Daily injections of anakinra markedly improved clinical and laboratory manifestations in patients with neonatal-onset multisystem inflammatory disease, with or without CIAS1 mutations. (ClinicalTrials.gov number, NCT00069329 [ClinicalTrials.gov].).

Tec family non-receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B-cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N-terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase-dependent phospholipase Cγ (PLCγ) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2-containing inositol phosphatase (SHIP)-dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby PtdIns-3,4,5-P 3 regulates receptordependent calcium signals through the function of Tec kinases.

Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.

We have recently demonstrated that the D3-phosphoinositide phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ) is critical for producing sustained calcium signals through its role in promoting the function of TEC family tyrosine kinases such as Bruton's tyrosine kinase. Although PtdIns-3,4,5-P 3 can potentially be synthesized by any of several types of phosphoinositide 3-kinases (PI3Ks), B cell receptor (BCR)-induced PtdIns-3,4,5-P 3 production is thought to occur primarily through the activation of the class Ia (p85/p110) PI3Ks. This process has been proposed to be mediated by an interaction between the Src family kinase LYN and the p85 subunit of PI3K and/or through p85 membrane recruitment mediated by CBL and/or CD19. However, calcium signaling and other PI3K-dependent signals are relatively preserved in a LYN kinase-deficient B lymphocyte cell line, suggesting that an alternative pathway for PI3K activation exists. As SYK/ZAP70 kinases are upstream from many BCR-initiated signaling events, we directly analyzed SYK-dependent accumulation of both PtdIns-3,4,5-P 3 and PtdIns-3,4-P 2 in B cell receptor signaling using both dominant negative and genetic knockout approaches. Both methods indicate that SYK is upstream of, and necessary for, a significant portion of BCR-induced PtdIns-3,4,5-P 3 production. Whereas CD19 does not appear to be involved in this SYK-dependent pathway, the SYK substrate CBL is likely involved as the dominant negative SYK markedly attenuates CBL tyrosine phosphorylation and completely blocks the BCR-dependent association of CBL with p85 PI3K. Engagement of surface immunoglobulin (BCR)1 on B cells results in early signaling events including tyrosine phosphorylation of ITAM motifs, activation of the non-receptor protein tyrosine kinases LYN and SYK, and calcium mobilization. The D3-phosphoinositides PtdIns-3,4,5-P 3 and PtdIns-3,4-P 2 are also produced after engagement of surface immunoglobulin and have been linked to a multitude of downstream signaling events, including cell survival/AKT activation, membrane ruffling, the activation of the SOS GEF, and TEC kinase-dependent activation of phospholipase C␥1/2 (1-6).D3-phosphoinositide production occurs through the action of members of the phosphoinositide 3-kinase (PI3K) family of lipid kinases. This family now consists of four distinct subtypes of enzymes, including the p85/p110 heterodimeric isoforms (designated class Ia, and reviewed in Refs. 7 and 8), the class Ib G␤␥-responsive isoforms (9, 10), the class II C2 domain containing isoforms (11-15), and the class III PtdIns-specific isoforms (16 -18). The different classes of PI3K enzymes show distinct substrate preferences. Type Ia and type Ib enzymes exhibit a clear preference for PtdIns-4,5-P 2 over PtdIns-4-P (see Refs. 19 and 20 and reviewed in Ref. 21). In contrast, type II enzymes show a preference for PtdIns and PtdIns-4-P over PtdIns-4,5-P 2 (11), whereas the type III enzymes strongly prefer PtdIns over either PtdIns-4-P or PtdIns-4,5-P 2 (16,22). Based on these preferences...

Engagement of the high affinity receptor for IgE (Fc⑀RI) on mast cells and basophils results in Fc⑀RI ␤ and ␥ subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon Fc⑀RI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of Fc⑀RI ␤ and ␥ chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of Fc⑀RI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated Fc⑀RI and activated Syk to the proteasome for degradation.The activation of protein-tyrosine kinases (PTKs) 1 is an essential event in the transduction of intracellular signals from immune receptors (IR), including the T and B cell antigen receptors (TCR and BCR, respectively), the high affinity receptor for IgE (Fc⑀RI), and the widely distributed receptors for IgG.These IRs contain multiple subunits: some, distinct for each receptor, are used for ligand binding, whereas others share conserved cytoplasmic motifs that are critical for the process of cell activation (immune receptor tyrosine-based activation motif, ITAM) (1-6). The IRs lack intrinsic kinase activity; however, within seconds of their engagement, PTKs are activated leading to phosphorylation of various substrates, including IR subunits (7-12). ITAM phosphorylation by the Src family PTKs provides docking sites for the tandem pair of Src homology 2 (SH2) domains of a second class of PTKs belonging to the Syk family (3-6). This family includes only two members: Syk, which is present in most hematopoietic cells and ZAP-70, which is exclusively expressed in T and NK cells. As documented by several studies, the expression of Syk and ZAP-70 is essential for lymphocyte development and signal transduction via IRs (13-16). The association of phosphorylated ITAMs with SH2 domains of Syk family PTKs leads to the activation of Syk and ZAP-70 mainly by autophosphorylation (17), thus allowing the propagation of IR signaling.We and others have demonstrated that Syk and ZAP-70 as well as IR subunits are subjected to an additional covalent modification following IR engagement in that they become modified by ubiquitin (Ub) (18 -22). Moreover, we have also provided evidence suggesting a direct correlation between IRinduced Syk and ZAP-70 ubiquitination and degradation (21).Ubiquitination, which consists in the covalen...

SummaryTyrosine phosphorylation of the Cbl protooncogene has been shown to occur after engagement of a number of different receptors on hematopoietic cells. However, the mechanisms by which these receptors induce Cbl tyrosine phosphorylation are poorly understood. Here we demonstrate that engagement of the high affinity IgE receptor (FceR1) leads to the tyrosine phosphorylation of Cbl and analyze how this occurs. We show that at least part of FcetLIinduced Cbl tyrosine phosphorylation is mediated by the Syk tyrosine kinase, and that the Sykdependent tyrosine phosphorylation of Cbl occurs mainly distal to the Cbl proline-rich region within the COOH-terminal 250 amino acids. Furthermore, we show by coprecipitation that Cbl is present in a complex with Syk before receptor engagement, that the proline-rich region of Cbl and a region of Syk comprised of the two SH2 domains and intradomain linker are required for formation of the complex, and that little or no tyrosine-phosphorylated Cbl is detected in complex with Syk. Overexpression of truncation mutants of Cbl capable of binding Syk has the effect of blocking tyrosine phosphorylation of endogenous Cbl. These results define a potentially important intramolecular interaction in mast cells and suggest a complex function for Cbl in intracellular signaling pathways.

The insulin receptor is a transmembrane protein found on multiple cell types. This receptor is synthesized as a 190-kDa proreceptor which is cleaved to produce mature alpha and beta subunits. The proreceptor contains 18 potential sites for N-linked glycosylation: 14 on the alpha subunit and 4 on the beta subunit. The codons for asparagine in the first four sites at the amino terminus of the alpha subunit were mutated to code for glutamine. This mutant receptor cDNA was stably transfected into NIH 3T3 cells. The insulin receptor produced in these cells remained in the proreceptor form; no mature alpha and beta subunits were produced. The proreceptor was slightly smaller on SDS-PAGE gels than the wild-type proreceptor and contained four less oligosaccharide chains by tryptic peptide mapping. The carbohydrate chains on the mutant proreceptor remained endoglycosidase H sensitive. However, in the presence of brefeldin A, these oligosaccharide chains could be processed to endoglycosidase H resistant chains. By immunofluorescence, the mutant proreceptor was shown to be localized to the endoplasmic reticulum. No insulin receptors could be found on the cell-surface either with cell surface labeling with biotin or with 125I-insulin binding. Thus, glycosylation of the first four N-linked glycosylation sites of the insulin receptor is necessary for the proper processing and intracellular transport of the receptor. This is in contrast to glycosylation at the four sites on the beta subunit which appear not to be important for processing but necessary for signal transduction. Therefore, N-linked glycosylation of the insulin receptor at specific sites has multiple distinctive roles.

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