SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

Divergent modulation of Src-family kinase regulatory interactions with ATP-competitive inhibitors.

Multidomain protein kinases, central controllers of signal transduction, use regulatory domains to modulate catalytic activity in a complex cellular environment. Additionally, these domains regulate noncatalytic functions, including cellular localization and protein-protein interactions. Src-family kinases (SFKs) are promising therapeutic targets for a number of diseases and are an excellent model for studying the regulation of multidomain kinases. Here, we demonstrate that the regulatory domains of the SFKs Src and Hck are divergently affected by ligands that stabilize two distinct inactive ATP-binding site conformations. Conformation-selective, ATP-competitive inhibitors differentially modulate the ability of the SH3 and SH2 domains of Src and Hck to engage in intermolecular interactions and the ability of the kinase-inhibitor complex to undergo post-translational modification by effector enzymes. This surprising divergence in regulatory domain behavior by two classes of inhibitors that each stabilize inactive ATP-binding site conformations is found to occur through perturbation or stabilization of the αC helix. These studies provide insight into how conformation-selective, ATP-competitive inhibitors can be designed to modulate domain interactions and post-translational modifications distal to the ATP-binding site of kinases.

Subcellular localization of a membrane-associated transglutaminase activity in rat liver.

Fractionation of rat liver by homogenization and differential centrifugation revealed that only about 83% of the transglutaminase activity in the tissue is in a soluble form, and that the remainder is associated with the particulate fraction. This latter activity remained with the membranes even after they were extensively washed to remove 99% of such soluble enzymes as lactate dehydrogenase and aldolase. Subsequent fractionation of the membranes by isopycnic density gradient centrifugation in sucrose resulted in a single band of transglutaminase activity at a density of 1.194 g/cm3. This activity was coincident with the major band of plasma membranes, which was identified by its content of 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and leucine aminopeptidase activities. After treatment with digitonin and fractionation on sucrose gradients, the transglutaminase activity and the plasma membrane marker enzyme activities were found at a new density of 1.210 g/cm3, while the enzyme markers for the other membrane fractions remained unchanged. From these data, we conclude that approximately 17% of the transglutaminase activity in rat liver is specifically associated with the plasma membranes.

Soup-to-nuts: a television approach to nutrition education.

Soup-to-Nuts is a fresh approach to nutrition education. Television is a relatively untapped medium in the classroom for teaching nutrition concepts. These open-ended lessons facilitate discussion and encourage critical analysis of food behavior. Soup-to-Nuts can serve as a model for other audiovisual nutrition education efforts which join the expertise on nutrition and media professionals.

Identification of pseudoisoenzymic subforms of muscle carbonic anhydrase.

Rabbit muscle carbonic anhydrase III, a recently discovered third isoenzyme (possibly muscle specific) of carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) (Register, A.M., Koester, M.K. and Noltmann, E.A. (1978) J. Biol. Chem. 253, 4143--4152) has been subjected to isoelectric focusing. When monomer samples, shown to be homogeneous by both ion-exchange and molecular sieve chromatography, were analyzed by this technique, three subspecies were produced, which were similar in amino acid composition and specific CO2 hydratase activity. In addition to having either monomer or dimer status, the subspecies differed in the extent of oxidation of their sulhydryl groups and in their isoelectric pH values (9.3, 8.8, and 8.4, respectively). Also, the presence of dithiothreitol will affect their relative concentrations. These subforms are therefore designated as pseudoisoenzymes and are considered to be neither genetically nor functionally separate enzyme species.

Induced reattachment in periodontic-endodontic lesions by root demineralization in situ.

A new method of root preparation to enhance reattachment of gingival tissues to root dentin has been developed through extensive animal research. The improved technique may aid periodontists and endodontists in the management of deep combined lesions. This article reviews essentials of experimental animal data and presents case reports of three different clinical approaches to combined lesion closure attempts utilizing surgical and chemical root preparation.

Accelerated reattachment with cementogenesis to dentin, demineralized in situ. II. Defect repair.

Three surgical experiments, with histologic evaluation, were performed to study induced gingival reattachment to tooth root dentin demineralized in situ during flap surgery in adult mongrel dogs. Experiments demonstrated aspects of: (1) Weekly histochemical and morphological sequences of repair; (2) Repair of chronically inflamed bony defects simulating periodontal pockets; and (3) Six and 12 month repair of reattached surgical defects. Flap reattachment with cementogenesis was induced by in situ root demineralization using citric acid at pH 1.0 applied for 2 minutes. Results demonstrate: (1) The production of anchoring cementum pins extending into dentin tubules widened by demineralization; (2) Reattachment with cementogenesis of inflamed gingiva to roots exposed to chronically-infected surgical defects for 3 months; (3) Success in repairing chronic interproximal one-walled and labial one surface defects by reattachment; (4) Relative failure to repair bifurcation and horizontal bone defects by flap reattachment; and (5) Complete alveolar bone repair over most labial defects by 1 year, with maintenance of a periodontal ligament between induced bone and cementum. These findings, together with previous reports of induced reattachment to demineralized roots, provide further evidence for mechanisms and consistency and suggest that this regenerative phenomenon may be useful in repairing osseous defects in periodontal therapy.

Accelerated reattachment with cementogenesis to dentin, demineralized in situ. I. Optimum range.

This study confirms an original report describing accelerated reattachment with cementogenesis to root dentin, surgically exposed and demineralized in situ. It additionally describes results of 250 experiments on over 1000 teeth in mongrel dogs and cats designed to identify an optimum range of demineralization related to type of agent, pH, and time of application. This optimum range consistently induces flap reattachment with cementogenesis, while demineralization rates above and below this range enhance reattachment relative to undermineralized controls, but do not consistently induce new cementum. Although most controls demonstrated some reattachment with partial cementogenesis, none produced complete repair as did properly demineralized root surfaces and approximately one-third showed spithelial migration to the apical borders of the wound. No demineralized teeth demonstrated this control result. Small species differences in the response to root demineralization and the degree of hypermineralization of roots adjacent to chronic periodontal pockets, may make optimum ranges determined in this animal study slightly low for human pocket reaattachment techniques.