Graham B. Bloomberg

Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3

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.

Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity

The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration.

Synthesis of a branched cyclic peptide using a strategy employing Fmoc chemistry and two additional orthogonal protecting groups

Abstract A novel branched cyclic peptide has been made by solid-phase synthesis (Fmoc/t-Bu chemistry) using two additional orthogonal strategies (Allyl and Dde) and resin-bound cyclization.

Identification of different specificity requirements between SGK1 and PKBα

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.

The Development and Use of Phospho-Specific Antibodies to Study Protein Phosphorylation

The reversible phosphorylation of proteins is a key mechanism whereby signalling cascades involved in the response to extracellular stimuli bring about changes in cellular function. These proteins include the kinases/phosphatases that form such signaling pathways as well as the transcription factors involved in inducible changes in gene expression (1). Phosphorylation induces changes in the function of these proteins either by induction of allosteric conformational changes in the protein itself or in the regulation of its interaction with other cellular factors.

Conformational properties of the prion octa-repeat and hydrophobic sequences

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.