface receptor tyrosine kinases. Activation of the enzyme is effected in part by the binding of two Src homology-2 domains (SH2) of the 85 kDa regulatory subunit to specific phosphotyrosine-containing peptide motifs within activated cytoplasmic receptor domains. The solution structure of the uncomplexed C-terminal SH2 (C-SH2) domain of the p85 alpha subunit of PI 3-kinase has been determined by means of multinuclear, double and triple-resonance NMR experiments and restrained molecular-dynamics simulated-annealing calculations. The solution structure clearly indicates that the uncomplexed C-SH2 domain conforms to the consensus polypeptide fold exhibited by other SH2 domains, with an additional short helical element at the N terminus. In particular, the C-SH2 structure is very similar to both the p85 alpha N-terminal SH2 domain (N-SH2) and the Src SH2 domain with a root mean square difference (rmsd) for 44 C alpha atoms of 1.09 and 0.89 A, respectively. The canonical BC, EF and BG loops are less well-defined by the experimental restraints and show greater variability in the ensemble of C-SH2 conformers. The lower level of definition in these regions may reflect the presence of conformational disorder, an interpretation supported by the absence or broadening of backbone and side-chain NMR resonances for some of these residues. NMR experiments were performed, where C-SH2 was titrated with phosphotyrosine-containing peptides corresponding to p85 alpha recognition sites in the cytoplasmic domain of the platelet-derived growth-factor receptor. The ligand-induced chemical-shift perturbations indicate the amino-acid residues in C-SH2 involved in peptide recognition follow the pattern predicted from homologous complexes. A series of C-SH2 mutants was generated and tested for phosphotyrosine peptide binding by surface plasmon resonance. Mutation of the invariant Arg36 (beta B5) to Met completely abolishes phosphopeptide binding. Mutation of each of Ser38, Ser39 or Lys40 in the BC loop to Ala reduces the affinity of C-SH2 for a cognate phosphopeptide, as does mutation of His93 (BG5) to Asn. These effects are consistent with the involvement of the BC loop and BG loops regions in ligation of phosphopeptide ligands. Mutation of Cys57 (beta D5) in C-SH2 to Ile, the corresponding residue type in the p85 alpha N-SH2 domain, results in a change in peptide binding selectivity of C-SH2 towards that demonstrated by p85 alpha N-SH2. This pattern of p85 alpha phosphopeptide binding specificity is interpreted in terms of a model of the p85 alpha/PDGF-receptor interaction.

Amino Acid Sequence↗

PubMed1975

Structural requirements for the enzymatic phosphorylation of phosvitin.

Some structural features required for the enzymatic phosphorylation of phosvitin by purified rat liver cytosol phosvitin kinase have been investigated by testing the activity of such an enzyme toward phosphopeptides differing in size and chemical composition, obtained by pronase or acid hydrolysis of phosvitin. The results obtained can be summarized as follows: (a) Phosvitin kinase phosphorylates even fairly simple phosphopeptides (mol.wt 1000-2000) at rates comparable with intact phosvitin. (b) Acetylation of both phosvitin and pronase phosphopeptides completely prevents their phosphorylation indicating that some lysine residues are strictly required for the phosvitin kinase reaction. (c) Accordingly polyphosphorylserine blocks Ser(P)n which are very actively phosphorylated in phosvitin and pronase phosphopeptides, do not undergo any more enzymatic phosphorylation once isolated as such in a form free of other amino acids. (d) The activity of phosvitin kinase toward substrates probably devoid of Ser(P)n blocks suggests that there are not required for the protein kinase reaction. However, they apparently enhance the phosphorylation rate of the peptide substrates, likely by making easier their binding to the enzyme. It is proposed therefore that the peptidic unit able to undergo phosphorylation by rat liver cytosol phosvitin kinase consists of one or more phosphorylserine residues having in their close proximity a lysine residue playing a critical role in the mechanism of transphosphorylation.

Amino Acids↗

PubMed1983

Phosphorylation of different sites of acetyl CoA carboxylase by ATP-citrate lyase kinase and cyclic AMP-dependent protein kinase.

Native acetyl CoA carboxylase was phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase and ATP-citrate lyase kinase to 1 and 0.5 mol/subunit respectively. Both protein kinases added together increased acetyl CoA carboxylase phosphorylation additively. Partial proteolysis of 32P-acetyl CoA carboxylase followed by electrophoretic analysis showed that the 32P-phosphopeptides generated from acetyl CoA carboxylase phosphorylated with lyase kinase were different from the peptides obtained from the enzyme phosphorylated by cyclic AMP-dependent protein kinase. Mapping of tryptic 32P-phosphopeptides by high performance liquid chromatography showed that the major phosphopeptides phosphorylated by ATP-citrate lyase kinase were different from the major phosphopeptides phosphorylated by cyclic AMP-dependent protein kinase. The results suggest that at least one different site on acetyl CoA carboxylase is preferentially phosphorylated by each protein kinase.

Acetyl-CoA Carboxylase↗

PubMed1995

Phosphorylation and inactivation of rat heart glycogen synthase by cAMP-dependent and cAMP-independent protein kinases.

The regulation of cardiac muscle glycogen metabolism is not well understood. Previous studies have indicated that heart glycogen synthase is heavily phosphorylated in vivo on multiple sites. Using purified enzymes, we have investigated the effect of phosphorylation of different sites on the activity of rat heart glycogen synthase. A convenient procedure was developed for the purification of rat heart glycogen synthase. The enzyme was phosphorylated by selected kinases, and glycogen synthase activity, extent of phosphorylation, and phosphopeptide maps were analyzed. Rat heart glycogen synthase, purified to apparent homogeneity (M(r) 87,000 on SDS-PAGE), had a specific activity of 18 U/mg protein and had an activity ratio of 0.74 (activity in the absence divided by the activity in the presence of glucose 6-P). cAMP-dependent protein kinase, glycogen synthase kinase 3, Ca2+/calmodulin-dependent protein kinase II, protein kinase C, and phosphorylase kinase phosphorylated the enzyme with a concomitant decrease in the activity ratio to values ranging from 0.1 to 0.4. Casein kinase II phosphorylated but did not inactivate glycogen synthase. Six tryptic phosphopeptides, obtained from heart glycogen synthase phosphorylated by the various kinases, were separated by reverse-phase chromatography. The phosphopeptide(s) obtained with each kinase eluted at the same position(s) as corresponding phosphopeptides obtained from rat skeletal muscle glycogen synthase. The study shows that the pattern of phosphorylation and effects on activity are very similar for cardiac and skeletal muscle glycogen synthase. It is suggested that the well known differences in heart and glycogen metabolism may be due to the interplay of kinases and phosphatases which could lead to different phosphorylation and activity states of glycogen synthase.

Animals↗

PubMed2003

Susceptibility of the hydroxyl groups in serine and threonine to beta-elimination/Michael addition under commonly used moderately high-temperature conditions.

The beta-elimination/Michael addition reaction has been employed for the modification of O-acylated and phosphorylated Ser and Thr residues in a variety of derivatives. The modified Ser and Thr can be analyzed by amino acid composition analysis, N-terminal Edman degradation sequence analysis, and tandem mass spectrometric sequencing which generally allows the identification and localization of the phosphorylation or glycosylation sites. However, the reactivity of the free hydroxyl group on serine and threonine by sodium hydroxide-induced beta-elimination has not been critically examined. In this study, two analogous phosphopeptides, KMpSTLSYR and KMSpTLSYR, were subjected to beta-elimination under the widely used conditions previously reported, followed by sulfite or ethanethiol addition. After treatment of the phosphopeptides in 0.1 N NaOH/0.6 M Na(2)SO(3) at 37 degrees C for 24 h, matrix-assisted laser desorption ionization-time of flight mass spectrometric analyses of the products revealed an appreciable mass peak with an additional observed mass of 64 compared to the expected mass from the conversion of phosphate to sulfite. Similarly, treatment of the phosphopeptides in 0.52 N NaOH/1.36 M ethanethiol at 50 degrees C for 18 h or for even as short as 1h also yielded additional 44 mass of ethylthiogroup in excess of the expected mass for the modified phosphopeptide. Electrospray ionization tandem mass spectrometric analysis confirms that the modification occurred on the hydroxyl group of Ser and Thr in addition to P-Ser and P-Thr. On the other hand, modification on the free hydroxyl group of Ser or Thr was not detected under the mild condition of 0.1 N NaOH/0.6 M Na(2)SO(3) at 25 degrees C for 24 h as previously reported. This finding suggests that temperatures above 25 degrees C and excessive alkalinity should be avoided to prevent the beta-elimination of the hydroxyl group of Ser and Thr in peptides. This is of particular concern when employing highly sensitive tandem mass spectrometric methods for the identification and localization of Ser and Thr as modification sites by the beta-elimination/Michael addition reaction. The additional modification site(s) may complicate the interpretation of data and lead to an erroneous conclusion.

Hydroxyl Radical↗

PubMed2003

Identification of an in vitro insulin receptor substrate-1 phosphorylation site by negative-ion muLC/ES-API-CID-MS hybrid scan technique.

Recently, we reported a fast on-line alkaline micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric approach for sensitive phosphopeptide screening of a tryptic digested protein and subsequent characterization of the identified phosphopeptide. Based on this study, we now applied an improved method for the identification of phosphorylation sites in insulin receptor substrate 1, an important mediator in insulin signal transduction which was phosphorylated in vitro by protein kinase C-zeta. The approach consists of an on-line alkaline negative-ion micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric hybrid scan experiment using a triple-quadrupole mass spectrometer with fractionation and subsequent off-line nanoES-MS (ion trap) analysis of the phosphopeptide-containing fractions. During the liquid chromatography (LC)/ES-MS experiment, the phosphopeptides of the enzymatic digest mixture of the studied insulin receptor substrate 1 fragment were detected under high skimmer potential (API-CID) using phosphorylation-specific m/z 79 marker ions as well as the intact m/z-values of the peptides which were recorded under low skimmer potential. Subsequently, the targeted fractions were analyzed by off-line nanoES-MS/MS and MS(3). Using this approach, serine 318 was clearly identified as a major in vitro protein kinase C-zeta phosphorylation site in the insulin receptor substrate -1 fragment. Together, our results indicate that the applied strategy is useful for unequivocal and fast analysis of phosphorylation sites in low abundant signaling proteins.

Amino Acid Sequence↗

PubMed2002

Phosphoprotein isotope-coded affinity tags: application to the enrichment and identification of low-abundance phosphoproteins.

The use of a phosphoprotein isotope-coded affinity tag (PhIAT), which employs differential isotopic labeling and biotinylation, has been shown capable of enriching and identifying mixtures of low-abundance phosphopeptides. A denatured solution of beta-casein was labeled using the PhIAT method, and after proteolytic digestion, the labeled peptides were isolated using immobilized avidin. The recovered peptides were separated by capillary reversed-phase liquid chromatography and identified by tandem mass spectrometry. PhIAT-labeled peptides corresponding to known O-phosphorylated peptides from beta-casein were identified along with the phosphorylated peptides from alphas1-casein and alphas2-casein, known low-level (<5%) contaminants of commercially available beta-casein. All of the casein-phosphorylated residues identified by the present PhIAT approach correspond to previously documented sites of phosphorylation. The results illustrate the efficacy of the PhIAT-labeling strategy to not only enrich mixtures for phosphopeptides but also, more importantly, permit the detection and identification of low-level phosphopeptides. In addition, the differences in the phosphorylation state could be determined between phosphopeptides in comparative samples by stoichiometric conversion using the light and heavy isotopic versions of the PhIAT reagents. Overall, our results exemplify the application of the PhIAT approach and demonstrate its utility for proteome-wide phosphoprotein identification and quantitation.

Affinity Labels↗

PubMed2005

Quantitative analysis of protein phosphorylation in mouse brain by hypothesis-driven multistage mass spectrometry.

Determination of site-specific changes in the levels of protein phosphorylation in mammals presents a formidable analytical challenge. Here, we demonstrate a strategy for such analyses utilizing a combination of stable isotope chemical labeling and tandem mass spectrometry. Phosphoproteins of interest are isolated from two sets of animals that have undergone differential drug treatments, separated by SDS-PAGE, excised, and subjected to in-gel enzymatic digestion. Using a simple chemical labeling step, we introduce stable, isotopically distinct mass tags into each of the two sets of peptides that originate from the samples under comparison, mix the samples, and subject the resulting mixture to a procedure based on our previously reported hypothesis-driven multistage MS (HMS-MS) method (Chang, E. J.; Archambault, V.; McLachlin, D. T.; Krutchinsky, A. N.; Chait, B. T. Anal. Chem. 2004, 76, 4472-4483). The method takes advantage of the dominant loss of H3PO4 during MS/MS from singly charged phosphopeptide ions produced by matrix-assisted laser desorption/ionization (MALDI) in the ion trap mass spectrometer. In the present work, quantitation is achieved by isolating the range of m/z values that include both isotopic forms of the putative phosphopeptide and measuring the relative intensities of the two resulting -98-Da fragment ion peaks. This MS/MS measurement can be repeated on the same MALDI sample for all potential phosphopeptide ion pairs that we hypothesize might be produced from the protein under study. Use of MS/MS for quantitation greatly increases the sensitivity of the method and allows us to measure relatively low levels of phosphorylation, phosphopeptides, or both that are not easily observable by single-stage MS. We apply the current method to the determination of changes in the levels of phosphorylation in DARPP-32 from the mouse striatum upon treatment of animals with psychostimulant drugs.

Amino Acid Sequence↗

PubMed1999

Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry: potentials and limitations.

Precursor ion scans have proven to be extremely useful for the characterization of unseparated peptide mixtures. In conjunction with the nanoelectrospray source, precursor ion scans provide a sensitive tool for the detection of posttranslationally modified peptides and have been used to determine phosphorylation sites of proteins digested in solution. In this report, we extend our previous work to the determination of protein phosphorylation sites of gel-isolated proteins. The in-gel digestion procedure developed in our laboratory for protein microsequencing was found to be suitable for phosphorylation mapping as well. The risk of losing hydrophilic peptides in the desalting step was decreased by using column packing material designed for the purification of oligonucleotides and by adjusting the pH conditions to the needs of phosphopeptide analysis. With this method, the tryptic phosphopeptides of beta-casein were detected after in-gel digestion at a sensitivity of 250 fmol of protein applied to the gel. The phosphorylation sites of two other proteins, Src-delta U and Op18, have similarly been mapped. Subpicomole to low-picomole amounts of protein starting material are needed in general, although we and others have reported attomole sensitivity for the detection of model phosphopeptides using precursor ion scans. This indicates that the success in determining phosphorylation sites depends crucially on the digestion, extraction, and detection efficiency for individual phosphopeptides.

Amino Acid Sequence↗

PubMed1994

Phosphorylation sites in ligand-induced and ligand-independent activation of the progesterone receptor.

Steroid hormone receptors are phosphoproteins that undergo hyperphosphorylation upon binding of hormone. The mechanism and the role of this reaction remain poorly understood. Two-dimensional analysis of ligand-free progesterone receptor (PR) tryptic digests showed the existence of seven main phosphopeptides. Incubation of the cells with the progestin R5020 led to a global increase in the levels of PR phosphorylation. However, the same phosphopeptides were seen, and their levels of labeling relative to each other were unchanged. A similar result was observed after incubation of cells with the antiprogestin RU486. The antiprogestin ZK98299 demonstrated only half of the activity of RU486 in terms of receptor hyperphosphorylation, but the same phosphopeptides, proportionally labeled to the same extent, were observed by chromatography electrophoresis. Ligand-induced DNA binding did not play a role in receptor hyperphosphorylation since the mutant delta 547-592, which is devoid of the first zinc finger region, exhibited the same phosphopeptides, labeled to the same extent, as did wild-type receptor after incubation of cells with hormone. These results suggest that the same kinase(s) act in vivo on ligand-free and on agonist or antagonist-bound progesterone receptor. Binding of different ligands produces different conformational changes in the ligand binding domain of the receptor which enhance, to varying extents, affinity of the receptor for the kinase(s). The DNA binding region also plays a role in the interaction with the kinase(s), although binding to DNA per se is not necessary for the hyperphosphorylation of the receptor to take place.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate phospholemman, an insulin and adrenaline-regulated membrane phosphoprotein, at specific sites in the carboxy terminal domain.

Phospholemman, a transmembrane, 72 residue protein enriched in striated muscle and heart [Palmer, Scott and Jones (1991) J. Biol. Chem. 266, 11126-11130], is phosphorylated in response to insulin [Walaas, Horn and Walaas (1991) Biochim. Biophys. Acta 1094, 92-102]. The present study is aimed at identifying the phosphorylation sites of this protein. A synthetic peptide, GTFRSS63IRRLS68TRRR (in the single letter code) and consisting of phospholemman residues 58-72, is a substrate for both protein kinase C and cyclic AMP (cAMP)-dependent protein kinase, with Km values of 6-7 microM for both enzymes. Amino acid sequencing of the phosphopeptide shows that protein kinase C phosphorylates both Ser-63 and Ser-68, while cAMP-dependent protein kinase phosphorylates Ser-68. Thermolytic phosphopeptide mapping of 32P-labelled phospholemman from rat diaphragms shows that treatment with insulin results in labelling of phosphopeptides containing both Ser-63 and Ser-68, whereas treatment with adrenaline results in labelling of the phosphopeptide containing Ser-68. Hence, insulin and adrenaline regulate the phosphorylation of phospholemman, presumably through protein kinase C and cAMP-dependent protein kinase, respectively, on partly overlapping phosphorylation sites.

Amino Acid Sequence↗

PubMed1998

Insulin stimulation of phosphorylation of elongation factor 1 (eEF-1) enhances elongation activity.

To examine the role of phosphorylation of the elongation factor eEF-1 in regulation of translation, 32P-labeled 3T3-L1 cells were deprived of serum, then incubated in the presence or absence of 10 nM insulin for 15 min. eEF-1 was purified by affinity chromatography on tRNA-Sepharose and shown to be phosphorylated on the alpha, beta and delta subunits. Phosphorylation of eEF-1alpha was stimulated sixfold in response to insulin, beta was stimulated fourfold and delta was threefold. The rate of elongation assayed with eEF-1 from insulin-stimulated cells was over twofold greater than with eEF-1 from serum-deprived cells. When eEF-1 from insulin-treated cells was subjected to two-dimensional tryptic phosphopeptide mapping, nine phosphopeptides were obtained with the alpha subunit, one with the beta subunit and three with the delta subunit. When compared with phosphopeptide maps of alpha, beta and delta subunits of eEF-1 phosphorylated in vitro by the insulin-stimulated multipotential protein kinase, the maps of the beta and delta subunits were identical. Five phosphopeptides obtained with the alpha subunit in vivo were identical to those obtained with S6 kinase in vitro; the remainder were unique. To examine whether protein kinase C had a role in phosphorylation of eEF-1 in response to insulin, protein kinase C was down-regulated by prolonged exposure of 3T3-L1 cells to 4beta-phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the alpha, beta and delta subunits was stimulated 2.5-fold in response to insulin, with elongation activity stimulated to a similar extent, suggesting that protein kinase C had no effect on stimulation of elongation in response to insulin. Thus, stimulation of eEF-1 activity in response to insulin appears to be mediated primarily by multipotential S6 kinase. This data is consistent with previous studies on stimulation of initiation via phosphorylation of initiation factors by multipotential S6 kinase [Morley, S. J. & Traugh, J. A. (1993) Biochemie (Paris) 95, 985-989].

3T3 Cells↗

PubMed1980

Site-specific phosphorylation of the alpha subunit of eukaryotic initiation factor eIF-2 by the heme-regulated and double-stranded RNA-activated eIF-2 alpha kinases from rabbit reticulocyte lysates.

The site specificity of phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) by the heme-regulated and double-stranded RNA-activated eIF-2alpha kinases were compared by phosphopeptide mapping. eIF-2alpha was maximally phosphorylated in vitro with [gamma-(32)P]ATP and either crude or partially purified preparations of the kinases. (32)P-Labeled eIF-2alpha was isolated by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. The fixed, stained, and dried polypeptide band was excised and then exhaustively digested directly in the gel slice with one of several proteases (trypsin, chymotrypsin, subtilisin, or thermolysin); the resultant [(32)P]phosphopeptides were analyzed by one-dimensional chromatography or by two-dimensional chromatography and high-voltage electrophoresis. In addition, limited proteolysis of [(32)P]eIF-2alpha contained in fixed, dried, and stained gel slices was achieved with Staphylococcus aureus protease V8, chymotrypsin, or subtilisin, and the partial (32)P-labeled cleavage products were analyzed by gel electrophoresis. Each protease produced distinct and reproducible [(32)P]phosphopeptide profiles after partial or exhaustive proteolysis of [(32)P]eIF-2alpha. With a given protease, identical [(32)P]phosphopeptide patterns were obtained whether eIF-2alpha was phosphorylated by the heme-regulated or the double-stranded RNA-activated kinase. These data indicate that, in vitro, the kinases phosphorylate sites on eIF-2alpha that are identical or proximally located in the primary sequence. In this report we also provide preliminary evidence that the two eIF-2alpha kinases activated in lysates by heme deficiency or double-stranded RNA phosphorylate site(s) of endogenous eIF-2alpha that are similar, if not identical, to the sites phosphorylated in vitro with partially purified eIF-2alpha kinase(s) and eIF-2.

Animals↗

PubMed1995

Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209.

Initiation factor 4E (eIF-4E) binds to the m7GTP-containing cap of eukaryotic mRNA and facilitates the entry of mRNA into the initiation cycle of protein synthesis. eIF-4E is a phosphoprotein, and the phosphorylated form binds to mRNA caps 3-4-fold more tightly than the nonphosphorylated form. A previous study indicated that the major phosphorylation site was Ser-53 (Rychlik, W., Russ, M. A., and Rhoads, R. E. (1987) J. Biol. Chem. 262, 10434-10437). In the present study, we synthesized the phosphopeptide expected to result from tryptic digestion of eIF-4E, O-phosphoseryllysine. Surprisingly, the tryptic and synthetic phosphopeptides did not comigrate electrophoretically. Accordingly, we redetermined the phosphorylation site by isolating a chymotryptic phosphopeptide on reverse phase high performance liquid chromatography. The peptide was sequenced by Edman degradation and corresponded to 198QSHADTATKSGSTTKNRF215. The site of phosphorylation was determined to be Ser-209 by four methods: the increase in the ratio of dehydroalanine to serine derivatives during Edman degradation, the release of 32P, the further digestion of the chymotryptic phosphopeptide with trypsin, Glu-C, and Asp-N, and site-directed mutagenesis of eIF-4E cDNA. The S209A variant was not phosphorylated in a rabbit reticulocyte lysate system, whereas the wild-type, S53A, and S207A variants were. This site falls within the consensus sequence for phosphorylation by protein kinase C.

Amino Acid Sequence↗

PubMed1996

A potential SH3 domain-binding site in the Crk SH2 domain.

The Src homology 2 (SH2) domain of the mammalian adaptor protein Crk-II contains a proline-rich insert, predicted to lie within an extended DE loop, which is dispensable for phosphopeptide binding. Using the yeast two-hybrid system, this region of the Crk-II SH2 domain was found to interact with a subset of SH3 domains, notably the Abl SH3 domain. Furthermore, this proline-rich insert was found to modify the efficiency with which Crk-II was phosphorylated by the p140(c-abl) tyrosine kinase. In vitro, the interaction of full-length non-phosphorylated Crk-II with a glutathione S-transferase-Abl SH3 domain fusion protein was very weak. However, phosphorylation of Crk-II on Tyr-221 which induces an intramolecular association with the SH2 domain, or addition of a phosphopeptide corresponding to the Crk-II Tyr-221 phosphorylation site, stimulated association of Crk-II with the Abl SH3 domain. NMR spectroscopic analysis showed that binding of the Tyr-221 phosphopeptide to the Crk SH2 domain induced a chemical shift change in Val-71, located in the proline-rich insert, indicative of a change in the structure of the proline-rich loop in response of Crk SH2-pTyr-221 interaction. These results suggest that the proline-rich insert in the Crk SH2 domain constitutes an SH3 domain-binding site that can be regulated by binding of a phosphopeptide ligand to the Crk SH2 domain.

Amino Acid Sequence↗

PubMed1998

Common in vitro substrate specificity and differential Src homology 2 domain accessibility displayed by two members of the Src family of protein-tyrosine kinases, c-Src and Hck.

Hck and Src are members of the Src family of protein- tyrosine kinases that carry out distinct and overlapping functions in vivo (Lowell, C. A., Niwa, M., Soriano, P., and Varmus, H. E. (1996) Blood 87, 1780-1792). In an attempt to understand how Hck and Src can function both independently and in concert, we have compared 1) their in vitro substrate specificity and 2) the accessibility of their Src homology 2 (SH2) domain. Using several synthetic peptides, we have demonstrated that Hck and Src recognize similar structural features in the substrate peptides, suggesting that both kinases have the intrinsic ability to carry out overlapping cellular functions by phosphorylating similar cellular proteins in vivo. Using a phosphotyrosine-containing peptide that has previously been shown to bind the SH2 domain of Src family kinases with high affinity, we found that although Src could bind to the phosphopeptide, Hck showed no interaction. The inability of Hck to bind the phosphopeptide was not a result of a stable intramolecular interaction between its SH2 domain and C-terminal regulatory phosphotyrosine residue (Tyr-520), as most Hck molecules in the purified Hck preparation were not tyrosine-phosphorylated. In contrast to intact Hck, a recombinant truncation analog of Hck was able to bind the phosphopeptide with an affinity similar to that of the Src SH2 domain, suggesting that conformational constraints are imposed on intact Hck that limit accessibility of its SH2 domain to the phosphopeptide. Furthermore, the difference in SH2 domain accessibility is a potential mechanism that enables Src and Hck to perform their respective unique functions by 1) targeting them to different subcellular compartments, whereupon they phosphorylate different cellular proteins, and/or 2) facilitating direct binding to their cellular substrates.

Amino Acid Sequence↗

PubMed1999

Analysis of tyrosine phosphorylation-dependent interactions between stimulatory effector proteins and the B cell co-receptor CD22.

The B cell-restricted transmembrane glycoprotein CD22 is rapidly phosphorylated on tyrosine in response to cross-linking of the B cell antigen receptor, thereby generating phosphotyrosine motifs in the cytoplasmic domain which recruit intracellular effector proteins that contain Src homology 2 domains. By virtue of its interaction with these effector proteins CD22 modulates signal transduction through the B cell antigen receptor. To define further the molecular mechanism by which CD22 mediates its co-receptor function, phosphopeptide mapping experiments were conducted to determine which of the six tyrosine residues in the cytoplasmic domain are involved in recruitment of the stimulatory effector proteins phospholipase Cgamma (PLCgamma), phosphoinositide 3-kinase (PI3K), Grb2, and Syk. The results obtained indicate that the protein tyrosine kinase Syk interacts with multiple CD22-derived phosphopeptides in both immunoprecipitation and reverse Far Western assays. In contrast, the Grb2.Sos complex was observed to bind exclusively to the fourth phosphotyrosine motif (Y828ENV) from CD22 and does so via a direct interaction based on Far Western and reverse Far Western blotting. Although both PLCgamma and PI3K were observed to bind to multiple phosphopeptides in precipitation experiments, subsequent studies using reverse Far Western blot analysis demonstrated that only the carboxyl-terminal phosphopeptide of CD22 (Y863VTL) binds directly to either one. This finding suggests that PLCgamma and PI3K may be recruited to CD22 either through a direct interaction with Tyr863 or indirectly through an association with one or more intermediate proteins.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Identification of three previously unknown in vivo protein phosphorylation sites in thylakoid membranes of Arabidopsis thaliana.

The proteins in plant photosynthetic thylakoid membranes undergo light-induced phosphorylation, but only a few phosphoproteins have been characterized. To access the unknown sites of in vivo protein phosphorylation the thylakoid membranes were isolated from Arabidopsis thaliana grown in normal light, and the surface-exposed peptides were cleaved from the membranes by trypsin. The peptides were methylated and subjected to immobilized metal affinity chromatography, and the enriched phosphopeptides were sequenced using tandem nanospray quadrupole time-of-flight mass spectrometry. Three new phosphopeptides were revealed in addition to the five known phosphorylation sites in photosystem II proteins. All phosphopeptides are found phosphorylated at threonine residues implementing a strict threonine specificity of the thylakoid kinases. For the first time protein phosphorylation is found in photosystem I. The phosphorylation site is localized to the first threonine in the N terminus of PsaD protein that assists in the electron transfer from photosystem I to ferredoxin. A new phosphorylation site is also revealed in the acetylated N terminus of the minor chlorophyll a-binding protein CP29. The third novel phosphopeptide, composed of 25 amino acids, belongs to a nuclear encoded protein annotated as "expressed protein" in the Arabidopsis database. The protein precursor has a chloroplast-targeting peptide followed by the mature protein with two transmembrane helices and a molecular mass of 14 kDa. This previously uncharacterized protein is named thylakoid membrane phosphoprotein of 14 kDa (TMP14). The finding of the novel phosphoproteins extends involvement of the redox-regulated protein phosphorylation in photosynthetic membranes beyond the photosystem II and its light-harvesting antennae.

Amino Acid Sequence↗