Unexpected signals in a system subject to kinetic proofreading.

When multivalent ligands attach to IgEs bound to the receptors with high affinity for IgE on mast cells, the receptors aggregate, tyrosines on the receptors become phosphorylated, and a variety of cellular responses are stimulated. Prior studies, confirmed here, demonstrated that the efficiency with which later events are generated from earlier ones is inversely related to the dissociation rate of the aggregating ligand. This finding suggests that the cellular responses are constrained by a "kinetic proofreading" regimen. We have now observed an apparent exception to this rule. Doses of the rapidly or slowly dissociating ligands that generated equivalent levels of tyrosine-phosphorylated receptors comparably stimulated a putatively distal event: transcription of the gene for monocyte chemoattractant protein 1. Possible explanations of this apparent anomaly were explored.

Interaction between the unphosphorylated receptor with high affinity for IgE and Lyn kinase.

Chinese hamster ovary fibroblasts previously transfected with the high affinity receptor for IgE (FcepsilonRI) were further transfected with the alpha subunit of the receptor for interleukin 2 (Tac) or with chimeric constructs in which the cytoplasmic domain of Tac was replaced with the C-terminal cytoplasmic domain of either the beta subunit or the gamma subunit of FcepsilonRI. Whereas native Tac failed to affect the aggregation-induced phosphorylation of FcepsilonRI, both chimeric constructs substantially inhibited this reaction. Alternatively, the FcepsilonRI-bearing fibroblasts were transfected with two chimeric constructs in which the cytoplasmic domain of Tac was replaced with a modified short form of Lyn kinase. The Lyn in both of the chimeric constructs had been mutated to remove the sites that are normally myristoylated and palmitoylated, respectively; one of the constructs had in addition been altered to be catalytically inactive. The catalytically active construct enhanced, and the inactive construct inhibited, aggregation-induced phosphorylation of the receptors. All of the chimeric constructs were largely distributed outside the detergent resistant microdomains, and whereas aggregation caused them to move to the domains in part, their aggregation was neither necessary nor enhanced their effects. These results and others indicate that the receptor and Lyn interact through protein-protein interactions that neither are dependent upon either the post-translational modification of the kinase with lipid moieties nor result exclusively from their co-localization in specialized membrane domains.

A quantitative approach to signal transduction.

The high affinity receptor for IgE (FcepsilonRI), is one of a family of immunoreceptors whose antigen-induced clustering leads to a variety of cellular responses. The signaling pathways are enormously complex but by focusing on only the most initial steps, it is now possible to sketch plausible molecular models that relate the interaction of multivalent antigens with the receptor-bound IgE to the earliest cellular events. In this paper, we describe how we have combined quantitative experimentation and mathematical modeling to probe this system further. We also discuss some of the formidable challenges that remain before we can claim reasonably complete understanding of even these early events.

Tyrosine phosphorylation of Vav stimulates IL-6 production in mast cells by a Rac/c-Jun N-terminal kinase-dependent pathway.

This study investigates whether the guanine nucleotide exchange activity of Vav is linked to cytokine production in mast cells. Overexpression of Vav in the RBL-2H3 mast cell line resulted in the constitutive tyrosine phosphorylation and activation of Vav. We analyzed the functional effect of Vav overexpression on cytokine production. IL-2 and IL-6 mRNA levels were dramatically increased in Vav-overexpressing cells and correlated with increased NF-AT activity. Little or no effect was observed on the mRNA levels of IL-3, IL-4, GM-CSF, TNF-alpha, and TGF-beta. FcepsilonRI engagement did not further enhance IL-2 and IL-6 mRNA levels and only slightly enhanced NF-AT activity, but dramatically increased the mRNA levels of other tested cytokines. To understand the signal transduction required, we focused primarily on IL-6 induction by measuring mitogen-activated protein kinase activity and analyzing the effects of mutant or dominant negative forms of Vav, Rac1, and c-Jun N-terminal kinase-1 (JNK1). Vav overexpression resulted in the constitutive activation of JNK1 with little or no effect on p38 mitogen-activated protein kinase and ERK2. This was dependent on Vav-mediated activation of Rac1 as a Dbl domain-mutated Vav, inactive Rac N17, and inactive JNK1 down-regulated the Vav-induced JNK1 or IL-6 responses. Vav expression, but not expression of domain-mutated Vav, increased IL-6 secretion from nonimmortalized bone marrow-derived mast cells upon FcepsilonRI engagement. We conclude that Vav phosphorylation contributes to IL-6 induction in mast cells.

Polyethylene glycol-mediated infection of non-permissive mammalian cells with semliki forest virus: application to signal transduction studies.

Semliki Forest Virus (SFV) vectors allow the subcloning of a gene of interest directly in the expression vector, thus avoiding the need to select and purify viral recombinants, making this viral expression system attractive over many others for mammalian protein expression. We now describe a novel and generally applicable method for infection of non-permissive mammalian cells with SFV, that greatly enhances the utility of this expression system. We demonstrate that the hygroscopic polymer poly (ethylene glycol), PEG, promotes the infectivity of cells by SFV under conditions that did not promote cell-cell fusion. We also found that the PEG-induced infection and expression of an exogenous protein (green fluorescent protein, GFP) did not elevate the basal tyrosine kinase activity, induce a stress-activated responses, or result in aberrant cell responses. Expression of GFP tagged-Vav, an activator of stress-activated protein kinase (SAPK/JNK), resulted in the expected induction of JNK activity and in the normal redistribution of Vav in response to engagement of the high affinity receptor for IgE (FcepsilonRI). Thus, our findings that PEG allows the infection of non-permissive cells by SFV makes this system extremely attractive for expression of proteins in mammalian cells, and studies on signal transduction and cellular localization in immune and non-immune cells.

The unique domain as the site on Lyn kinase for its constitutive association with the high affinity receptor for IgE.

Aggregation of the high affinity receptor for IgE (FcepsilonRI) leads to the phosphorylation of tyrosines on the beta and gamma chains of the receptor by the Src family kinase Lyn. We have studied the interaction between Lyn and the FcepsilonRI in vivo using a transfection-based approach. FcepsilonRI were stably transfected into Chinese hamster ovary cells. The small amount of endogenous Src family kinase was sufficient to phosphorylate receptor tyrosines upon extensive aggregation of FcepsilonRI but not after addition of dimers of IgE. Upon stable co-transfection of Lyn kinase into the cells, dimers were now able to stimulate receptor phosphorylation and the response to more extensive aggregation was enhanced. In contrast, co-transfection with catalytically inactive Lyn inhibited the aggregation-induced phosphorylation by the endogenous kinase, and a quantitatively similar inhibition was observed in cells transfected with the SH4-containing unique domain of Lyn. Consistent with the results of others using alternative approaches, our additional studies using a yeast two-hybrid system detected a direct interaction between intact Lyn or its unique domain and the C-terminal cytoplasmic domain of the beta chain but not with the receptor's other cytoplasmic domains.

Tyrosine phosphorylation of protein kinase C-delta in response to the activation of the high-affinity receptor for immunoglobulin E modifies its substrate recognition.

The delta isoform of protein kinase C is phosphorylated on tyrosine in response to antigen activation of the high-affinity receptor for immunoglobulin E. While protein kinase C-delta associates with and phosphorylates this receptor, immunoprecipitation of the receptor revealed that little, if any, tyrosine-phosphorylated protein kinase C-delta is receptor associated. In vitro kinase assays with immunoprecipitated tyrosine-phosphorylated protein kinase C-delta showed that the modified enzyme had diminished activity toward the receptor gamma-chain peptide as a substrate but not toward histones or myelin basic protein peptide. We propose a model in which the tyrosine phosphorylation of protein kinase C-delta regulates the kinase specificity toward a given substrate. This may represent a general mechanism by which in vivo protein kinase activities are regulated in response to external stimuli.

Cloning and functional characterization of the rat glutamine synthetase gene.

Glutamine synthetase catalyzes the formation of glutamine from glutamate and ammonia. It plays a central role in both amino acid neurotransmitter metabolism and ammonia detoxification in the central nervous system. Glutamine synthetase expression is regulated in developmental, hormonal, and in tissue- and cell-specific manners. We have cloned a full-length cDNA coding for rat glutamine synthetase, and have found an AT-rich area of conservation in the 3' untranslated regions between rat, mouse, and chicken, which may play a part in the regulation of the stability of the glutamine synthetase message. We have also cloned and mapped the gene coding for rat glutamine synthetase, and identified, by sequence analysis, areas potentially important for the regulation of glutamine synthetase transcription. Transient transfection of a variety of cell lines with deletion constructs of the glutamine synthetase promoter driving a chloramphenicol acetyltransferase reporter gene functionally demonstrates regions of the promoter containing elements important for transcriptional regulation.

Evidence that the B2 chain of laminin is responsible for the neurite outgrowth-promoting activity of astrocyte extracellular matrix.

Extracellular matrix (ECM) derived from cerebral cortical astrocytes stimulates neurite outgrowth from pheochromocytoma (PC12) cells in the absence of the classical nerve growth factor (NGF). We have shown here that astrocyte ECM can also stimulate neurite outgrowth from primary cultures of central nervous system (CNS) neurons. Using PC12 cells for a quantitative assay, we also demonstrated that the neurite growth-promoting activity increased as the astrocytes matured in vitro: ECM from older astrocytes (3-12 weeks in vitro) exhibited two-fold more neurite growth-promoting activity than ECM for younger astrocytes (5 days to 2 weeks in vitro). We applied various antibodies to identify the neurite growth-promoting factor of astrocyte ECM and found that anti-laminin inhibited neurite outgrowth by 50%, whereas anti-fibronectin and anti-NGF had no effect. Immunoblots, using laminin chain-specific antibodies, and cDNA hybridization of laminin mRNA demonstrated that cultured astrocytes synthesize only the B2 chain of laminin. This suggests that the B2 chain of laminin suffices to stimulate neurite outgrowth.