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PubMed1992

Effect of casein, casein phosphopeptides and calcium intake on ileal 45Ca disappearance and temporal systolic blood pressure in spontaneously hypertensive rats.

Paracellular 45Ca absorption and temporal systolic blood pressure (SBP) measurements were recorded in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats fed on casein (C) and soya-bean-protein isolate (S) diets, containing 20 (H), 5 (H) and 0.5 (L) g Ca/kg. Similar measurements were also taken in SHR rats only fed on C-M and S-M diets supplemented with 30 g caseinophosphopeptides (CPP)/kg. Absorption of 45Ca from the ileal loop was equivalent in both SHR and WKY animals and largely affected by the level of dietary Ca. In addition, animals fed on C diets exhibited significantly (P < 0.05) greater ileal absorption of 45Ca compared with S-fed animals. This result was attributed to the presence of CPP and a greater (P < 0.05) proportion of soluble 45Ca in the contents of the ileum. Animals fed on S diets supplemented with CPP confirmed this finding. The SBP of SHR rats was higher (P < 0.01) than WKY controls after 9-10 weeks of age. The temporal pattern of observed hypertension was independent of dietary influence in the SHR. The severity of hypertension in SHR rats was affected only by dietary Ca deficiency, and not by Ca supplementation or CPP enhancement of Ca bioavailability. These findings suggest that tryptic digestion products of casein in milk can enhance Ca bioavailability by increasing Ca solubility; however, this action had no effect in reducing hypertension in SHR.

Animals↗

PubMed2002

High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site.

3-phosphoinositide dependent protein kinase-1 (PDK1) plays a key role in regulating signalling pathways by activating AGC kinases such as PKB/Akt and S6K. Here we describe the 2.0 A crystal structure of the PDK1 kinase domain in complex with ATP. The structure defines the hydrophobic pocket termed the "PIF-pocket", which plays a key role in mediating the interaction and phosphorylation of certain substrates such as S6K1. Phosphorylation of S6K1 at its C-terminal PIF-pocket-interacting motif promotes the binding of S6K1 with PDK1. In the PDK1 structure, this pocket is occupied by a crystallographic contact with another molecule of PDK1. Interestingly, close to the PIF-pocket in PDK1, there is an ordered sulfate ion, interacting tightly with four surrounding side chains. The roles of these residues were investigated through a combination of site-directed mutagenesis and kinetic studies, the results of which confirm that this region of PDK1 represents a phosphate-dependent docking site. We discuss the possibility that an analogous phosphate-binding regulatory motif may participate in the activation of other AGC kinases. Furthermore, the structure of PDK1 provides a scaffold for the design of specific PDK1 inhibitors.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed1989

Solid-phase synthesis of phosphopeptides.

We report the solid-phase synthesis of peptides containing O-phosphoserine. Coupling was with commercially available Fmoc-amino acid pentafluorophenyl esters, with base used at each cycle to cleave Fmoc. Phosphorylation of those serine residues left unprotected on the peptide-resin was achieved with dibenzylphosphochloridate, and finally trifluoroacetic acid was used to remove side-chain protecting groups (including the benzyl groups used for the phosphate), and to cleave the peptide from the resin in the same step. This synthetic strategy enables the preparation of peptides with individual, selectively phosphorylated residues. Alternative approaches to introduce protected phosphate and continue with coupling of further amino acids were less advantageous due to the lability of the phosphate group to base and to steric hindrance.

Amino Acid Sequence↗

PubMed1981

Analysis of site occupancies in [32P]phosphorylated pyruvate dehydrogenase complexes by aspartyl-prolyl cleavage of tryptic phosphopeptides.

Activity of mammalian pyruvate dehydrogenase complexes from all sources so far tested is regulated by phosphorylation involving three sites. To facilitate understanding of the precise biological roles of the individual phosphorylation sites a method has now been developed, using pig heart [32P]phosphorylated complexes, which enables unambiguous measurement of their occupancies. Established methods of tryptic digestion give two peptides that contain the three phosphorylation sites: TA contains sites 1 and 2; TB contains site 3. Thus, while occupancy of site 3 may be determined unequivocally by tryptic digestion, occupancies of sites 1 and 2 cannot. The present paper shows that peptide TA may be specifically and quantitatively cleaved by formic acid at an Asp-Pro bond located between the two phosphorylation sites. Equal amounts of two new peptides each containing a different phosphorylation site (site 1 or site 2) are produced. The 32P-labelled peptides may be completely separated and quantified by high-voltage paper electrophoresis at pH 2. A combination of tryptic digestion (determination of 32P in site 3) and formic acid cleavage of peptide TA (determination of 32P in sites 1 and 2) thus enables unequivocal assignment of occupancies of individual phosphorylation sites to greater than 95% accuracy. This method has been used to show that during phosphorylation and inactivation of pig heart complexes (inactivated to between 1.5 % and 90%) greater than 98% of the observed inactivation was primarily attributable to phosphorylation of site 1; the contribution of site 2 was less than 2% if at all. Relative initial rates of phosphorylation site 1 X site 2 X site 3 were approximately 90:3:1.

Animals↗

PubMed1997

Identification of phosphopeptide ligands for the Src-homology 2 (SH2) domain of Grb2 by phage display.

We report here on the identification of phosphopetide ligands which interact with the Src-homology 2 (SH2) domain of the adapter protein Grb2 by screening a random peptide library established on phage. Phage were phosphorylated in vitro at an invariant tyrosine residue by a mixture of phosphotyrosine kinases c-Src, Blk and Syk. Selection of binding motifs was carried out by interaction of the library with the recombinant SH2 domain of Grb2 expressed as a glutathione S-transferase (GST) fusion protein. Several subsequent cycles of selection led to the enrichment of phage which bound to the GST-Grb2 SH2 domain only when previously phosphorylated. Sequence analysis revealed that all of the selected phage displayed peptides with the consensus motif Y*M/ENW (Y* denotes phosphotyrosine). One of these peptides, bearing the Y*ENW motif, bound the Grb2 SH2 domain with a threefold higher affinity than the peptide motif Y*VNV derived from the natural ligand Shc. Thus, phage display can be employed to rapidly identify high affinity ligands to SH2 domains.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Phosphoinositide, phosphopeptide and pyridone interactions of the Abl SH2 domain.

Signaling proteins are localized and regulated by Src homology 2 domains which recognize phosphotyrosine-containing sequences. Recently, noncanonical ligands have been proposed for Src homology 2 domains including that of Abl and its breakpoint cluster region fusion, which causes chronic myelogenous leukemia. Here, the Abl Src homology 2 domain's binding sites and affinities for phosphotyrosine- and phosphoserine-containing motifs, phosphoinositides as well as a pyridone-based peptidomimetic inhibitor were determined using nuclear magnetic resonance spectroscopy in order to define their roles. The cognate Crk peptide ligand was bound with an affinity of 69 microM and, like the higher affinity peptidomimetic, engages the phosphotyrosine and +3 hydrophobic pockets while putative phosphoserine-containing breakpoint cluster region ligands are ruled out. Surprisingly, phosphatidylinositol 4, 5 bisphosphate interacts with an overlapping site through an electrostatic mechanism that does not appear to involve hydrophobic insertion into micelles. The conserved Arg36 residue in the FLVRES motif is required for both phosphotyrosine binding and for localization to phosphatidylinositol 4, 5 bisphosphate-containing liposomes, while Arg59 in the betaD strand is necessary for the phosphoinositide interaction. Thus the Src homology 2 domain of Abl, a myristoylated and membrane-localized protein, is able to interact directly with phosphoinositides through a multifunctional basic site that overlaps the phosphotyrosine pocket.

Animals↗

PubMed1958