Patients with small cell lung cancer (SCLC) die because of chemoresistance. Fibroblast growth factor‐2 (FGF‐2) increases the expression of antiapoptotic proteins, XIAP and Bcl‐XL, and triggers chemoresistance in SCLC cells. Here we show that these effects are mediated through the formation of a specific multiprotein complex comprising B‐Raf, PKCε and S6K2. S6K1, Raf‐1 and other PKC isoforms do not form similar complexes. RNAi‐mediated downregulation of B‐Raf, PKCε or S6K2 abolishes FGF‐2‐mediated survival. In contrast, overexpression of PKCε increases XIAP and Bcl‐XL levels and chemoresistance in SCLC cells. In a tetracycline‐inducible system, increased S6K2 kinase activity triggers upregulation of XIAP, Bcl‐XL and prosurvival effects. However, increased S6K1 kinase activity has no such effect. Thus, S6K2 but not S6K1 mediates prosurvival/chemoresistance signalling.

A novel ribosomal S6 kinase, termed p70 S6 kinase β (p70β), which has a highly conserved amino acid sequence compared with that of p70/p85 S6 kinase (p70α) within the catalytic, kinase extension, and autoinhibitory pseudosubstrate domains, was identified. However, the amino acid sequence of p70β differs from that of p70α in the noncatalytic amino-terminal region and in the carboxyl-terminal tail, which contains a proline-rich region. The majority of the regulatory phosphorylation sites identified in p70α are conserved in p70β. Two isoforms of p70β, referred to as β1 (495 amino acids) and β2 (482 amino acids), could be expressed from the single gene either by alternative mRNA splicing or by the use of alternative start codons. Here we report the characterization of p70β2. Similarly to p70α, the catalytic activity of p70β toward ribosomal protein S6 could be rapidly activated by serum, insulin, and phorbol ester in transiently transfected cells. The p70β kinase was found to be significantly less sensitive to wortmannin and rapamycin than p70α. These results indicate that p70β has the potential to participate in the regulation of protein synthesis and the cell cycle.

Class IA phosphatidylinositol 3‐kinase (PI 3‐kinase) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, Rukl, which forms complexes with the PI 3‐kinase holoenzyme in vitro and in vivo . This interaction involves the proline‐rich region of Ruk and the SH3 domain of the p85α regulatory subunit of the class IA PI 3‐kinase. In contrast to many other adaptor proteins that activate PI 3‐kinase, interaction with Rukl substantially inhibits the lipid kinase activity of the enzyme. Overexpression of Rukl in cultured primary neurons induces apoptosis, an effect that could be reversed by co‐expression of constitutively activated forms of the p110α catalytic subunit of PI 3‐kinase or its downstream effector PKB/Akt. Our data provide evidence for the existence of a negative regulator of the PI 3‐kinase signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between Ruk, CIN85 and CD2AP/CMS suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions.

NY‐CO‐58/KIF2C has been identified as a tumor antigen by screening antibody responses in patients with colorectal cancer. However, expression had not consequently been examined, and nothing was known about its ability to induce spontaneous T cell responses, which have been suggested to play a role in the development of colorectal cancer. We analyzed 5 colorectal cancer cell lines, and tumor samples and adjacent healthy tissues from 176 patients with epithelial cancers for the expression of NY‐CO‐58/KIF2C by RT‐PCR and Western Blot. T cell responses of 43 colorectal cancer patients and 35 healthy donors were evaluated by ELISpot following stimulation with 30mer peptides or full‐length protein. All cell lines and tumor samples from colorectal cancer patients expressed NY‐CO‐58/KIF2C on the protein and RNA level, and expression levels correlated strongly with Ki‐67 expression (r = 0.69; p = 0.0003). Investigating NY‐CO‐58/KIF2C‐specific T cell responses, CD8⁺ T cells directed against 1 or more peptides were found in less than 10% of patients, whereas specific CD4⁺ T cells were detected in close to 50% of patients. These T cells were of high avidity, recognized the naturally processed antigen and secreted IFN‐γ and TNF‐α. Depletion of CD4⁺CD25⁺ T cells before stimulation significantly increased the intensity of the preexisting response. NY‐CO‐58/KIF2C is significantly overexpressed in colorectal and other epithelial cancers and expression levels correlate with the proliferative activity of the tumor. Importantly, NY‐CO‐58/KIF2C was able to induce spontaneous CD4⁺ T cell responses of the Th1‐type, which were tightly controlled by peripheral T regulatory cells.

In all living organisms, coenzyme A (CoA) is an essential cofactor with a unique design allowing it to function as an acyl group carrier and a carbonyl-activating group in diverse biochemical reactions. It is synthesized in a highly conserved process in prokaryotes and eukaryotes that requires pantothenic acid (vitamin B5), cysteine and ATP. CoA and its thioester derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. A novel unconventional function of CoA in redox regulation has been recently discovered in mammalian cells and termed protein CoAlation. Here, we report for the first time that protein CoAlation occurs at a background level in exponentially growing bacteria and is strongly induced in response to oxidizing agents and metabolic stress. Over 12% of Staphylococcus aureus gene products were shown to be CoAlated in response to diamide-induced stress. In vitro CoAlation of S. aureus glyceraldehyde-3-phosphate dehydrogenase was found to inhibit its enzymatic activity and to protect the catalytic cysteine 151 from overoxidation by hydrogen peroxide. These findings suggest that in exponentially growing bacteria, CoA functions to generate metabolically active thioesters, while it also has the potential to act as a low-molecular-weight antioxidant in response to oxidative and metabolic stress.