The Abl-family non-receptor tyrosine kinases are essential regulators of the cytoskeleton. They transduce diverse extracellular cues into cytoskeletal rearrangements that have dramatic effects on cell motility and morphogenesis. Recent biochemical and genetic studies have revealed several mechanisms that Abl-family kinases use to mediate these effects. Abl-family kinases stimulate actin polymerization through the activation of cortactin, hematopoietic lineage cell-specific protein (HS1), WASp-and WAVE-family proteins, and Rac1. They also attenuate cell contractility by inhibiting RhoA and altering adhesion dynamics. These pathways impinge on several physiological processes, including development and maintenance of the nervous and immune systems, and epithelial morphogenesis. Elucidating how Abl-family kinases are regulated, and where and when they coordinate cytoskeletal changes, is essential for garnering a better understanding of these complex processes.

The RhoA (Rho) GTPase is a master regulator of dendrite morphogenesis. Rho activation in developing neurons slows dendrite branch dynamics, yielding smaller, less branched dendrite arbors. Constitutive activation of Rho in mature neurons causes dendritic spine loss and dendritic regression, indicating that Rho can affect dendritic structure and function even after dendrites have developed. However, it is unclear whether and how endogenous Rho modulates dendrite and synapse morphology after dendrite arbor development has occurred. We demonstrate that a Rho inhibitory pathway involving the Arg tyrosine kinase and p190RhoGAP is essential for synapse and dendrite stability during late postnatal development. Hippocampal CA1 pyramidal dendrites develop normally in arg Ϫ/Ϫ mice, reaching their mature size by postnatal day 21 (P21). However, dendritic spines do not undergo the normal morphological maturation in these mice, leading to a loss of hippocampal synapses and dendritic branches by P42. Coincident with this synapse and dendrite loss, arg Ϫ/Ϫ mice exhibit progressive deficits in a hippocampus-dependent object recognition behavioral task. p190RhoGAP localizes to dendritic spines, and its activity is reduced in arg Ϫ/Ϫ hippocampus, leading to increased Rho activity. Although mutations in p190rhogap enhance dendritic regression resulting from decreased Arg levels, reducing gene dosage of the Rho effector ROCKII can suppress the dendritic regression observed in arg Ϫ/Ϫ mice. Together, these data indicate that signaling through Arg and p190RhoGAP acts late during synaptic refinement to promote dendritic spine maturation and synapse/dendrite stability by attenuating synaptic Rho activity.

The protein kinase BRAF is a key component of the RAS-RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAF V600E in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAF V600 -expressing cell lines and inhibiting tumor growth in BRAF V600E bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAF V600E -mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206).The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAF V600E mutation.Cancer Res; 72(3); 779-89. Ó2011 AACR.

The Rho family GTPases RhoA (Rho), Rac1, and Cdc42 are essential effectors of integrin-mediated cell attachment and spreading. Rho activity, which promotes formation of focal adhesions and actin stress fibers, is inhibited upon initial cell attachment to allow sampling of the new adhesive environment. The Abl-related gene (Arg) tyrosine kinase mediates adhesion-dependent inhibition of Rho through phosphorylation and activation of the Rho inhibitor p190RhoGAP-A (p190). p190 phosphorylation promotes its binding to p120RasGAP (p120). Here, we elucidate the mechanism by which p120 binding regulates p190 activation after adhesion. We show that p190 requires its p120-binding domain to undergo Arg-dependent activation in vivo. However, p120 binding does not activate p190RhoGAP activity in vitro. Instead, activation of p190 requires recruitment to the cell periphery. Integrin-mediated adhesion promotes relocalization of p190 and p120 to the cell periphery in wild-type fibroblasts, but not in arg(-/-) fibroblasts. A dominant-negative p120 fragment blocks p190:p120 complex formation, prevents activation of p190 by adhesion, and disrupts the adhesion-dependent recruitment of p190 to the cell periphery. Our results demonstrate that integrin signaling through Arg activates p190 by promoting its association with p120, resulting in recruitment of p190 to the cell periphery where it inhibits Rho.

Integrins are heterodimeric extracellular matrix receptors that are essential for the proper development of the vertebrate nervous system. We report here that selective loss of integrin β1 in excitatory neurons leads to reductions in the size and complexity of hippocampal dendritic arbors, hippocampal synapse loss, impaired hippocampus-dependent learning, and exaggerated psychomotor sensitivity to cocaine in mice. Our biochemical and genetic experiments demonstrate that the intracellular tail of integrin β1 binds directly to Arg kinase, and that this interaction stimulates activity of the Arg substrate p190RhoGAP, an inactivator of the RhoA GTPase. Moreover, genetic manipulations that reduce integrin β1 signaling through Arg recapitulate the integrin β1 knockout phenotype in a gene-dose sensitive manner. Together, these results describe a novel integrin β1-Arg-p190RhoGAP pathway that regulates dendritic arbor size, promotes synapse maintenance, supports proper hippocampal function, and mitigates the behavioral consequences of cocaine exposure.