Inhibition of Lyn function in mast cell activation by SH3 domain binding peptides.

While Lyn tyrosine kinase has been shown to be necessary for IgE-receptor (FcepsilonRI)-mediated mast cell activation, the mechanism of Lyn activation is not yet understood. Using a micro-electroporation technique to quantitatively introduce peptides into the cytosol of tumor mast cells, we show that proline-rich peptides that preferentially bind Src family SH3 domains block receptor-induced repetitive calcium spikes in a concentration dependent manner. The Src family member Lyn was the likely target, since a series of phage displaying derived peptides with increased Lyn SH3 domain binding specificity inhibited FcepsilonRI-mediated calcium signaling at concentrations consistent with binding to Lyn rather than other Src-type kinases. Furthermore, SH3 binding peptides prevented the plasma membrane translocation of a fluorescently labeled Syk tandem SH2 domain, which binds to phosphorylated FcepsilonRI, suggesting that the peptides specifically block the Lyn-mediated step by which FcepsilonRI cross-linking leads to receptor phosphorylation. Our study suggests that the binding of proline-rich peptides, or corresponding cellular interaction partners, to Lyn SH3 domain suppresses the Lyn-mediated phosphorylatation of FcepsilonRI and calcium signaling.

The effect of acrylamide and other sulfhydryl alkylators on the ability of dynein and kinesin to translocate microtubules in vitro.

Chronic exposure to acrylamide leads to a dying-back axonopathy afflicting the longest axons of all tested mammalian and avian species. Prior to the onset of acrylamide-induced axonal degeneration, alterations in axonal fast transport have been consistently reported to be more severe for the retrograde than the anterograde direction. The putative retrograde motor protein, dynein, is compromised by exposure to the sulfhydryl-alkylating agent N-ethylmaleimide (NEM) at concentrations far below those required to inactivate kinesin, the putative anterograde motor protein. Since acrylamide is capable of alkylating protein sulfhydryl moieties, we tested whether a direct exposure of purified kinesin or dynein to acrylamide would result in an impairment of either enzyme's ability to translocate microtubules. Motor activity was assayed by sequentially adsorbing either kinesin or dynein to acid-washed coverslips, treating with an alkylating agent or control solution, adding microtubules and ATP, and finally imaging and quantifying the binding and gliding of microtubules using video-enhanced differential interference contrast (VE-DIC) microscopy. In comparison to controls, incubation of dynein with NEM, ethacrynic acid, or iodoacetic acid resulted in dose-dependent decreases in the amount and rate of microtubule gliding, but increases in irreversible high-affinity microtubule binding. In contrast, exposure of dynein to 1-100 mM solutions of acrylamide did not significantly alter either the binding or gliding of microtubules (a molar/hour exposure to acrylamide equivalent to 50 times that which causes retrograde transport deficits in vivo). Likewise, kinesin motility parameters were not significantly affected by acrylamide concentrations up to 100 mM while NEM solutions > 100 microM led to significant losses in the ability of kinesin to bind MT. These data indicate that acrylamide does not significantly interact with bound (adsorbed) kinesin or dynein, implying that the mechanism by which acrylamide interferes with fast axonal transport in vivo is by interaction with other factor(s) that govern the movement of vesicles.

In vitro acrylamide exposure alters growth cone morphology.

Acrylamide intoxication leads to degeneration of the longest axons of the central and peripheral nervous systems in humans and laboratory animals. Axonal derangements resulting from in vivo acrylamide exposure are first noted within synapses of the longest axons before involving more proximally located axonal segments or shorter axons, thus illustrating the specificity of acrylamide for the terminal axonal regions. As a possible model system for investigating the mechanism of toxicity of acrylamide on the distal axon, we exposed neurite-extending chick dorsal root ganglion (DRG) cells to acrylamide in vitro and then examined growth cones for alterations in morphology and function. Exposing DRG explants to media containing from 0.125 to 1.0 mM acrylamide for 16 hr leads to specific and dose-responsive alterations of growth cone morphology including: a nearly total loss of filopodial elements, the preservation of highly active but two-dimensional lamellar structures, an inappropriate extension of the axonal cytoskeleton into the forward region of most growth cones, and a frequent breakdown of the central and peripheral growth cone domains. The sulfhydryl alkylating agents ethacrynic acid, iodoacetamide, and iodoacetic acid were tested and none produced acrylamide-like morphological alterations at any dose. DRG cultures were also exposed to the neurotoxic acrylamide analogs glycidamide, N-hydroxy-methacrylamide (HM-ACR), and methacrylamide (M-ACR). At concentrations of 0.25 to 1.0 mM, glycidamide exposure resulted in acrylamide-like growth cone alterations. HM-ACR exposure also resulted in growth cones that were acrylamide-like but only at concentrations > 1.5 mM. M-ACR did not produce acrylamide-like growth cones at doses of up to 16.6 mM. Thus, in vitro exposure of DRG explants to acrylamide and two neurotoxic acrylamide analogs leads to reproducible and specific morphological alterations that are dose-dependent and separable from the effects of sulfhydryl alkylation.

Axonal transport: beyond kinesin and cytoplasmic dynein.

In vitro and in vivo studies of specific neuronal fast and slow transport components are presently reshaping our understanding of how the processes of vesicular and cytoskeletal transport are regulated in axons and dendrites. Evidence suggests that vesicles possess an inherent directionality, possibly the result of their motor receptor proteins responding to intracellular cues, which then allows movement with either kinesin or cytoplasmic dynein.

The exposure of murine macrophages to alpha 2-macroglobulin 'fast' forms results in the rapid secretion of eicosanoids.

The exposure of [3H]arachidonate-radiolabelled murine peritoneal macrophages to alpha 2-macroglobulin-methylamine or alpha 2-macroglobulin-trypsin but not native alpha 2-macroglobulin (alpha 2M) results in the rapid secretion of [3H]eicosanoids. Resident peritoneal macrophages stimulated with 0.1 microM alpha 2M-methylamine exhibited an enhanced secretion within 10 min. The ability of alpha 2M 'fast' forms to stimulate secretion of [3H]eicosanoids was similar to that observed in the presence of the murine macrophage chemoattractant platelet-activating factor. As observed for total [3H]eicosanoid secretion, alpha 2M 'fast' forms also rapidly enhanced the secretion of the cAMP-elevating prostanoid, prostaglandin E2, from resident peritoneal macrophages. Stimulated secretion of prostaglandin E2 in response to 0.1 microM alpha 2M-methylamine was less rapid than that observed using 0.1 microM platelet-activating factor. Similar amounts of secreted prostaglandin E2 were present in media of macrophage cultures after 1 h exposure to the two stimuli. In the presence of 0.1 microM alpha 2M-methylamine, secreted prostaglandin E2 remained elevated, compared to the appropriate buffer control, for at least 24 h. The present results indicate that receptor recognition of alpha 2M 'fast' forms by macrophages results in the rapid stimulation of eicosanoid secretion and suggest that secretion of prostaglandin E2 and other eicosanoids may be involved in the ability of alpha 2 M 'fast' forms to regulate various macrophage functional responses.