Drosophila rolling pebbles: a multidomain protein required for myoblast fusion that recruits D-Titin in response to the myoblast attractant Dumbfounded.

The fusion of myoblasts leading to the formation of myotubes is an integral part of skeletal myogenesis in many organisms. In Drosophila, specialized founder myoblasts initiate fusion through expression of the receptor-like attractant Dumbfounded (Duf), which brings them into close contact with other myoblasts. Here, we identify Rols7, a gene expressed in founders, as an essential component for fusion during myotube formation. During fusion, Rols7 localizes in a Duf-dependent manner at membrane sites that contact other myoblasts. These sites are also enriched with D-Titin, which functions to maintain myotube structure and morphology. When Rols7 is absent or its localization is perturbed, the enrichment of D-Titin fails to occur. Rols7 integrates the initial event of myoblast attraction with the downstream event of myotube structural organization by linking Duf to D-Titin.

Involvement of a putative protein-tyrosine phosphatase and I kappa B-alpha serine phosphorylation in nuclear factor kappa B activation by tumor necrosis factor.

Inhibitors of phosphotyrosyl protein phosphatases, pervanadate and phenylarsine oxide, abrogate tumor necrosis factor (TNF)-induced nuclear factor kappa B (NF-kappa B) nuclear translocation in transformed cell lines (U-937 and Jurkat) and primary fibroblasts (MRC-5 and REF). The inhibitors also abrogate NF-kappa B activation by the phosphoseryl/threonyl protein phosphatase inhibitor okadaic acid in U-937 cells. Inhibition of NF-kappa B activation is not due to a general inhibitory effect since neither pervanadate nor phenylarsine oxide treatment affected the constitutive DNA-binding activity of the transcription factors octamer-1 and cAMP response element-binding protein in U-937 cells, nor did these compounds inhibit the TNF-induced phosphorylation of proteins, viz. hsp-27, eukaryotic initiation factor 4e, and pp19, in MRC-5 fibroblasts. Overexpression of the protein-tyrosine phosphatase HPTP alpha resulted in a constitutive nuclear NF-kappa B-like DNA-binding activity in REF cells. Conversely, treatment of human protein-tyrosine phosphatase alpha-overexpressing cells with phenylarsine oxide led to a loss of the constitutive NF-kappa B activity. The presence of a tyrosine phosphorylation site on the inhibitor of NF-kappa B (I kappa B-alpha) suggested that it could be a target for TNF/okadaic acid-induced tyrosine dephosphorylation. However, no tyrosine phosphorylation was detected on I kappa B-alpha fron unstimulated cells, while TNF/okadaic acid-treated cells showed increased phosphorylation of I kappa B-alpha exclusively at serine residue(s). Treatment of cells with pervanadate inhibited TNF-induced I kappa B-alpha phosphorylation and degradation, whereas the serine protease inhibitors tosylphenylalanyl chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone prevented TNF-induced I kappa B-alpha degradation and NF-kappa B nuclear translocation, but not the TNF-induced phosphorylation of I kappa B-alpha. The data suggest that TNF and okadaic acid induce the activation of a putative protein-tyrosine phosphatase(s), leading to I kappa B-alpha serine phosphorylation and degradation and NF-kappa B nuclear translocation.

Differential induction of nuclear NF-kappa B by protein phosphatase inhibitors in primary and transformed human cells. Requirement for both oxidation and phosphorylation in nuclear translocation.

Phosphoseryl/threonyl protein phosphatase inhibitors, viz. okadaic acid and calyculin-A, failed to induce nuclear factor-kappa B (NF-kappa B) nuclear translocation in several primary human cells although a marked and rapid induction was observed in their simian virus 40 transformed counterparts. Inability to induce NF-kappa B cannot be due to a non-activatable system since NF-kappa B was strongly activated by tumor necrosis factor in all the five primary cell types tested. It is also unlikely that the differential induction was due to differential sensitivity of primary and transformed cells to phosphatase inhibitors as the intracellular phosphatase activities of both cell types were equally inhibited by these inhibitors. However, pretreatment with hydrogen peroxide or buthionine sulfoximine, chemicals known to directly or indirectly elevate the intracellular free-radical levels, enabled okadaic acid to induce nuclear translocation of NF-kappa B in primary cells. Conversely cysteine, an antioxidant and precursor of the free radical scavenger, glutathione, inhibited the induction of NF-kappa B by tumor necrosis factor in primary cells, and by okadaic acid or tumor necrosis factor in transformed cells. These data, taken together, suggest that free radical-dependent oxidation and protein phosphorylation are not independent modes of NF-kappa B induction, but are both required for the release of NF-kappa B from I kappa B. Furthermore, the differential induction of NF-kappa B nuclear translocation by okadaic acid in primary and transformed human cells, reported herein, reflects intrinsic differences in the intracellular oxidative state between the two groups of cells. The induction of NF-kappa B by tumor necrosis factor in primary cells suggests that this cytokine fulfills the requirement for oxidation, possibly by inducing the production of free radicals.

Arachidonic acid regulates the binding of human interferon in human skin fibroblasts.

The induction of the antiviral state in human fibroblasts by human interferon is inhibited by arachidonic acid, its analogues 5,8,11,14-eicosatetraynoic and 5,8,11-eicosatriynoic acids, as well as by sodium arachidonate. The fatty acids myristic or oleic acid and sodium palmitate do not inhibit the antiviral action of interferon. Experiments were conducted to investigate the mechanism by which arachidonic acid could inhibit the action of interferon. No correlation between cellular lipoxygenase activities and the inhibition of antiviral action of interferon was observed in the fatty acid treated cells. Likewise, the cyclooxygenase inhibitors indomethacin and oxyphenylbutazone do not inhibit the interferon-induced antiviral state. Taken together, the inhibition of interferon action by arachidonates is unlikely to be mediated by cyclooxygenase or lipoxygenase-generated intermediates, even though arachidonates are known to affect the activity of these enzymes in vitro. Measurement of interferon receptors in the fatty acid treated cells showed that arachidonic acid, sodium arachidonate and its analogues decreased the number of human type I interferon receptors available for binding, and inhibited the transcription of the interferon-induced 6-16 gene and the induction of cellular (2'-5')-oligoadenylate synthetase, suggesting the mechanism of inhibition is mediated at the level of the interferon receptor. The significance of the finding that arachidonic acid, a common fatty acid of cells and serum, can affect the antiviral action of interferon is discussed.