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Phosphatase Treatment (phosphatase + treatment)
Selected AbstractsPhosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometryFEBS JOURNAL, Issue 8 2001Cyrille Garnier HSP90 is one of the most abundant proteins in the cytosol of eukaryotic cells. HSP90 forms transient or stable complexes with several key proteins involved in signal transduction including protooncogenic protein kinases and nuclear receptors, it interacts with cellular structural elements such as actin-microfilament, tubulin-microtubule and intermediate filaments, and also exhibits conventional chaperone functions. This protein exists in two isoforms ,-HSP90 and ,-HSP90, and it forms dimers which are crucial species for its biological activity. PAGE, ESI-MS and MALDI-MS were used to study HSP90 purified from pig brain. The two protein isoforms were clearly distinguished by ESI-MS, the , isoform being ,,six times more abundant than the , isoform. ESI-MS in combination with ,,phosphatase treatment provided direct evidence of the existence of four phosphorylated forms of native pig brain ,-HSP90, with the diphosphorylated form being the most abundant. For the , isoform, the di-phosphorylated was also the most abundant. MALDI mass spectra of HSP90 samples after chemical cross-linking showed a high percentage of ,,, homodimers. In addition, evidence for the existence of higher HSP90 oligomers was obtained. [source] Molecular cloning and expression of Tenebrio molitor ultraspiracle during metamorphosis and in vivo induction of its phosphorylation by 20-hydroxyecdysoneINSECT MOLECULAR BIOLOGY, Issue 3 2000M. Nicolaļ Abstract Using a RT-PCR approach, the Tenebrio molitor homologue of Drosophila Ultraspiracle (TmUSP) was characterized. Its DNA binding domain shows a degree of identity with those of the other insect USPs. However, the ligand binding domain is closer to those of retinoid X receptors. Using an antibody raised against DmUSP, Western blot analysis of proteins from epidermis and other tissues revealed five immunoreactive bands, corresponding to different phosphorylated forms of a unique polypeptide, as shown by ,-phosphatase treatment. The nuclear form of TmUSP seems unphosphorylated. An in vivo 20-hydroxyecdysone treatment increases considerably and rapidly the phosphorylated forms of TmUSP. This post-translational modification may play a role in the 20-hydroxyecdysone response. [source] Microtubule-associated protein tau in human prostate cancer cells: Isoforms, phosphorylation, and interactions,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2009Skye Souter Abstract Tau is a microtubule-associated protein whose function has been investigated primarily in neurons. Recently, tau expression has been correlated with increased drug resistance in various cancers of non-neuronal tissues. In this report, we investigate the tau expressed in cancerous prostate lines ALVA-31, DU 145, and PC-3. Prostate cancer tau is heat-stable and highly phosphorylated, containing many of the modifications identified in Alzheimer's disease brain tau. RT-PCR and phosphatase treatment indicated that all six alternatively spliced adult brain tau isoforms are expressed in ALVA-31 cells, and isoforms containing exon 6 as well as high molecular weight tau isoforms containing either exon 4A or a larger splice variant of exon 4A are also present. Consistent with its hyperphosphorylated state, a large proportion of ALVA-31 tau does not bind to microtubules, as detected by confocal microscopy and biochemical tests. Finally, endogenous ALVA-31 tau can interact with the p85 subunit of phosphatidylinositol 3-kinase, as demonstrated by co-immunoprecipitations and in vitro protein-binding assays. Our results suggest that tau in prostate cancer cells does not resemble that from normal adult brain and support the hypothesis that tau is a multifunctional protein. J. Cell. Biochem. 108: 555,564, 2009. © 2009 Wiley-Liss, Inc. [source] Rice sucrose-phosphate synthase: Identification of an isoform specific for heterotrophic tissues with distinct metabolite regulation from the mature leaf enzymePHYSIOLOGIA PLANTARUM, Issue 4 2000Gabriela C. Pagnussat Immunohistological analyses for rice (Oryza sativa) sucrose-phosphate synthase (SPS, UDP-glucose d -fructose-6-phosphate-2-glucosyltransferase, EC 2.4.1.14) show that the protein is differently localized in photosynthetic and etiolated leaves. Very little is known about SPS regulation in heterotrophic tissues; therefore, we studied the biochemical properties of the enzyme from etiolated seedlings and embryo. Two SPS forms (SPS-1 and SPS-2) were partially purified from etiolated seedlings. The effects of Glc-6-P (activator) and Pi (inhibitor) on SPS activities allowed us to differentiate the two forms. SPS-1 showed high sensitivity to Pi which also strongly decreased enzyme activation by Glc-6-P. SPS-2 was highly activated by Glc-6-P and showed low sensitivity to Pi. In vitro alkaline phosphatase treatment suggested that SPS-1 could be regulated as leaf SPS in darkness and that SPS-2 is present in a dephosphorylated state or is not regulated by protein phosphorylation. The relative MM value (116 kDa) estimated for both SPS forms in SDS-PAGE is identical to the rice leaf SPS polypeptide. Taken together, these data led us to conclude that SPS-2 is an enzyme form only present in non-photosynthetic tissues. [source] Local energetic regulation of sarcoplasmic and myosin ATPase is differently impaired in rats with heart failureTHE JOURNAL OF PHYSIOLOGY, Issue 21 2008Frederic Joubert Local control of ATP/ADP ratio is essential for efficient functioning of cellular ATPases. Since creatine kinase (CK) activity and mitochondrial content are reduced in heart failure (HF), and cardiomyocyte ultrastructure is altered, we hypothesized that these changes may affect the local energetic control of two major cardiac ATPases, the sarcoplasmic reticulum (SR) Ca2+ -ATPase (SERCA) and the myosin ATPase. Heart failure was induced by aortic stenosis in rats. Electron microscopy confirmed that failing cardiomyocytes had intracellular disorganization, with fewer contacts between mitochondria and myofibrils. Despite normal SERCA protein content, spontaneous Ca2+ release measurements using Fluo-4 on saponin-permeabilized cardiomyocytes showed a lower SR loading in HF even when endogenous CK and mitochondria were fully activated. Similarly, in permeabilized fibres, SR Ca2+ loading supported by SR-bound CK and mitochondria was significantly reduced in HF (by 49% and 40%, respectively, 43% when both systems were activated, P < 0.05). Alkaline phosphatase treatment had no effect, but glycolytic substrates normalized calcium loading in HF to the sham level. The control by CK and mitochondria of the local ATP/ADP ratio close to the myosin ATPase (estimated by rigor tension) was also significantly impaired in HF fibres (by 32% and 46%, respectively). However, while the contributions of mitochondria and CK to local ATP regeneration were equally depressed in HF for the control of SERCA, mitochondrial contribution was more severely impaired than CK (P < 0.05) with respect to myofilament regulation. These data show that local energetic regulation of essential ATPases is severely impaired in heart failure, and undergoes a complex remodelling as a result of a decreased activity of the ATP-generating systems and cytoarchitecture disorganization. [source] | ||||||||||