Sceptrin, a marine natural compound, inhibits cell motility in a variety of cancer cell lines.

Sceptrin, a natural compound produced by various marine sponges, was tested for its effect on cell motility. We report for the first time that sceptrin inhibits cell motility in several cancer cell lines. The compound shows no toxicity at concentrations that are double the amount of sceptrin required for maximal inhibitory effect. Both random and factor-induced migration were impaired, suggesting that sceptrin targets a central process of cell motility machinery. Activity of de novo synthesized sceptrin was indistinguishable from sceptrin purified from Agelas nakamurai, and the inhibitory activity was found to be, at least partially, due to sceptrin's capability to inhibit cell contractility. Additionally, sceptrin was found to bind to monomeric actin, further suggesting a mechanism involving the actin cytoskeleton. Close analogues of sceptrin were synthesized, tested for their effect on cell motility, and found to be either equimolar or less potent compared to the parental compound. Inadvertent cell motility is a key contributing factor in various human diseases, including cancer and chronic inflammation. Marine compounds isolated from sponges have been proven to be an excellent source of metabolites that show biological activities. Given the recently achieved total synthesis of sceptrin in multigram quantities, sceptrin could prove to be an attractive lead molecule for further preclinical testing and development for therapeutic purposes, as well as a useful research tool to elucidate the mechanisms involved in cell motility.

Essential function for the GTPase TC21 in homeostatic antigen receptor signaling.

T cell antigen receptors (TCRs) and B cell antigen receptors (BCRs) transmit low-grade signals necessary for the survival and maintenance of mature cell pools. We show here that TC21, a small GTPase encoded by Rras2, interacted constitutively with both kinds of receptors. Expression of a dominant negative TC21 mutant in T cells produced a rapid decrease in cell viability, and Rras2(-/-) mice were lymphopenic, possibly as a result of diminished homeostatic proliferation and impaired T cell and B cell survival. In contrast, TC21 was overexpressed in several human lymphoid malignancies. Finally, the p110delta catalytic subunit of phosphatidylinositol-3-OH kinase (PI(3)K) was recruited to the TCR and BCR in a TC21-dependent way. Consequently, we propose TC21 directly links antigen receptors to PI(3)K-mediated survival pathways.

Role of the diacylglycerol kinase alpha-conserved domains in membrane targeting in intact T cells.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGKalpha is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGKalpha domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr(335). This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.

Abl functions as a negative regulator of Met-induced cell motility via phosphorylation of the adapter protein CrkII.

HGF, the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, which are processes that play a crucial role in cell motility during normal development and malignant transformation. We and others have shown earlier that the adapter protein CrkII and its associated proteins positively regulate cell migratory events in response to both haptotactic and chemotactic stimuli, including HGF. Here, we demonstrate for the first time that phosphorylation of CrkII serves as a negative feedback loop to regulate motile responses upon Met stimulation. Thus, we found that the treatment of cells with HGF induces tyrosine phosphorylation of CrkII at Y221, which in turn results in inhibition of CrkII signaling via formation of an intramolecular pY221-SH2-domain interaction. Accordingly, expression of a mutant form of CrkII, CrkII-Y221F, which is resistant to phosphorylation at this negative regulatory site, enhanced Met-induced cell motility. Furthermore, we demonstrate here that the Met-induced CrkII phosphorylation depends on the Abl tyrosine kinase activity. As a corollary, we found that Abl inhibitors, such as the STI571 compound, significantly enhanced Met-induced cell motility, but failed to do so in cells that expressed the CrkII-Y221F mutant protein. Taken together, these results demonstrate that the Abl tyrosine kinase functions as a negative regulator of Met-induced cell migration, and that it does so by inducing CrkII phosphorylation at the site Y221.

Caspase-8 promotes cell motility and calpain activity under nonapoptotic conditions.

Significant caspase-8 activity has been found in normal and certain tumor cells, suggesting that caspase-8 possesses an alternative, nonapoptotic function that may contribute to tumor progression. In this article, we report that caspase-8 promotes cell motility. In particular, caspase-8 is required for the optimal activation of calpains, Rac, and lamellipodial assembly. This represents a novel nonapoptotic function of caspase-8 acting at the intersection of the caspase-8 and calpain proteolytic pathways to coordinate cell death versus cell motility signaling.

Regulation of diacylglycerol kinase alpha by phosphoinositide 3-kinase lipid products.

Diacylglycerol kinase alpha (DAGK alpha), like all type I DAGKs, has calcium regulatory motifs that act as negative regulators of enzyme activity and localization. Accordingly, DAGK alpha is activated by phospholipase C-coupled receptors in a calcium-dependent manner. One of the first functions attributed to DAGK alpha in lymphocytes was that of regulating interleukin 2-induced cell cycle entry. Interleukin-2 nonetheless exerts its action in the absence of cytosolic calcium increase. We have studied alternative receptor-derived signals to explain calcium-independent DAGK alpha activation, and show that DAGK alpha is stimulated by Src-like kinase-dependent phosphoinositide 3 kinase (PI3K) activation in lymphocytes. Our results demonstrate that, in vivo, the increase in cellular levels of PI3K products is sufficient to induce DAGK alpha activation, allowing DAGK alpha relocation to the intact lymphocyte plasma membrane. This activation is isoform-specific, because other type I DAGKs are not subject to this type of regulation. These studies are the first to describe a pathway in which, in the absence of receptor-regulated calcium increase, DAGK alpha activation and membrane localization is a direct consequence of PI3K activation.