Suramin inhibits initiation of defense signaling by systemin, chitosan, and a β-glucan elicitor in suspension-cultured Lycopersicon peruvianum cells

Systemin-mediated defense signaling in tomato (Lycopersicon esculentum) plants is analogous to the cytokine-mediated inflammatory response in animals. Herein, we report that the initiation of defense signaling in suspension-cultured cells of Lycopersicon peruvianum by the peptide systemin, as well as by chitosan and β-glucan elicitor from Phytophtora megasperma, is inhibited by the polysulfonated naphtylurea compound suramin, a known inhibitor of cytokine and growth factor receptor interactions in animal cells. Using a radioreceptor assay, we show that suramin interfered with the binding of the systemin analog 125I-Tyr-2,Ala-15-systemin to the systemin receptor with an IC50 of 160 μM. Additionally, labeling of the systemin receptor with a photoaffinity analog of systemin was inhibited in the presence of suramin. Receptor-mediated tyrosine phosphorylation of a 48-kDa mitogen-activated protein kinase and alkalinization of the medium of suspension-cultured cells in response to systemin and carbohydrate elicitors were also inhibited by suramin. The inhibition of medium alkalinization by suramin was reversible in the presence of high concentrations of systemin and carbohydrate elicitors. Calyculin A and erythrosin B, intracellular inhibitors of phosphatases and plasma membrane proton ATPases, respectively, both induce medium alkalinization, but neither response was inhibited by suramin. The polysulfonated compound heparin did not inhibit systemin-induced medium alkalinization. NF 007, a suramin derivative, induced medium alkalinization, indicating that neither NF 007 nor heparin interact with elicitor receptors like suramin. The data indicate that cell-surface receptors in plants show some common structural features with animal cytokine and growth factor receptors that can interact with suramin to interfere with ligand binding.

An in vitro assay reveals essential protein components for the “catch” state of invertebrate smooth muscle

“Catch,” a state where some invertebrate muscles sustain high tension over long periods of time with little energy expenditure (low ATP hydrolysis rate) is similar to the “latch” state of vertebrate smooth muscles. Its induction and release involve Ca2+-dependent phosphatase and cAMP-dependent protein kinase, respectively. Molecular mechanisms for catch remain obscure. Here, we describe a quantitative microscopic in vitro assay reconstituting the catch state with proteins isolated from catch muscles. Thick filaments attached to glass coverslips and pretreated with ≈10−4 M free Ca2+ and soluble muscle proteins bound fluorescently labeled native thin filaments tightly in catch at ≈10−8 M free Ca2+ in the presence of MgATP. At ≈10−4 M free Ca2+, the thin filaments moved at ≈4 μm/s. Addition of cAMP and cAMP-dependent protein kinase at ≈10−8 M free Ca2+ caused their release. Rabbit skeletal muscle F-actin filaments completely reproduced the results obtained with native thin filaments. Binding forces >500 pN/μm between thick and F-actin filaments were measured by glass microneedles, and were sufficient to explain catch tension in vivo. Synthetic filaments of purified myosin and twitchin bound F-actin in catch, showing that other components of native thick filaments such as paramyosin and catchin are not essential. The binding between synthetic thick filaments and F-actin filaments depended on phosphorylation of twitchin but not of myosin. Cosedimentation experiments showed that twitchin did not bind directly to F-actin in catch. These results show that catch is a direct actomyosin interaction regulated by twitchin phosphorylation.

CD95/Fas induces cleavage of the GrpL/Gads adaptor and desensitization of antigen receptor signaling

The balance between cell survival and cell death is critical for normal lymphoid development. This balance is maintained by signals through lymphocyte antigen receptors and death receptors such as CD95/Fas. In some cells, ligating the B cell antigen receptor can protect the cell from apoptosis induced by CD95. Here we report that ligation of CD95 inhibits antigen receptor-mediated signaling. Pretreating CD40-stimulated tonsillar B cells with anti-CD95 abolished B cell antigen receptor-mediated calcium mobilization. Furthermore, CD95 ligation led to the caspase-dependent inhibition of antigen receptor-induced calcium mobilization and to the activation of mitogen-activated protein kinase pathways in B and T cell lines. A target of CD95-mediated caspase 3-like activity early in the apoptotic process is the adaptor protein GrpL/Gads. GrpL constitutively interacts with SLP-76 via its C-terminal SH3 domain to regulate transcription factors such as NF-AT. Cleavage of GrpL removes the C-terminal SH3 domain so that it is no longer capable of recruiting SLP-76 to the membrane. Transfection of a truncated form of GrpL into Jurkat T cells blocked T cell antigen receptor-induced activation of NF-AT. These results suggest that CD95 signaling can desensitize antigen receptors, in part via cleavage of the GrpL adaptor.

Kinesin molecular motors: Transport pathways, receptors, and human disease

Kinesin molecular motor proteins are responsible for many of the major microtubule-dependent transport pathways in neuronal and non-neuronal cells. Elucidating the transport pathways mediated by kinesins, the identity of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. Kinesin-II recently was found to be required for transport in motile and nonmotile cilia and flagella where it is essential for proper left-right determination in mammalian development, sensory function in ciliated neurons, and opsin transport and viability in photoreceptors. Thus, these pathways and proteins may be prominent contributors to several human diseases including ciliary dyskinesias, situs inversus, and retinitis pigmentosa. Kinesin-I is needed to move many different types of cargoes in neuronal axons. Two candidates for receptor proteins that attach kinesin-I to vesicular cargoes were recently found. One candidate, sunday driver, is proposed to both link kinesin-I to an unknown vesicular cargo and to bind and organize the mitogen-activated protein kinase components of a c-Jun N-terminal kinase signaling module. A second candidate, amyloid precursor protein, is proposed to link kinesin-I to a different, also unknown, class of axonal vesicles. The finding of a possible functional interaction between kinesin-I and amyloid precursor protein may implicate kinesin-I based transport in the development of Alzheimer's disease.