Differential effects of W mutations on p145c-kit tyrosine kinase activity and substrate interaction.

The c-kit gene, mapped to the dominant white spotting (W) locus of the mouse (Chabot, B., Stephenson, D. A., Chapman, V. M., Besmer, P., and Bernstein, A. (1988) Nature 335, 88-89; Geissler, E. N., Ryan, M. A., and Housman, D. E. (1988) Cell 55, 185-192), encodes a receptor tyrosine kinase, p145c-kit. Germline mutations at the W locus lead to loss of function alterations in p145c-kit, and result in mice with developmental defects of varying severity in the melanocytic, hematopoietic stem cell, and primordial germ cell lineages. To investigate in more detail the effect of W mutations on p145c-kit signaling, three mutations, W42, Wv, and W41, that confer severe, intermediate, and mild phenotypic characteristics, respectively, were introduced into the human p145c-kit tyrosine kinase domain. These mutations attenuated the intrinsic tyrosine kinase activity of the receptor to different degrees. In addition, they had differential effects on the interaction of the p145c-kit substrates, phospholipase C gamma, GTPase-activating protein, and the receptor-binding subunit of phosphatidylinositol 3'-kinase, p85. Notably, the Wv mutation, while retaining significant receptor tyrosine kinase activity, was unable to bind phospholipase C gamma and GTPase-activating protein, but could still associate with p85. These results suggest that the location of W mutations may be an important determinant of the specificity of substrate association and phosphorylation, and may explain, at least in part, the cell type-specific defects associated with certain W alleles.     

The preparation of hydrophilic derivatives of band 3 protein by acylation of the human red blood cell membrane

In situ reaction of erythrocyte membranes with dicarboxylic anhydrides leads to solubilization of hydrophobic integral proteins. Removal of peripheral proteins and bulk lipid by appropriate sedimentation and dialysis steps yields hydrophilic band 3 protein derivatives. These acyl compounds display size heterogeneity upon gel filtration. A chromatographically homogeneous acyl band 3 protein is obtained if the acylation is conducted in the presence of detergent and the detergent subsequently removed. Hydrophilic acyl derivatives of band 3 protein can be subjected to conventional analytical techniques without the use of detergents.     

Untersuchungen zur Bestimmung des Geschlechts

Ohne Zusammenfassung Jakob Baron v.Üxküll zu seinem 70. Geburtstag gewidmet.     

Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-1?C]glucose 1-phosphate, [U-1?C]sucrose, [U-1?C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-1?C]sucrose plus unlabelled equimolar glucose 1-phosphate. C1?-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced 1?C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-1?C]glucose 1-phosphate or adenosine-[U-1?C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C1?C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.     

Proteome-wide overexpression of host proteins for identification of factors affecting tombusvirus RNA replication: an inhibitory role of protein kinase C

To identify host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model positive-stranded RNA virus, we overexpressed 5,500 yeast proteins individually in Saccharomyces cerevisiae, which supports TBSV replication. In total, we identified 141 host proteins, and overexpression of 40 of those increased and the remainder decreased the accumulation of a TBSV replicon RNA. Interestingly, 36 yeast proteins were identified previously by various screens, greatly strengthening the relevance of these host proteins in TBSV replication. To validate the results from the screen, we studied the effect of protein kinase C1 (Pkc1), a conserved host kinase involved in many cellular processes, which inhibited TBSV replication when overexpressed. Using a temperature-sensitive mutant of Pkc1p revealed a high level of TBSV replication at a semipermissive temperature, further supporting the idea that Pkc1p is an inhibitor of TBSV RNA replication. A direct inhibitory effect of Pkc1p was shown in a cell-free yeast extract-based TBSV replication assay, in which Pkc1p likely phosphorylates viral replication proteins, decreasing their abilities to bind to the viral RNA. We also show that cercosporamide, a specific inhibitor of Pkc-like kinases, leads to increased TBSV replication in yeast, in plant single cells, and in whole plants, suggesting that Pkc-related pathways are potent inhibitors of TBSV in several hosts.     

Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra

We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC??C and 3D C??NCA with sequential ¹³C acquisitions, 3D NHH and 3D NC??H with sequential ¹H acquisitions and 3D CANH and 3D C??NH with broadband ¹³C?C¹⁵N mixing are demonstrated using microcrystalline samples of the ??1 immunoglobulin binding domain of protein G (GB1) and the chicken ??-spectrin SH3 domain.     

Numerical design of RN<Stack><Subscript><Emphasis Type="Italic">n</Emphasis></Subscript><Superscript>ν</Superscript></Stack> symmetry-based RF pulse schemes for recoupling and decoupling of nuclear spin interactions at high MAS frequencies

An approach for the efficient implementation of RN _n ^ν symmetry-based pulse schemes that are often employed for recoupling and decoupling of nuclear spin interactions in biological solid state NMR investigations is demonstrated at high magic-angle spinning frequencies. RF pulse sequences belonging to the RN _n ^ν symmetry involve the repeated application of the pulse sandwich {R _ϕ R _−ϕ}, corresponding to a propagator U _RF = exp(−i4ϕI _z), where ϕ = πν/N and R is typically a pulse that rotates the nuclear spins through 180° about the x-axis. In this study, broadband, phase-modulated 180° pulses of constant amplitude were employed as the initial ‘R’ element and the phase-modulation profile of this ‘R’ element was numerically optimised for generating RN _n ^ν symmetry-based pulse schemes with satisfactory magnetisation transfer characteristics. At representative MAS frequencies, RF pulse sequences were implemented for achieving ¹³C–¹³C double-quantum dipolar recoupling and through bond scalar coupling mediated chemical shift correlation and evaluated via numerical simulations and experimental measurements. The results from these investigations are presented here. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.