Regulation of the localisation and function of the oncogene LYRIC/AEG-1 by ubiquitination at K486 and K491.

The pivotal role of LYRIC/AEG-1 in malignant transformation, tumourigenesis and chemo-resistance has previously been demonstrated in different cell types and sub-cellular compartments. The localisation of LYRIC/AEG-1 appears crucial to its function and is regulated by three lysine-rich nuclear localisation signal regions, one of which was previously demonstrated to be modified by ubiquitin. Here we show that mutation of LYRIC/AEG-1 at K486 and K491 results in a loss of ubiquitination.

Functional proteomic analysis of long-term growth factor stimulation and receptor tyrosine kinase coactivation in Swiss 3T3 fibroblasts.

In Swiss 3T3 fibroblasts, long-term stimulation with PDGF, but not insulin-like growth factor 1 (IGF-1) or EGF, results in the establishment of an elongated migratory phenotype, characterized by the formation of retractile dendritic protrusions and absence of actin stress fibers and focal adhesion complexes. To identify receptor tyrosine kinase-specific reorganization of the Swiss 3T3 proteome during phenotypic differentiation, we compared changes in the pattern of protein synthesis and phosphorylation during long-term exposure to PDGF, IGF-1, EGF, and their combinations using 2DE-based proteomics after (35)S- and (33)P-metabolic labeling. One hundred and five differentially regulated proteins were identified by mass spectrometry and some of these extensively validated.

Site recognition and substrate screens for PKN family proteins.

The PRKs [protein kinase C-related kinases; also referred to as PKNs (protein kinase Ns)] are a kinase family important in diverse functions including migration and cytokinesis. In the present study, we have re-evaluated and compared the specificity of PKN1 and PKN3 and assessed the predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and PKN3 phosphorylate serine residues in sequence contexts that have an arginine residue in position -3.

Mammal-restricted elements predispose human RET to folding impairment by HSCR mutations.

The maturation of human RET is adversely affected by a range of missense mutations found in patients with Hirschsprung's disease (HSCR), a complex multigenic disease. Here we show that two N-terminal cadherin-like domains, CLD1 and CLD2 (CLD(1-2)), from human RET adopt a clam-shell arrangement distinct from that of classical cadherins. CLD1 structural elements and disulfide composition are unique to mammals, indicating an unexpected structural diversity within higher and lower vertebrate RET CLD regions.

Involvement of heterogeneous ribonucleoprotein F in the regulation of cell proliferation via the mammalian target of rapamycin/S6 kinase 2 pathway.

The S6 kinases (S6Ks) have been linked to a number of cellular processes, including translation, insulin metabolism, cell survival, and RNA splicing. Signaling via the phosphotidylinositol 3-kinase and mammalian target of rapamycin (mTOR) pathways is critical in regulating the activity and subcellular localization of S6Ks. To date, nuclear functions of both S6K isoforms, S6K1 and S6K2, are not well understood.

The identification and characterization of novel PKCepsilon phosphorylation sites provide evidence for functional cross-talk within the PKC superfamily.

PKCepsilon (protein kinase Cepsilon) is a phospholipid-dependent serine/threonine kinase that has been implicated in a broad array of cellular processes, including proliferation, survival, migration, invasion and transformation. Here we demonstrate that, in vitro, PKCepsilon undergoes autophosphorylation at three novel sites, Ser(234), Ser(316) and Ser(368), each of which is unique to this PKC isoform and is evolutionarily conserved. We show that these sites are phosphorylated over a range of mammalian cell lines in response to a number of different stimuli.

Novel phosphorylation site markers of protein kinase C delta activation.

Protein kinase C delta (PKCdelta) is a Ser/Thr kinase which regulates numerous cellular processes, including proliferation, differentiation, migration and apoptosis. Here, we demonstrate that PKCdelta undergoes in vitro autophosphorylation at three sites within its V3 region (S299, S302, S304), each of which is unique to this PKC isoform and evolutionarily conserved. We demonstrate that S299 and S304 can be phosphorylated in mammalian cells following phorbol ester stimulation and that S299-phosphorylated PKCdelta is localised to both the plasma and nuclear membranes.

Labeling and identification of LNCaP cell surface proteins: a pilot study.

Background: Membrane proteins provide the interface between the cell and its environment and are responsible for cell adhesion, mobility, and intracellular signaling. Previous studies have focused on the LNCaP whole cell proteome and transcriptome but little is known about proteins at the prostate cell membrane and how they change in response to androgens.

Materials And Methods: Following treatment with R1881 or vehicle, membrane proteins of the prostate cancer LNCaP cell line were tagged with biotin using EZ-link sulfo-NHS-LC-biotin.

PLCgamma is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly.

Nuclear envelope assembly is an essential event in each cell cycle but the proteins and lipids involved in its regulation remain mostly unknown. Assembly involves membrane fusions but neither specific SNAREs nor Rab GTPases have been identified in its control. We report that a precursor membrane population (MV1) required for NE assembly has a unique lipid composition consisting prominently of poly-phosphatidylinositides.

Proteomic identification of a role for the von Hippel Lindau tumour suppressor in changes in the expression of mitochondrial proteins and septin 2 in renal cell carcinoma.

The von Hippel Lindau (VHL) tumour suppressor gene, VHL, plays a central role in development of sporadic conventional renal cell carcinomas (RCCs). Studying VHL function may, therefore, increase understanding of the pathogenesis of RCC and identify markers/therapeutic targets. Comparison of 2-DE protein profiles of VHL-defective RCC cells (UMRC2) transfected with control vector or wild-type VHL showed differences in 30 proteins, including several novel changes.

Proteomic analysis of primary cell lines identifies protein changes present in renal cell carcinoma.

New markers/targets for renal cell carcinoma (RCC) are needed to enable earlier detection and monitoring of disease and therapeutic targeting. To identify such molecules, normal and RCC-derived primary cell lines have been used as a simplified model system for studying changes that accompany tumorigenesis. Short-term cultures allow enrichment of relevant cell types from tissue samples, which is balanced against the potential for in vitro changes.

RhoE function is regulated by ROCK I-mediated phosphorylation.

The Rho GTPase family member RhoE regulates actin filaments partly by binding to and inhibiting ROCK I, a serine/threonine kinase that induces actomyosin contractility. Here, we show that ROCK I can phosphorylate multiple residues on RhoE in vitro. In cells, ROCK I-phosphorylated RhoE localizes in the cytosol, whereas unphosphorylated RhoE is primarily associated with membranes.

Androgen receptor is targeted to distinct subcellular compartments in response to different therapeutic antiandrogens.

Purpose: Antiandrogens are routinely used in the treatment of prostate cancer. Although they are known to prevent activation of the androgen receptor (AR), little is known about the mechanisms involved. This report represents the first study of the localization of wild-type AR following expression at physiologic relevant levels in prostate cells and treatment with androgen and antiandrogens.

Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

Exosomes are 40-100 nm vesicles released by numerous cell types and are thought to have a variety of roles depending on their origin. Exosomes derived from antigen presenting cells have been shown to be capable of initiating immune responses in vivo and eradicating established tumours in murine models. Tumour-derived exosomes can be utilised as a source of tumour antigen for cross-priming to T-cells and are thus of interest for use in anti-tumour immunotherapy.

Identification of proteins regulated by interferon-alpha in resistant and sensitive malignant melanoma cell lines.

Treatment of patients with malignant melanoma with interferon-alpha achieves a response in a small but significant subset of patients. Currently, although much is known about interferon biology, little is known about either the particular mechanisms of interferon-alpha activity that are crucial for response or why only some patients respond to interferon-alpha therapy. Two melanoma cell lines (MeWo and MM418) that are known to differ in their response to the antiproliferative activity of interferon-alpha, have been used as a model system to investigate interferon-alpha action.

Identification of pro-interleukin 16 as a novel target of MAP kinases in activated T lymphocytes.

T lymphocyte activation is controlled by a coordinated web of tyrosine and serine kinases. There is a large body of information about tyrosine kinase substrates in T cells but analysis of serine kinase substrates has been more difficult. Recently we described an antiserum that recognizes serine-phosphorylated peptides corresponding to the substrate sequences for AGC serine kinases.

Proteomic changes in renal cancer and co-ordinate demonstration of both the glycolytic and mitochondrial aspects of the Warburg effect.

Renal cell carcinoma (RCC) is the tenth most common cancer although the incidence is increasing. The main clinical problems stem from the relatively late presentation of many patients due to the often asymptomatic nature of the illness, and the relative insensitivity of metastatic disease to conventional chemotherapy and radiotherapy. Despite increasing knowledge of some of the genetic changes underlying sporadic renal cancer such as those involving the Von Hippel Lindau (VHL) gene, many of the underlying pathophysiological changes are ill-defined and there remains a need for the identification of disease markers for use in diagnosis and prognosis or as potential therapeutic targets.

Approaches to define antigen receptor-induced serine kinase signal transduction pathways.

In the present report we describe the properties of a novel phospho-specific antiserum that has opened a route to the characterization of antigen receptor-activated serine kinase pathways in lymphocytes. The basis for the present work was that Ser-21 in glycogen synthase kinase 3alpha is robustly phosphorylated following antigen receptor triggering. We predicted accordingly that antigen receptors would also stimulate phosphorylation of other proteins with a similar sequence.

Spatially specific expression of Hoxb4 is dependent on the ubiquitous transcription factor NFY.

Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. We have performed a detailed analysis of the mouse Hoxb4 intron enhancer to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance.

Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue preparation and sample limitations.

Laser capture microdissection (LCM) is now well established as a tool for facilitating the enrichment of cells of interest from tissue sections, overcoming the problem of tissue heterogeneity. LCM has been used extensively in combination with analysis at the DNA and RNA levels, but only a small number of studies have employed LCM with subsequent protein analysis, albeit with promising results. This study focuses on the potential of LCM in combination with two-dimensional polyacrylamide gel electrophoresis.