Robin M. Scaife

A Mouse with a Loss-of-function Mutation in the c-Cbl TKB Domain Shows Perturbed Thymocyte Signaling without Enhancing the Activity of the ZAP-70 Tyrosine Kinase

The unique tyrosine kinase binding (TKB) domain of Cbl targets phosphorylated tyrosines on activated protein tyrosine kinases (PTKs); this targeting is considered essential for Cbl proteins to negatively regulate PTKs. Here, a loss-of-function mutation (G304E) in the c-Cbl TKB domain, first identified in Caenorhabditis elegans, was introduced into a mouse and its effects in thymocytes and T cells were studied. In marked contrast to the c-Cbl knockout mouse, we found no evidence of enhanced activity of the ZAP-70 PTK in thymocytes from the TKB domain mutant mouse. This finding contradicts the accepted mechanism of c-Cbl–mediated negative regulation, which requires TKB domain targeting of phosphotyrosine 292 in ZAP-70. However, the TKB domain mutant mouse does show aspects of enhanced signaling that parallel those of the c-Cbl knockout mouse, but these involve the constitutive activation of Rac and not enhanced PTK activity. Furthermore, the enhanced signaling in CD4+CD8+ double positive thymocytes appears to be compensated by the selective down-regulation of CD3 on mature thymocytes and peripheral T cells from both strains of mutant c-Cbl mice.

The multi-adaptor proto-oncoprotein Cbl is a key regulator of Rac and actin assembly

The induction of protein tyrosine kinase signaling pathways is a principal mechanism for promoting cellular activation. Biochemical and genetic analyses have implicated the multi-adaptor proto-oncogene protein Cbl as a key negative regulator of activated protein tyrosine kinases. By inhibiting the function of Cbl as a multi-domain adaptor protein, through expression of a truncated form (480-Cbl), we demonstrate that Cbl is a potent negative regulator of actin assembly in response to receptor tyrosine kinase (RTK) activation. Expression of 480-Cbl dramatically enhances RTK-dependent induction of actin dorsal ruffles, which correlates with a pronounced increase in Rac activation. By contrast, mitogenic signaling by RTK targets, such as PI 3-kinase and MAP kinases, as well as RTK-mediated tyrosine phosphorylation do not appear to be affected by 480-Cbl expression. Further, we determined that Cbl undergoes a striking RTK-activation-dependent translocation to sites of active actin dorsal ruffle nucleation. Hence, the selective regulation of RTK signaling to the actin cytoskeleton appears to result from recruitment of signaling proteins on a Cbl template bound to the actin cytoskeleton.

Selective and irreversible cell cycle inhibition by diphenyleneiodonium.

Because cell proliferation is subject to checkpoint-mediated regulation of the cell cycle, pharmacophores that target cell cycle checkpoints have been used clinically to treat human hyperproliferative disorders. It is shown here that the flavoprotein inhibitor diphenyleneiodionium can block cell proliferation by targeting of cell cycle checkpoints. Brief exposure of mitotically arrested cells to diphenyleneiodonium induces a loss of the mitotic cell morphology, and this corresponds with a decrease in the levels of the mitotic markers MPM2 and phospho-histone H3, as well as a loss of centrosome maturation, spindle disassembly, and redistribution of the chromatin remodeling helicase ATRX. Surprisingly, this mitotic exit resulted in a tetraploidization that persisted long after drug release. Analogously, brief exposure to diphenyleneiodonium also caused prolonged arrest in G1 phase. By contrast, diphenyleneiodonium exposure did not abrogate S phase, although it did result in a subsequent block of G2 cell cycle progression. This indicates that diphenyleneiodonium selectively targets components of the cell cycle, thereby either causing cell cycle arrest, or checkpoint override followed by cell cycle arrest. These irreversible effects of diphenyleneiodonium on the cell cycle may underlie its potent antiproliferative activity.

Paclitaxel inhibits osteoclast formation and bone resorption via influencing mitotic cell cycle arrest and RANKL‐induced activation of NF‐κB and ERK

Pathological bone destruction (osteolysis) is a hallmark of many bone diseases including tumor metastasis to bone, locally osteolytic giant cell tumor (GCT) of bone, and Pagets disease. Paclitaxel is frequently prescribed in the treatment of several malignant tumors where it has been shown to exert beneficial effects on bone lesions. However, the mechanism(s) through which paclitaxel regulates osteoclast formation and function remain ill defined. In the present study, we demonstrate that paclitaxel dose‐dependently inhibits receptor activator of nuclear factor‐kappa B ligand (RANKL)‐induced osteoclastogenesis in both RAW264.7 cells and mouse bone marrow macrophage (BMM) systems. In addition, paclitaxel treatment reduces the bone resorptive activity of human osteoclasts derived from GCT of bone, and attenuates lipopolysaccharide (LPS)‐induced osteolysis in a mouse calvarial model. Complementary cellular and biochemical analyses revealed that paclitaxel induces mitotic arrest of osteoclastic precursor cells. Furthermore, luciferase reporter gene assays and western blot analysis indicate that paclitaxel modulates key RANKL‐induced activation pathways that are essential to osteoclast formation including NF‐κB and ERK. Collectively, our findings demonstrate a role for paclitaxel in the regulation of osteoclast formation and function and uncover potential mechanism(s) through which paclitaxel alleviates pathological osteolysis. J. Cell. Biochem. 113: 946–955, 2012.

Microtubule Disassembly and Inhibition of Mitosis by a Novel Synthetic Pharmacophore

Microtubule drugs, which block cell cycle progression through mitosis, have seen widespread use in cancer chemotherapies. Although microtubules are subject to regulation by signal transduction mechanisms, their pharmacological modulation has so far relied on compounds that bind to the tubulin subunit. A new microtubule pharmacophore, diphenyleneiodonium, causing disassembly of the microtubule cytoskeleton is described here. Although this synthetic compound does not affect the assembly state of purified microtubules, it profoundly suppresses microtubule assembly in vivo, causes paclitaxel‐stabilized microtubules to cluster around the centrosomes, and selectively disassembles dynamic microtubules. Similar to other microtubule drugs, this new pharmacophore blocks mitotic spindle assembly and mitotic cell division. J. Cell. Biochem. 98: 102–114, 2006.