The production of the second messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate is mediated by activated phosphatidylinositol-specific phospholipase C (PLC) enzymes. Here we report the cloning of a bovine brain complementary DNA encoding an enzyme PLC-148 that is characterized by calcium-dependent and phosphatidylinositol-specific phospholipase C activity when expressed in mammalian cells. Bovine brain messenger RNA contains a 7.5-kilobase transcript corresponding to the isolated cDNA; a related transcript of the same size is present in mRNA from some but not all human cell lines tested. Southern blot analysis of the bovine genome indicated that one or possibly two genes hybridize to the cloned PLC-148 cDNA. There is a striking sequence similarity between specific regions of PLC-148 and the non-catalytic domain of the non-receptor tyrosine kinases. The newly characterized crk transforming gene of the avian sarcoma virus CT10 also contains extensive sequence similarities with PLC-148.

Background: IB kinase ␤ is a key regulator in the NB signaling pathway. Results: Crystal structure of a human IKK␤ asymmetric dimer shows one kinase active site phosphorylated and in the active conformation and the other unphosphorylated and inactive. Conclusion: Depending on the phosphorylation state, IKK␤ can adopt distinct dimeric geometry. Significance: High resolution structure of hIKK␤ provides structural basis for its activation and potential use of inhibitor design.

PKC-e was isolated from a murine brain eDNA library. The clone, 261PKC-e, encoded a polypeptide of 737 amino acids that is homologous to other PKCs. Northern analysis showed that the 7 kb mRNA for this eDNA is widely expressed. The protein when expressed in COS-1 cells displayed phorbol ester-binding activity. However in order to detect the kinase activity of PKC-e, it was necessary to employ a synthetic peptide substrate based upon the pseudosubstrate site. Subsequent analysis demonstrated that PKC-e, while showing certain properties characteristic of the PKC family, has a quite distinct substrate specificity and is independent of Ca 2 +.

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 Å 2 ) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.Injured neurons in mature organisms are unable to effectively regrow their axons after central nervous system damage. One of the many factors restricting axonal regeneration after injury is the growth-inhibiting components associated with damaged myelin. At least three of these components, Nogo-66, myelin-associated glycoprotein (MAG), 3 and oligodendrocyte myelin glycoprotein, either individually or collectively, have been shown to be potent inhibitors of neurite outgrowth (1, 2). All three signal inhibition through the Nogo receptor complex, composed of the ligand-binding Nogo-66 receptor (NgR) and two complementary co-receptors p75 and Lingo-1 that act as a signal-transducing pair on an axon's cell membrane (3, 4). Although both NgR and the p75 nerve growth factor receptor have well documented roles in the context of myelin inhibition, reports exploring the role of Lingo-1 are more recent.Human Lingo-1 is a central nervous system-specific transmembrane glycoprotein (Fig. 1) also known as LERN-1, which belongs to a larger family of LRR-Ig-containing proteins involved in central nervous system development and axonal growth (5). Its large extracellular or ectodomain is thought to be of functional importance in protein-protein recognition and is characterized by a tandem array of multiple LRRs and one Iglike domain. The first studies examining the role of Lingo-1 demonstrated that in cultured neurons Lingo-1 directly associates with NgR and p75 and that whenever myelin-NgR/p75-mediated growth inhibition is observe...

P 2 Y 12 receptor is a G i -coupled adenosine diphosphate (ADP) receptor with a critical role in platelet aggregation. It contains two potential N-linked glycosylation sites at its extra cellular amino-terminus, which may modulate its activity. Studies of both tunicamycin treatment and site-directed mutagenesis have revealed a dispensable role of the N-linked glycosylation in the receptor's surface expression and ligand binding activity. However, the non-glycosylated P 2 Y 12 receptor is defective in the P 2 Y 12 -mediated inhibition of the adenylyl cyclase activity. Thus the study uncovers an unexpected vital role of N-linked glycans in receptor's signal transducing step but not in surface expression or ligand binding. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Acidic mammalian chitinase (AMCase) is a mammalian chitinase that has been implicated in allergic asthma. One of only two active mammalian chinases, AMCase, is distinguished from other chitinases by several unique features. Here, we present the novel structure of the AMCase catalytic domain, both in the apo form and in complex with the inhibitor methylallosamidin, determined to high resolution by X-ray crystallography. These results provide a structural basis for understanding some of the unique characteristics of this enzyme, including the low pH optimum and the preference for the b-anomer of the substrate. A triad of polar residues in the second-shell is found to modulate the highly conserved chitinase active site. As a novel target for asthma therapy, structural details of AMCase activity will help guide the future design of specific and potent AMCase inhibitors.

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Protein kinase C theta (PKCtheta), a member of the Ca(2+)-independent novel subfamily of PKCs, is required for T-cell receptor (TCR) signaling and IL2 production. PKCtheta-deficient mice have impaired Th2 responses in a murine ova-induced asthma model, while Th1 responses are normal. As an essential component of the TCR signaling complex, PKCtheta is a unique T-cell therapeutic target in the specific treatment of T-cell-mediated diseases. We report here the PKCtheta autophosphorylation characteristics and elucidation of the catalytic mechanism of the PKCtheta kinase domain using steady-state kinetics. Key phosphorylated residues of the active PKCtheta kinase domain expressed in Escherichia coli were characterized, and mutational analysis of the kinase domain was performed to establish the autophosphorylation and kinase activity relationships. Initial velocity, product inhibition, and dead-end inhibition studies provided assignments of the kinetic mechanism of PCKtheta(362)(-)(706) as ordered, wherein ATP binds kinase first and ADP is released last. Effects of solvent viscosity and ATPgammaS on PKCtheta catalysis demonstrated product release is partially rate limiting. Our studies provide important mechanistic insights into kinase activity and phosphorylation-mediated regulation of the novel PKC isoform, PKCtheta. These results should aid the design and discovery of PKCtheta antagonists as therapeutics for modulating T-cell-mediated immune and respiratory diseases.