Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: insights from NMR mapping studies and site-directed mutagenesis.

Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.     

The structure of the IgE Cepsilon2 domain and its role in stabilizing the complex with its high-affinity receptor FcepsilonRIalpha

The stability of the complex between IgE and its high-affinity receptor, FcepsilonRI, on mast cells is a critical factor in the allergic response. The long half-life of the complex of IgE bound to this receptor in situ ( approximately 2 weeks, compared with only hours for the comparable IgG complex) contributes to the permanent sensitization of these cells and, hence, to the immediate response to allergens. Here we show that the second constant domain of IgE, Cepsilon2, which takes the place of the flexible hinge in IgG, contributes to this long half-life. When the Cepsilon2 domain is deleted from the IgE Fc fragment, leaving only the Cepsilon3 and Cepsilon4 domains (Cepsilon3-4 fragment), the rate of dissociation from the receptor is increased by greater than 1 order of magnitude. We report the structure of the Cepsilon2 domain by heteronuclear NMR spectroscopy and show by chemical shift perturbation that it interacts with FcepsilonRIalpha. By sedimentation equilibrium we show that the Cepsilon2 domain binds to the Cepsilon3-4 fragment of IgE. These interactions of Cepsilon2 with both FcepsilonRIalpha and Cepsilon3-4 provide a structural explanation for the exceptionally slow dissociation of the IgE-FcepsilonRIalpha complex.     

Disturbances in signal transduction mechanisms in Alzheimer's disease

Many of the treatments directed towards alleviation of symptoms in Alzheimer's disease assume that target receptor systems are functionally intact. However, there is now considerable evidence that this is not the case. In human post-mortem brain tissue samples, the function of the GTP-binding protein G_s in regulating adenylyl cyclase is severely disabled, whereas that of G_i is intact. This difference in the function of the two G-protein types is also found in G-protein regulation of high- and low-affinity receptor recognition site populations. Measurement of G-protein densities using selective antibodies has indicated that the dysfunction in G_s-stimulation of cAMP production correlates with the ratio of the large to small molecular weight isoforms of the G_s subunit. With respect to intracellular second messenger effects, there is a dramatic decrease in the density of brain receptor recognition sites for Ins(1,4,5)P₃ that is not accompanied by a corresponding change in the Ins(1,3,4,5)P₄ recognition site density. Protein kinase C function is also altered in Alzheimer's disease, a finding that may be of importance for the control of -amyloid production. These studies indicate that signal transduction processes are severely compromised in Alzheimer's disease. Some of these disturbances are also seen in cultured fibroblasts from Alzheimer's disease patients, indicating that they are neither restricted to areas of histopathological change, nor non-specific changes found late in the course of the disease. Cellular models to investigate the relation between amyloid production and deficits in signal transduction are also discussed.     

Enzymatic semisynthesis of porcine despentapeptide (B26-30) insulin using unprotected desoctapeptide (B23-30) insulin as a substrate. Model studies

Unprotected porcine desoctapeptide(B23-30) insulin (DOPI) and the synthetic Gly-Phe-Phe were used as substrates for the trypsin-catalyzed synthesis of despentapeptide(B26-30) insulin (DPPI). The DPPI synthesis was accompanied by a moderate oligomerization and by the formation of a side produce which was identified as a DOPI derivative having an extra peptide bond between the Gly(A1) and Arg(B22) and which was named des(23-63) proinsulin (1). Despite side reactions, the conditions were found where the overall DPPI yields were comparable to those obtained via di-Boc DOPI, and these procedures were faster and simpler since the Boc protection and deprotection steps were omitted. The reaction progress was directly monitored by HPLC.     

1-Methyl-4-Phenylpyridinium Uptake by Human and Rat Striatal Synaptosomes

1-Methyl-4-phenylpyridinium (MPP+) was taken up into human and rat striatal synaptosomes by a saturable system, similar to that for dopamine, with Km values of 0.24 and 0.17 microM, respectively, and similar Vmax values. Uptake of MPP+ and dopamine into both rat and human synaptosomes was inhibited by cocaine and amfonelic acid, with the latter being five to 10 times more potent than the former. MPP+ uptake was potently inhibited by dopamine in preparations from both species. In general, the characteristics of human and rat synaptosomal MPP+ uptake were very similar It seems unlikely that species differences in toxicity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or reaction to dopamine uptake blockers stem from this system.     

Assay of the acetylcholine receptor by affinity labeling

An accurate and sensitive assay for nicotinic acetylcholine receptor binding sites is described which is based on the specificities of receptor both for an affinity label, 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA), and for α-neurotoxins from Naja venoms. It has been demonstrated that MBTA reacts exclusively with one type of subunit of the acetylcholine receptors isolated from the electric tissue of Electrophorus electricus and Torpedo californica and that this reaction is blocked in the presence of Naja naja siamensis α-neurotoxin and of other ligands of the acetylcholine binding site. Thus, in this assay the difference in the extent of labeling by MBTA in the absence and presence of N. n. siamensis toxin is considered the specific labeling of receptor. Although this assay is more complicated than direct α-neurotoxin binding, it is justified by the wellestablished site specificity of the labeling. The specific activities of several different receptor preparations determined using this assay are one-half of those determined using toxin binding. It is possible to assay accurately as little as 0.25 μg of receptor in the presence of 100-fold as much other protein.