Pleckstrin homology (PH) domains comprised of loosely conserved sequences of ϳ100 amino acid residues are a functional protein motif found in many signal-transducing and cytoskeletal proteins. We recently demonstrated that the PH domains of Tec family protein-tyrosine kinases Btk and Emt (equal to Itk and Tsk) interact with protein kinase C (PKC) and that PKC down-regulates Btk by phosphorylation. In this study we have characterized the PKC-BtkPH domain interaction in detail. Using pure PKC preparations, it was shown that the Btk PH domain interacts with PKC with high affinity (K D ‫؍‬ 39 nM). Unlike other tested phospholipids, phosphatidylinositol 4,5-bisphosphate, which binds to several PH domains, competed with PKC for binding to the PH domain apparently because their binding sites on the amino-terminal portion of the PH domains overlap. The minimal PKC-binding sequence within the Btk PH domain was found to correspond roughly to the second and third ␤-sheets of the PH domains of known tertiary structures. On the other hand, the C1 regulatory region of PKC⑀ containing the pseudosubstrate and zinc finger-like sequences was found to be sufficient for strong binding to the Btk PH domain. Phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC that interacts with the C1 region of PKC, inhibited the PKC-PH domain interaction, whereas the bioinactive PMA (4-␣-PMA) was ineffective. The isoform of PKC, which has a single zinc finger-like motif instead of the two tandem zinc finger-like sequences present in conventional and novel PKC isoforms, does not bind PMA. Thus, as expected, PH domain binding with PKC was not interfered with by PMA. Further, inhibitors that are known to attack the catalytic domains of serine/threonine kinases did not affect this PKC-PH domain interaction. In contrast, the presence of physiological concentrations of Ca 2؉ induced less than a 2-fold increase in PKC-PH domain binding. These results indicate that PKC binding to PH domains involve the ␤2-␤3 region of the Btk PH domain and the C1 region of PKC, and agents that interact with either of these regions (i.e. phosphatidylinositol 4,5-bisphosphate binding to the PH domain and PMA binding to the C1 region of PKC) might act to regulate PKC-PH domain binding.

No abstract

Bruton's tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptorstimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors.Cross-linking of high-affinity IgE receptor (FcRI) found predominantly on mast cells and basophils stimulates signaling cascades that lead to exocytosis of inflammatory mediators of the allergic response (1). FcRI consists of an IgE-binding ␣ subunit, a ␤ subunit with four transmembrane domains, and a disulfidebonded pair of ␥ subunits (2). Similar to the signaling subunits of the T cell receptor and B cell receptor (BCR) systems, the ␤ and ␥ subunits have immunoreceptor tyrosine-based activation motif (ITAM) sequences in their cytoplasmic portions (3). A ␤ subunitassociated Src family protein-tyrosine kinase (PTK), Lyn, is activated on FcRI cross-linking (4) and phosphorylates tyrosine residues in the ITAM sequences. Phosphorylated ITAM sequences in ␤ and ␥ subunits recruit Lyn and Syk, another PTK with two tandemly arranged Src homology 2 (SH2) domains, respectively, in a phosphotyrosine-SH2 interaction-dependent manner (5-8).

Reactions of [Cp*MCl 2 ] 2 (M = Rh, Ir) with bidentate ligand (L = pyrazine; LA = diisocyanide) gave [Cp*MCl 2 (L or LA}] 2 , which were converted into tetranuclear complexes [Cp* 2 M 2 Cl 2 (L)(LA)] 2 (OTf) 4 containing different ligands on treatment with Ag(OTf).

No abstract

No abstract

No abstract

No abstract