MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the 'hydrophobic motif' Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2.

NDRG1 is phosphorylated by SGK1 (but not PKB) in vivo at three residues each contained within three nonapeptide repeats. Here, we demonstrate that this nonapeptide, like the NDRG1 protein, is phosphorylated by SGK1, but not by PKBα or RSK1 in vitro. The inability of PKBα and RSK1 to phosphorylate the nonapeptide was traced to residues n + 1, n + 2 and n − 4 (where n is the phosphorylation site). Changing them from Ser, Glu and Ser to Phe, Ala and Pro, respectively, transformed the nonapeptide into an excellent substrate for PKBα and RSK1. Our results identify a specific substrate for SGK1 and may facilitate detection of additional physiological substrates for this enzyme.

Protein-tyrosine phosphatase-L1 (PTPL1, also known as FAP-1, PTP1E, PTP-BAS, and PTPN13) is mutated in a significant number of colorectal tumors and may play a role in down-regulating signaling responses mediated by phosphatidylinositol 3-kinase, although the precise substrates are as yet unknown. In this study, we describe a 1.8 Å resolution crystal structure of a fully active fragment of PTPL1 encompassing the catalytic domain. PTPL1 adopts the standard PTP fold, albeit with an unusually positioned additional N-terminal helix, and shows an ordered phosphate in the active site. Interestingly, a positively charged pocket is located near the PTPL1 catalytic site, reminiscent of the second phosphotyrosine binding site in PTP1B, which is required to dephosphorylate peptides containing two adjacent phosphotyrosine residues (as occurs for example in the activated insulin receptor). We demonstrate that PTPL1, like PTP1B, interacts with and dephosphorylates a bis-phosphorylated insulin receptor peptide more efficiently than monophosphorylated peptides, indicating that PTPL1 may down-regulate the phosphatidylinositol 3-kinase pathway, by dephosphorylating insulin or growth factor receptors that contain tandem phosphotyrosines. The structure also reveals that four out of five PTPL1 mutations found in colorectal cancers are located on solvent-exposed regions remote from the active site, consistent with these mutants being normally active. In contrast, the fifth mutation, which changes Met-2307 to Thr, is close to the active site cysteine and decreases activity significantly. Our studies provide the first molecular description of the PTPL1 catalytic domain and give new insight into the function of PTPL1.

We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by SGK1 (serum- and glucocorticoid-induced kinase 1), but not by protein kinase Ba, and identified it as NDRG2 (N-myc downstream-regulated gene 2). SGK1 phosphorylated NDRG2 at Thr330, Ser332 and Thr348 in vitro. All three residues were phosphorylated in skeletal muscle from wild-type mice, but not from mice that do not express SGK1. SGK1 also phosphorylated the related NDRG1 isoform at Thr328, Ser330 and Thr346 (equivalent to Thr330, Ser332 and Thr348 of NDRG2), as well as Thr356 and Thr366. Residues Thr346, Thr356 and Thr366 are located within identical decapeptide sequences GTRSRSHTSE, repeated three times in NDRG1. These threonines were phosphorylated in NDRG1 in the liver, lung, spleen and skeletal muscle of wild-type mice, but not in SGK1-/- mice. Knock-down of SGK1 in HeLa cells using small interfering RNA also suppressed phosphorylation of the threonine residues in the repeat region of NDRG1. The phosphorylation of NDRG1 by SGK1 transformed it into an excellent substrate for GSK3 (glycogen synthase kinase 3), which could then phosphorylate Ser342, Ser352 and Ser362 in the repeat region. Incubation of HeLa cells with the specific GSK3 inhibitor CT 99021 increased the electrophoretic mobility of NDRG1 in HeLa cells, demonstrating that this protein is phosphorylated by GSK3 in cells. Our results identify NDRG1 and NDRG2 as physiological substrates for SGK1, and demonstrate that phosphorylation of NDRG1 by SGK1 primes it for phosphorylation by GSK3.

We have carried out a solution-state NMR study of synthetic peptides patterned on the first membrane span of normal human band 3, and the same region of the mutant band 3 present in Southeast Asian ovalocytosis (SAO) which has a nine amino acid deletion. In 1:1 (v/v) chloroform/methanol, the 42 residue normal peptide (R389-K430) consisted of three helical regions. The slow solvent exchange of backbone amide protons revealed the helix from P403 to A416 was more stable than the “cytoplasmic” N-terminal helix from P391 to A400. These helices were separated by a sharp bend at P403, which is probably located at the boundary between the cytoplasmic domain and the first transmembrane span. The SAO deletion (A400-A408) removed the bend at P403, to leave a stable helix from P391 to A416 containing the residuum of the normal first transmembrane helix and with a hydrophobic turn replaced by a polar turn in the SAO peptide. Insertion of fragments of normal band 3 and band 3 SAO into microsomal membranes was investigated using a cell free translation system. A fragment composed of the cytoplasmic domain and the putative first membrane domain of normal band 3 (B3(1)) inserted stably into the membrane. However, the corresponding fragment of band 3 SAO [SAO(1)] did not integrate stably into membranes. Our results suggest that in SAO band 3, the region of the first membrane span of normal band 3 does not integrate properly into the membrane because it lacks a sufficiently long hydrophobic segment, and the deletion also disrupts a conserved structural subdomain at the membrane surface.

The synthesis of the Fmoc-protected C-glycosyl tyrosines 1 and 2, together with two other related C-glycosyl tyrosines, has been achieved. Key reactions involved (i) the reaction of a glycal with an organozinc reagent (carrying an aryl iodide function) in the presence of a Lewis acid to establish the C-glycosyl linkage and (ii) subsequent cross coupling of the aryl iodide to an alanyl zinc reagent (in the presence of a Pd(0) catalyst) to complete the construction of the alpha-amino acid moiety. Using solid-phase peptide synthesis methods, two units of the mannosyl derivative 1 (shown as L-Tyr[C-Ac(4)-alpha-D-Man]) have been incorporated (with four units of glycine) into the linear hexapeptide 3 which was then converted to the C(2)-symmetric cyclic oligopeptide 4.

The transmembrane sector of sarcoplasmic reticulum Ca 2+ ‐ATPase comprises ten putative transmembrane spans (M1−M10) in current topology models. We report here the structure and properties of three synthetic peptides with a single Trp representing the M6 and M7 regions implicated in Ca 2+ binding : peptide M6 (amino acid residues 785−810), peptide M7‐L (amino acid residues 808−847) corresponding to loop 6‐7 and the majority of span M7, and peptide M7‐S (amino acid residues 818−847) which contains a shorter version of loop 6‐7 than M7‐L. After uptake of the peptides in the hydrophobic environment of dodecyl maltoside micelles, the peptides gain a significant amount of secondary structure, as indicated by their CD spectra. However, the α‐helical content of M6 is lower than would be expected for a classical transmembrane segment. For M7‐L peptide, the L6‐7 loop is subject to specific proteolytic cleavage by proteinase K, as in intact Ca 2+ ‐ATPase. The formation of the peptide‐detergent complexes was followed from the resulting fluorescence intensity changes, either enhancement using n ‐dodecyl β‐ D ‐maltoside or quenching using the recently introduced brominated analog of n ‐dodecyl β‐ D ‐maltoside : 7,8‐dibromododecyl β‐maltoside [de Foresta, B., Legros, N., Plusquellec, D., le Maire, M. & Champeil, P. (1996) Eur. J. Biochem. 241 , 343−354]. Our results indicate that M7‐L and M7‐S are completely taken up by the detergent micelles. In contrast, the M6 peptide, which is highly water soluble, is more loosely associated with the detergent, as is also demonstrated by size‐exclusion chromatography. The location of Trp in micelles was evaluated from the quenching observed in mixed micelles of n ‐dodecyl β‐ D ‐maltoside/7,8‐dibromododecyl β‐maltoside, using tryptophan octyl ester and solubilized Ca 2+ ‐ATPase as reference compounds. We conclude that W832 in M7 appears to be located near the surface of the micelle, in agreement with its membrane interfacial localization predicted in most Ca 2+ ‐ATPase topology models. In contrast, our data suggest that W794 in M6 has a deeper insertion in the micelle although not to the extent predicted by current models of Ca 2+ ‐ATPase and the rather short α‐helix span of M6 may lead to exposure of a significant part of the C‐terminal of this peptide to the micelle surface. The results are discussed in relation to the proposed roles of these membrane segments in active transport of Ca 2+ ions, in particular, the demonstration that M6 does not behave as a classical transmembrane helix may be correlated with the evidence, from site‐directed mutagenesis, that this transmembrane segment should be essential in Ca 2+ binding.

The membrane domain of the human red cell anion transport protein, band 3, is too large to be studied by solution nuclear magnetic resonance spectroscopy (NMR), and its amphiphilic nature requires the use of detergents for solubilization. An alternative approach is to divide the protein into smaller (trans-membrane or surface loop) domains for NMR study. We report the structure of a 46-residue synthetic peptide that corresponds to the cytoplasmic surface loop connecting the putative 12th and 13th trans-membrane spans (residues 796-841) in the 14 span model of band 3. This peptide was shown by circular dichroism (CD) to be 38% helical in 30% trifluoroacetic acid. Two regions of helix (one close to the N-terminus of the peptide and one close to the C-terminus of the peptide) were identified by NMR. Long-range nuclear Overhauser effect (NOE) cross-peaks showed the two helices to be in near proximity. The helices were separated by a proline-rich loop that exhibited local order but was mobile with respect to the rest of the peptide. We discuss how the NMR structure of this loop fits the current models of band 3 structure and topology and the results of recent mutagenesis experiments. A cyclic version of this peptide was synthesized and studied by CD, but NMR studies were not possible due to the low solubility of this peptide.

Conference Article| August 01 1998 Studies on the structure of a transmembrane region and a cytoplasmic loop of the human red cell anion exchanger (band 3, AE1) E. J. Chambers; E. J. Chambers 1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K. Search for other works by this author on: This Site PubMed Google Scholar D. Askin; D. Askin 1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K. Search for other works by this author on: This Site PubMed Google Scholar G. B. Bloomberg; G. B. Bloomberg 1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K. Search for other works by this author on: This Site PubMed Google Scholar S. M. Ring; S. M. Ring 2 Search for other works by this author on: This Site PubMed Google Scholar M. J. A. Tanner M. J. A. Tanner 1 1To whom correspondence should be addressed. Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd Received: April 02 1998 Online ISSN: 1470-8752 Print ISSN: 0300-5127 © 1998 Biochemical Society1998 Biochem Soc Trans (1998) 26 (3): 516–520. https://doi.org/10.1042/bst0260516 Article history Received: April 02 1998 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation E. J. Chambers, D. Askin, G. B. Bloomberg, S. M. Ring, M. J. A. Tanner; Studies on the structure of a transmembrane region and a cytoplasmic loop of the human red cell anion exchanger (band 3, AE1). Biochem Soc Trans 1 August 1998; 26 (3): 516–520. doi: https://doi.org/10.1042/bst0260516 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: SAO, southeast Asian ovalcytosis, H2DIDS, di-isothiocyanato-dihydrostilbene disulphonate This content is only available as a PDF. © 1998 Biochemical Society1998 Article PDF first page preview Close Modal You do not currently have access to this content.

We have used circular dichroism to study synthetic peptides from two important regions of the prion protein: the N-terminal octa-repeat domain and a highly conserved hydrophobic section. Our results show that the octa-repeat sequence in free solution can adopt a non-random, extended conformation with properties similar to the poly-l-proline type II left-handed helix. We also show that the conformation can be changed by temperature, organic solvents (e.g. acetonitrile) and on binding to phospholipid vesicles. We compared CD data from two peptides corresponding to the hydrophobic region between residues 106 and 136 which contained either methionine or valine at position 129. This variation represents a common polymorphism in humans which has been shown to influence predisposition towards iatrogenic and sporadic CJD. There was no detectable difference between the CD spectra of these peptides irrespective of the solvent conditions we used.

We have studied the structures of synthetic peptides which correspond to the proposed first and second membrane-spanning segments of the human red cell anion transporter (band 3). The peptides, which were acetylated at their N-termini and amidated at the C-termini, comprise the 20 amino acids of residues 405-424 and 21 amino acids of residues 436-456 of the human band 3 sequence. The solution structures of the peptides in trifluoroethanol were studied by two-dimensional NMR spectroscopy. Characteristic NOEs were observed indicating that the peptides adopted a predominantly alpha-helical structure in trifluoroethanol solution. Dynamical simulated annealing using the program XPLOR was employed for the structure calculations. The amide exchange rates in trifluoroethanol have also been measured and are consistent with an alpha-helical structure for the peptides.