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PubMed1972

Demonstration of a phosphopeptide intermediate in the Mg ++ -dependent, Na + - and K + -stimulated adenosine triphosphatase reaction of the erythrocyte membrane.

Human erythrocyte membranes are phosphorylated by [gamma-(32)P]ATP in association with the Mg(++)-dependent, Na(+) and K(+)-stimulated ATPase (EC 3.1.6.3) reaction. To delineate the membrane species involved, phosphorylated membranes were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, under conditions that minimize hydrolysis of acyl phosphate linkages. Three radioactive components were detected, of which only one was a phosphopeptide, of apparent molecular weight 105,000. The phosphate bound to this peptide undergoes rapid turnover and is discharged by hydroxylamine. In the presence of Mg(++), the phosphorylation of this peptide is specifically stimulated by Na(+) and blocked by ethylene diamine tetraacetate; its dephosphorylation is stimulated by K(+) and blocked by ouabain. We conclude that this phosphopeptide is an intermediate in the Mg(++)-dependent, Na(+)- and K(+)-stimulated ATPase reaction of the erythrocyte membrane.

Adenosine Triphosphatases↗

PubMed1996

Phosphorylation of the respiratory burst oxidase subunit p47phox as determined by two-dimensional phosphopeptide mapping. Phosphorylation by protein kinase C, protein kinase A, and a mitogen-activated protein kinase.

The respiratory burst oxidase is responsible for superoxide (O2) production by phagocytes and B lymphocytes. This multicomponent enzyme is dormant in resting cells but is activated on exposure of the cells to an appropriate stimulus. Upon activation, several serine residues on the cytosolic oxidase subunit p47phox become phosphorylated. Using two-dimensional tryptic phosphopeptide mapping, we studied the phosphorylation of p47phox in 32Pi-loaded Epstein-Barr virus-transformed B lymphoblasts expressing wild type p47phox or any of several P47phox Ser -> Ala mutants. We were able to identify the labeled peptides from wild type p47phox as those contain- ing Ser303/304 Ser315, Ser320, Ser328 and/or Ser359/370, and Ser345/348 ; no 32P-labeled Ser310-containing peptide was found. When purified p47phox, was phosphorylated in vitro by various protein kinases, varying phosphopeptide patterns were observed. Protein kinase C phosphorylated all the peptides except the one containing Ser345/348; protein kinase A phosphorylated the peptide containing Ser320 and one or both of the peptides containing Ser328 and Ser359/370; while mitogen-activated protein kinase phophorylated only the peptide containing Ser345/348. These findings suggest that these three kinases play distinct roles in the activation of the respiratory burst oxidase, each of them catalyzing the phosphorylation of a different group of serines in p47phox.

Amino Acid Sequence↗

PubMed1999

Heparin-induced conformational change in microtubule-associated protein Tau as detected by chemical cross-linking and phosphopeptide mapping.

In Alzheimer's disease, microtubule-associated protein tau becomes abnormally phosphorylated and aggregates into paired helical filaments. Sulfated glycosaminoglycans such as heparin and heparan sulfate were shown to accumulate in pretangle neurons, stimulate in vitro tau phosphorylation, and cause tau aggregation into paired helical filament-like filaments. The sulfated glycosaminoglycan-tau interaction was suggested to be the central event in the development of neuropathology in Alzheimer's disease brain (Goedert, M., Jakes, R., Spillantini, M. G., Hasegawa, M., Smith, M. J., and Crowther, R. A. (1996) Nature 383, 550-553). The biochemical mechanism by which sulfated glycosaminoglycans stimulate tau phosphorylation and cause tau aggregation remains unclear. In this study, disuccinimidyl suberate (DSS), a bifunctional chemical cross-linker, cross-linked tau dimers, tetramers, high molecular size aggregates, and two tau species of sizes 72 and 83 kDa in the presence of heparin. In the absence of heparin only dimeric tau was cross-linked by DSS. Fast protein liquid chromatography gel filtration revealed that 72- and 83-kDa species were formed by intramolecular cross-linking of tau by DSS. These observations indicate that heparin, in addition to causing aggregation, also induces a conformational change in tau in which reactive groups are unmasked or move closer leading to the DSS cross-linking of 72- and 83-kDa species. Heparin-induced structural changes in tau molecule depended on time of heparin exposure. Dimerization and tetramerization peaked at 48 h, whereas conformational change was completed within 30 min of heparin exposure. Heparin exposure beyond 48 h caused an abrupt aggregation of tau into high molecular size species. Heparin stimulated tau phosphorylation by neuronal cdc2-like kinase (NCLK) and cAMP-dependent protein kinase. Phosphopeptide mapping and phosphopeptide sequencing revealed that tau is phosphorylated by NCLK on Thr212 and Thr231 and by cAMP-dependent protein kinase on Ser262 only in the presence of heparin. Heparin stimulation of tau phosphorylation by NCLK showed dependence on time of heparin exposure and correlated with the heparin-induced conformational change of tau. Our data suggest that heparin-induced conformational change exposes new sites for phosphorylation within tau molecule.

Amino Acid Sequence↗

PubMed2003

Characterization of phosphopeptide motifs specific for the Src homology 2 domains of signal transducer and activator of transcription 1 (STAT1) and STAT3.

Signal transducers and activators of transcription (STAT) 1 and STAT3 are activated by overlapping but distinct sets of cytokines. STATs are recruited to the different cytokine receptors through their Src homology (SH) 2 domains that make highly specific interactions with phosphotyrosine-docking sites on the receptors. We used a degenerate phosphopeptide library synthesized on 35-microm TentaGel beads and fluorescence-activated bead sorting to determine the sequence specificity of the peptide-binding sites of the SH2 domains of STAT1 and STAT3. The large bead library allowed not only peptide sequencing of pools of beads but also of single beads. The method was validated through surface plasmon resonance measurements of the affinities of different peptides to the STAT SH2 domains. Furthermore, when selected peptides were attached to a truncated erythropoietin receptor and stably expressed in DA3 cells, activation of STAT1 or STAT3 could be achieved by stimulation with erythropoietin. The combined analysis of pool sequencing, the individual peptide sequences, and plasmon resonance measurements allowed the definition of SH2 domain binding motifs. STAT1 preferentially binds peptides with the motif phosphotyrosine-(aspartic acid/glutamic acid)-(proline/arginine)-(arginine/proline/glutamine), whereby a negatively charged amino acid at +1 excludes a proline at +2 and vice versa. STAT3 preferentially binds peptides with the motif phosphotyrosine-(basic or hydrophobic)-(proline or basic)-glutamine. For both STAT1 and STAT3, specific high affinity phosphopeptides were identified that can be used for the design of inhibitory molecules.

Amino Acid Motifs↗

PubMed2004

Effect of casein phosphopeptide and 25-hydroxycholecalciferol on tibial dyschondroplasia in growing broiler chickens.

1. An experiment was conducted to investigate the influence of dietary casein phosphopeptides and 25-hydroxycholecalciferol on the incidence of tibial dyschondroplasia (TD) in 14-d-old commercial broiler chickens. 2. Three hundred and twenty broiler chicks (one day old) were randomly allocated to one of 4 dietary treatments. A standard broiler diet was used as the control with the three experimental treatments receiving the control diet supplemented with 10 g casein phosphopeptide/kg, 14 g casein phosphopeptide/kg or 69 microg 25-hydroxycholecalciferol/kg. 3. Those birds fed the diets supplemented with 14g casein phosphopeptide/kg or 25-hydroxycholecalciferol had a lower incidence of TD than both the control and 10g casein phosphopeptide/kg treatments when assessed grossly. 4. The body weight of birds fed the 10 g casein phosphopeptide/kg diet or the 25-hydroxycholecalciferol diet was higher than birds fed the control diet. Although not significant, the body weight of birds fed the 14 g casein phosphopeptide/kg diet was also greater than the control birds. 5. The current experiment demonstrated that both casein phosphopeptide and 25-hydroxycholecalciferol can reduce the incidence of TD in the young broiler chicken. More research is required to explain the unexpected increase in body weight described above.

Animal Feed↗

PubMed1995

Prediction and analysis of SH2 domain-phosphopeptide interactions.

Src homology 2 (SH2) domains are small protein modules of approximately 100 amino acids that are found in many proteins involved in intracellular signal transduction. They mediate protein-protein interactions and modulate enzyme activity by their ability to bind to specific sequence patterns that contain a phosphorylated tyrosine. As the three-dimensional structures of the phosphatidylinositol (PI) 3-kinase, Lck, Src and Abl SH2 domains have been shown to be similar, we have modelled other SH2 domains that show distinct sequence specificity to allow comparative analysis of SH2-phosphopeptide interactions. The SH2 domains of PLC gamma-Nterm., Nck, Grb2, GAP and Abl have been model-built with high-affinity phosphopeptides fitted into the putative binding sites. For each SH2 domain a detailed analysis of the peptide-protein interaction was performed. It is apparent that specificity is mainly conferred by three to five residues downstream from the phosphotyrosine residue (Y*), especially, although not exclusively, peptide position Y* + 3. The SH2 pocket that binds the Y* + 3 residue is mainly composed of three sections: part of strand beta E going into loop EF, part of alpha B and loop BG. The residues that constitute the Y* + 3 binding pocket show variability that seems to determine which amino acid binds preferentially. Residue position beta E4 seems to play a vital role in the SH2 specificity. This study shows that the development of modelling protocols for SH2 domains whose structure has not been determined can prove very useful in predicting which residues are involved in conferring the affinity and binding specificity of these domains towards distinct phosphotyrosine-containing sequences.

Amino Acid Sequence↗

PubMed1997

Casein phosphopeptides improve zinc and calcium absorption from rice-based but not from whole-grain infant cereal.

BACKGROUND: Casein phosphopeptides (CPP) are phosphorus-rich peptide fragments of casein, assumed to contribute to the high bioavailability of calcium from milk. METHODS: The effect of casein phosphopeptides on calcium and zinc absorption from infant foods was investigated. Twenty-two men and women were given single test meals extrinsically labeled with Ca and Zn. Absorption was calculated from measurements on whole-body retention of the radioisotopes. Each subject was given either rice-based cereal (n = 11) or whole-grain cereal (n = 11) on three occasions together with 250 ml water and added 0, 1, and 2 g CPP in random order. One serving of rice-based cereal contained 481 mg Ca and 1.29 mg Zn; whole-grain cereal contained 541 mg Ca and 1.77 mg Zn. One and 2 g of CPP contributed with additional 69 and 138 mg Ca, respectively. RESULTS: From rice-based cereal, fractional calcium absorption was not affected by CPP addition (mean +/- SD): 16.0 +/- 4.0% (no CPP), 17.6 +/- 4.5% (1 g CPP), and 15.8 +/- 4.3% (2 g CPP), while the total quantity of calcium absorbed was significantly improved: 7 +/- 19 mg, 97 +/- 25 mg, and 98 +/- 26 mg, respectively (p = 0.0004). Fractional zinc absorption as well as total quantity of zinc absorbed were increased with addition of CPP: 19.4 +/- 9.0% (0.25 +/- 0.12 mg), 25.2 +/- 7.5% (0.33 +/- 0.10 mg) and 23.9 +/- 5.4% (0.31 +/- 0.07 mg) at the three CPP levels (p = 0.04). From whole-grain cereal, CPP had no effect on the percentage or actual quantity of calcium absorbed: 17.0 +/- 3.2% (92 +/- 18 mg), 17.2 +/- 4.5% (105 +/- 27 mg), and 15.0 +/- 4.6% (102 +/- 31 mg), respectively. Zinc absorption was also not influenced by CPP: 16.0 +/- 5.1% (0.28 +/- 0.09 mg), 15.3 +/- 3.1% (0.27 +/- 0.06 mg) and 18.1 +/- 4.4% (0.32 +/- 0.08 mg), respectively. CONCLUSIONS: CPP addition improved calcium and zinc absorption from rice-based cereal, while no effect was seen from whole-grain cereal.

Adult↗

PubMed1983

The phosphorylation of ribosomal protein S6 from progesterone-stimulated Xenopus laevis oocytes. Kinetic studies and phosphopeptide analysis.

Xenopus laevis oocytes were prelabeled with [32P]orthophosphate overnight before maturation was induced by progesterone stimulation. The phosphorylation status of ribosomal protein S6 from control oocytes and the temporal changes in S6 phosphorylation after progesterone treatment were analyzed by two-dimensional gel electrophoresis. S6 protein was separated in up to five distinct S6 species, which differed in their degree of phosphorylation. 32P labeling of S6, as judged from the shift of radioactivity into more highly phosphorylated S6 derivatives, continuously increased in progesterone-stimulated oocytes even at later times when germinal vesicle breakdown was completed. S6 protein of unstimulated oocytes was labeled to a lower degree. Trypsin cleavage of total S6 protein, isolated from control and maturing oocytes, gave rise to different complex phosphopeptide patterns reflecting the existence of various multiply phosphorylated S6 derivatives in both samples. Two of the more highly phosphorylated S6 derivatives showed considerable differences between the phosphopeptide elution profiles of control and stimulated oocytes indicating that dissimilar sites had been modified under both physiological conditions. Only phosphoserine was detected in the phosphoamino acid analysis of individual S6 derivatives.

Animals↗

PubMedDate not supplied

Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: a review.

Using laboratory, animal, and human in situ caries models, investigators have shown that casein phosphopeptide amorphous calcium phosphate complexes (CPP-ACP) exhibit an anticariogenic activity. The casein phosphopeptides (CPP) are produced from a tryptic digest of the milk protein casein by aggregation with calcium phosphate and purification by ultrafiltration. The CPP have a remarkable ability to stabilize calcium phosphate in solution and substantially increase the level of calcium phosphate in dental plaque. Through their multiple phosphoseryl residues, the CPP bind to forming clusters of amorphous calcium phosphate (ACP) in metastable solution, preventing their growth to the critical size required for nucleation and precipitation. The proposed mechanism of anticariogenicity for the CPP-ACP is that they localize ACP in dental plaque, which buffers the free calcium and phosphate lon activities, thereby helping to maintain a state of supersaturation with respect to tooth enamel depressing demineralization and enhancing remineralization. The CPP-ACP, unlike fluoride, can be added to sugar-containing foods and therefore have commercial potential as an additive to foods as well as to toothpastes and mouthwashes for the control of dental caries.

Amino Acid Sequence↗

PubMed1979

Phosphopeptides of enamel matrix.

Although the tripeptides Glu-O-Phosphoserine-Tyr and Glu-O-Phosphoserine-Leu have been identified in embryonic bovine enamel proteins, 1, 2 the issue of whether both sequences occur in each of the phosphopeptides, or whether certain sequences occur in specific peptides only, has recently been resolved by isolating homogeneous samples of E33 and E44. All three of the Ser residues of both peptides are phosphorylated. All three in E3 are in the sequence Glu-O-Phosphoserine-Leu, and all three in E4 are in the sequence Glu-O-Phosphoserine-Tyr. It was not possible to sequence either of the polypeptide chains directly by automatic peptide sequencing. However, a partial sequence of E4 was constructed from data derived from peptides isolated after cyanogen bromide, trypsin and chymotrypsin digestions. The presence of Glu, Tyr and Leu adjacent to and near the O-Phosphoserine [Ser(L)] residues and the 2 degrees, 3 degrees and higher ordered structures of the enamel phosphopeptides may be important in calcium binding and mineralization.

Amino Acid Sequence↗

PubMed1994

Specificity in recognition of phosphopeptides by src-homology 2 domains.

SH2 domains and SH3 domains, found in a number of protein-tyrosine kinases and substrates of protein-tyrosine kinases, provide specificity in downstream signaling. Both of these domains bind to relatively short linear sequences of peptides to provide specific interactions between proteins. The SH2 domains directly bind to phosphotyrosine residues of proteins in a specific sequence context. We have devised a phosphopeptide library technique that allows us to rapidly determine the sequence specificity of individual SH2 domains on the basis of amino acids selected at position +1, +2 and +3 C-terminal of the phosphotyrosine. The optimal motif for 22 distinct SH2 domains has been determined and used to predict likely sites of in vivo interaction. A second phosphopeptide library was devised in which the amino acids N-terminal of the phosphotyrosine were also varied. The residues N-terminal of phosphotyrosine had little influence on binding to the N-SH2 domain of the 85 kDa subunit of phosphoinositide 3-kinase. These results indicate that for this SH2 domain, specificity is determined by sequences carboxy-terminal of the phosphotyrosine moiety. Knowledge of the specificity of SH2 domains allows predictions about likely downstream targets on the basis of primary sequence of proteins. Some of these predictions will be discussed.

Amino Acid Sequence↗

PubMed2002

Probing the phosphopeptide specificities of protein tyrosine phosphatases, SH2 and PTB domains with combinatorial library methods.

Protein tyrosine phosphatases, SH2 and PTB domains are crucial elements for cellular signal transduction and regulation. Much effort has been directed towards elucidating their specificity in the past decade using a variety of approaches. Combinatorial library methods have contributed significantly to the understanding of substrate and ligand specificity of phosphoprotein recognizing domains. This review gives a brief overview of the structural characteristics of protein tyrosine phosphatases, SH2 and PTB domains and their binding to phosphopeptides. The chemical synthesis of peptides containing phosphotyrosine or phosphotyrosine mimics and the various formats of synthesis and deconvolution of combinatorial libraries are explained in detail. Examples are given as how different combinatorial libraries have been used to study the interaction of phosphopeptides with SH2 domains and phosphatases. The intrinsic advantages and difficulties of library synthesis, screening and deconvolution are pointed out. Finally, some experimental results on the substrate specificity of protein tyrosine phosphatase 1B and the SH2 domain of the adaptor protein Grb-2 are summarized and discussed.

Binding Sites↗

PubMed2001

Selective inhibition of fibroblast growth factor (FGF)-stimulated mitogenesis by a FGF receptor-1-derived phosphopeptide.

The activated fibroblast growth factor receptor (FGFR)-1 is phosphorylated on five tyrosine residues outside the catalytic site. Although one such residue, Tyr730, is flanked by potential binding sites for phosphotyrosine-interacting molecules, a physiological role for this region is still controversial. We report that a cell-permeant phosphopeptide mimic of this site, FGFR730(p)Y, inhibits FGF-mediated mitogenesis in cells with no effect on responses stimulated by other growth factors. A similar phosphopeptide corresponding to the phospholipase Cgamma binding site on the receptor had no effect on the mitogenic response. The FGFR730(p)Y peptide did not inhibit phosphorylation of p90/FRS2 or Erk, suggesting that it does not act by inhibiting the Erk-kinase cascade. However, the FGFR730(p)Y peptide bound Shc in a manner requiring both phosphorylated tyrosine and a putative PTB domain binding determinant. These data suggest that the peptide might inhibit mitogenesis by competing with the corresponding site on the FGFR for the ability to bind SHC.

3T3 Cells↗

PubMed1976

Effects of epinephrine and insulin on phosphopeptide metabolism in adipocytes.

Isolated adipocytes, incubated in the presence of extracellular 32Pi to steady state 32P incorporation into cellular phosphopeptides, were exposed to hormones for 5 min. Epinephrine (10(-6) M) stimulated 32P incorporation into at least 12 major phosphopeptides, distributed in the cytoplasm, endoplasmic reticulum, and plasma membrane. Quantitatively pre-eminent among these were peptides of molecular weight 123,000 and 69,000, each located both in the cytoplasm and endoplasmic reticulum. The effect of epinephrine (10(-7) M) on 32P incorporation into these two peptides was augmented by theophylline (10(-3) M) in a synergistic fashion. Norepinephrine, dibutyryl N6,O2'-dibutyryl adenosine 3':5'-monophosphate, adrenocorticotropic hormone (ACTH) (synthetic 1 to 24 fragment), and glucagon mimicked the effect of epinephrine. Insulin modified adipocyte peptide phosphorylation in two ways. When present as the sole hormone, insulin (100 microunits/ml) consistently and selectively stimulated the 32P incorporation into a peptide of molecular weight 123,000 (endoplasmic reticulum, cytoplasm) without significant alteration in the 32P content of any other major peptide. A second effect of insulin was evident when epinephrine (10(-6) M) was present simultaneously. Insulin significantly inhibited the epinephrine-stimulated phosphorylation of the molecular weight 69,000 (endoplasmic reticulum, cytoplasm) and 26,000 (plasma membrane) peptides. Nevertheless, persistence of insulin-stimulated phosphorylation of the 123,000 peptide in the presence of epinephrine was shown by a 32P content of this peptide that was greater in the presence of both hormones than with either individually. These findings indicate that in intact adipocytes: (a) epinephrine acutely alters the phosphorylation of a large number of adipocyte peptides, partly at least, via activation of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase; (b) insulin opposes several epinephrine-stimulated phosphorylations in a manner consitent with its ability to lower epinephrine-stimulated intracellular cyclic AMP accumulation in adipocytes; and (c) insulin, in addition, exerts a unique stimulatory effect on adipocyte peptide phosphorylation that is independent of its effects on cyclic AMP metabolism and may be medicated by the generation of an as yet undefined intracellular "messenger" unique to insulin.

Adipose Tissue↗

PubMed1989

Mapping of a small phosphopeptide at the carboxyterminus of the viral myb protein by monoclonal antibodies.

Several myb-specific monoclonal antibodies were produced and their antigen recognition sites characterized using a series of bacterially expressed truncated myb proteins. The monoclonal antibodies were used for analysing the in vivo phosphorylation site of the oncogene protein from avian myeloblastosis virus (AMV), p48v-myb. The p48v-myb protein labeled metabolically with [32P]orthophosphate was isolated from the AMV-transformed chicken myeloblast cell line BM-2 by immunoaffinity chromatography. Phosphoamino acid analysis indicated that it was phosphorylated mainly on serine and to a lesser extent (less than 5%) on threonine residues. Indirect immunoprecipitation of phosphopeptides from trypsin-digested [32P]-labeled purified p48v-myb protein by use of the myb-specific monoclonal antibodies allowed the mapping of a small phosphopeptide at the carboxyterminus of p48v-myb.

Animals↗

PubMed1993

Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit.

Tyrosine-phosphorylated peptides based on the regions of polyoma virus middle t antigen and the platelet-derived growth factor receptor that bind phosphoinositide 3-kinase are shown to activate this enzyme 2-3-fold in vitro. The concentrations of the peptides required to activate the enzyme are at least 10-1000-fold higher than the dissociation constants of these peptides for the individual SH2 domains of the 85-kDa subunit (KD < 100 nM). Doubly phosphorylated peptides are more effective than singly phosphorylated peptides. The results suggest that a fraction of the cellular phosphoinositide 3-kinase has SH2 domains with relatively low affinity for phosphopeptides and that binding of phosphopeptides to these enzymes causes activation. Thus, SH2 domains may be involved not only in recruiting the enzyme but also in regulating activity.

Amino Acid Sequence↗

PubMed1994

Preparation and characterization of two phosphopeptide fractions from calf thymus nuclei; similarity to corresponding fractions in the DNA prepared from the nuclei.

In this study two phosphopeptide (PP) fractions from purified, calf thymus nuclei, prepared under protective conditions were characterized. The nuclei were lysed and dialyzed, and the material in the dialysates was fractionated by anion exchange column chromatography. This revealed the presence of two main phosphopeptide (PP) fractions, numbers 1 and 5. Amino acid analysis of the fraction 1, occurring in the greatest amount, showed that, after deionization, it contained the metal ion complexes of phosphoserine, aspartic and glutamic acids in large amounts, and nine other amino acids. In the same way the two main PP fractions, P1 and P5, were obtained during the dialysis of the EDTA-reacted, highly purified DNA (N-DNA) prepared from calf thymus nuclei. Fraction 1 from nuclei had practically the same amino acid composition as fraction P1 from EDTA-reacted N-DNA. In both cases, the second main fraction obtained (fraction 5) had nearly the same amino acid composition as fraction 1, but the total Pser molar ratio, was about twice as high as for fraction 1. In contrast to the low molecular weights of 900-1400 daltons observed for PPs isolated directly from the dialysates in water without exposure to HCOOH, the high molecular weight of 10,260 daltons was obtained for the PP fraction 1, after exposure to HCOOH used in the gradient for the column chromatography.

Amino Acids↗

PubMed1994

Characterization of protein tyrosine phosphatase SH-PTP2. Study of phosphopeptide substrates and possible regulatory role of SH2 domains.

The src homology 2 (SH2) domain containing protein-tyrosine-phosphatase SH-PTP2, was over-expressed in Escherichia coli for a kinetic study employing a set of synthetic 13- to 14-mer phosphopeptide substrates. The full-length SH-PTP2 protein, as well as a truncated form, lacking the two amino terminus SH2 domains (SH-PTP2(delta SH2)), exhibited Michaelis-Menten kinetics, and demonstrated striking substrate preferences on phosphopeptides having sequences based on sites of intracellular protein tyrosine phosphorylation. For example, while a KM of 59 microM and kcat/KM of 1.1 x 10(5) were obtained using SH-PTP2(delta SH2) and PDGFRY1021, a phosphorylation site within the platelet-derived growth factor receptor, other peptides revealed no detectable phosphate release. PDGFRY1009, modeled after a sequence identified as an in vivo binding site for SH-PTP2, was also a good substrate for this enzyme. The truncated form, lacking the SH2 domains demonstrated higher catalytic efficiency than the full-length enzyme. Interestingly, soluble SH2 domains were found to inhibit the catalytic activity of SH-PTP2 in a concentration-dependent manner. There was also evidence of a non-phosphotyrosine-mediated association between the two domains. These observations suggested that the SH2 domains have a direct role in regulating the catalytic activity of SH-PTP2.

Amino Acid Sequence↗