Proteomic analysis of an imatinib-resistant K562 cell line highlights opposing roles of heat shock cognate 70 and heat shock 70 proteins in resistance

Understanding the molecular basis of resistance to imatinib, a tyrosine kinase inhibitor used as front-line therapy in chronic myeloid leukemia, remains a challenge for successful treatment. In an attempt to identify new mechanisms of resistance, we performed a comparative proteomic analysis of an imatinib-resistant cell line generated from the erythroblastic cell line K562 (K562-r) for which no known mechanism of resistance has been detected. Bidimensional gel electrophoresis was carried out to compare the protein expression pattern of imatinib-sensitive and of imatinib-resistant K562 cells. Among the 400 matched spots on five pairs of gels, only 14 spots had a significantly increased or decreased expression leading to the identification of 24 proteins identified as scaffold proteins, metabolic enzymes, DNA translation and maturation, and chaperon proteins. Among the chaperon family, only Hsp70 and Hsc70 are overexpressed in K562-r, results confirmed by Western blotting. We recently reported the participation of Hsp70 overexpression in imatinib resistance whereas a role for Hsc70 has yet to be determined. Hsc70 is not involved in imatinib resistance as the inhibition of its expression by siRNA does not restore sensitivity to imatinib. In contrast, the induced decreased expression of Hsc70 was accompanied by a greater overexpression of Hsp70. This proteomic study therefore suggests opposing roles of Hsp70 and Hsc70 in imatinib resistance.     

Fungal virulence studies come of age

Sophisticated molecular biological research has revealed many virulence attributes in at least four pathogenic fungi, but the future study of fungal virulence requires investigators to distinguish between molecules that directly interact with the host, molecules that regulate these, and molecules that are always required for fungal growth and survival, independent of the host.     

Transduction cascades initiated by fibroblast growth factor 1 on Xenopus oocytes expressing MDA-MB-231 mRNAs. Role of Grb2, phosphatidylinositol 3-kinase, Src tyrosine kinase, and phospholipase Cgamma

Xenopus oocytes expressing fibroblast growth factor receptors (FGFRs) from the hormone-independent breast cancer cells, MDA-MB-231, are used as a biological system to analyze the signalling cascades initiated by FGF1. FGF1 induces ERK2 phosphorylation and G2/M transition. These events are dependent on the Shc/Grb2/Ras pathway, on Src and PI3Kinase (PI3K), as shown by the use of SH2 domains or dominant negative proteins, and on PLC gamma and calcium as demonstrated by a PLC gamma inhibitory peptide and BAPTA-AM. FGF1 mobilizes Ins(1,4,5)P3-sensitive calcium stores, as recorded through the inhibition by caffeine of a chloride calcium-dependent current in expressing oocytes. This study shows that the transduction cascades induced by FGF1 on FGFRs from MDA-MB-231 cells represent the sum of Ras, Src, PI3K, and PLC gamma pathways. It emphasizes the mitogenic effect of the PLC gamma-calcium cascade.     

c-Mos proteolysis is independent of the CA(2+) rise induced by 6-DMAP in Xenopus oocytes

In Xenopus oocytes, metaphase II arrest is due to a cytostatic factor (CSF) that involves c-Mos, maintaining a high MPF (cdk1/cyclin B) activity in the cell. At fertilization, a rise in intracellular calcium triggers the proteolysis of both cyclin B and c-Mos. The kinase inhibitor 6-dimethylaminopurine (6-DMAP) is also able to release matured Xenopus oocytes from metaphase II block. This is characterized by c-Mos proteolysis without degradation of cyclin B. We hypothesized that 6-DMAP induced an increase in intracellular calcium. Using the calcium-sensitive fluorescent dye Fura-2, we observed a systematic increase in intracellular calcium following 6-DMAP application. In matured oocytes previously microinjected with the calcium chelator BAPTA, no calcium changes occurred after 6-DMAP addition; however, c-Mos was still proteolysed. In oocytes at the GVBD stage, c-Mos proteolysis occurred in response to 6-DMAP but not to calcium ionophore treatment. We suggest that c-Mos proteolysis is rather controlled by a phosphorylation-dependent process.     

A non-canonical Grb2-PLC-gamma1-Sos cascade triggered by lipovitellin 1, an apolipoprotein B homologue

The injection of the Grb2 adapter in Xenopus oocytes promotes G2/M transition without stimulation from a receptor only the first day after the oocytes removal from the ovaries. This cell cycle reinitiation is Ras-dependent and requires the SH2 and SH3 domains of Grb2. The SH2 domain of Grb2 binds the tyrosine phosphorylated lipovitellin1, a homologue of the human apolipoprotein B. The N-SH3 domain of Grb2 is linked to a proline-rich sequence of the C2 domain of PLC-γ1, PLC-γ1 itself is linked, through its SH3 domain, to the C-terminal proline-rich region of Sos. When Grb2–PLC-γ1–Sos is associated, PLC-γ1 is not phosphorylated on Y783 but shows a phospholipase activity. Inhibition of lipovitellin 1 or PLC-γ1 avoids Grb2-induced cell cycle reinitiation. Therefore, the Grb2–lipovitellin 1 association is the starting point of a novel signaling pathway, where PLC-γ1 binds Grb2 and recruits Sos.     

Integrating sequence and structural biology with DAS

Background  

The Distributed Annotation System (DAS) is a network protocol for exchanging biological data. It is frequently used to share annotations of genomes and protein sequence.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.