Phosphorylation of a novel site on the {beta}4 integrin at the trailing edge of migrating cells promotes hemidesmosome disassembly.

Hemidesmosomes (HDs) are multiprotein structures that anchor epithelial cells to the basement membrane. HD components include the alpha6beta4 integrin, plectin, and BPAGs (bullous pemphigoid antigens). HD disassembly in keratinocytes is necessary for cells to migrate and can be induced by EGF through beta4 integrin phosphorylation. We have identified a novel phosphorylation site on the beta4 integrin: S(1424). Preventing phosphorylation by mutating S-->A(1424) results in increased incorporation of beta4 into HDs and resistance to EGF-induced disassembly. In contrast, mutating S-->D(1424) (mimicking phosphorylation) partially mobilizes beta4 from HDs and potentiates the disassembly effects of other phosphorylation sites. In contrast to previously described sites that are phosphorylated upon growth factor stimulation, S(1424) already exhibits high constitutive phosphorylation, suggesting additional functions. Constitutive phosphorylation of S(1424) is distinctively enriched at the trailing edge of migrating keratinocytes where HDs are disassembled. Although most of this S(1424)-phosphorylated beta4 is found dissociated from HDs, a substantial amount can be associated with HDs near the cell margins, colocalizing with plectin but always excluding BPAGs, suggesting that phospho-S(1424) might be a mechanism to dissociate beta4 from BPAGs. S(1424) phosphorylation is PKC dependent. These data suggest an important role for S(1424) in the gradual disassembly of HDs induced by cell retraction.

Proteasome inhibition causes regression of leukemia and abrogates BCR-ABL-induced evasion of apoptosis in part through regulation of forkhead tumor suppressors.

BCR-ABL plays an essential role in the pathogenesis of chronic myeloid leukemia (CML) and some cases of acute lymphocytic leukemia (ALL). Although ABL kinase inhibitors have shown great promise in the treatment of CML, the persistence of residual disease and the occurrence of resistance have prompted investigations into the molecular effectors of BCR-ABL. Here, we show that BCR-ABL stimulates the proteasome-dependent degradation of members of the forkhead family of tumor suppressors in vitro, in an in vivo animal model, and in samples from patients with BCR-ABL-positive CML or ALL. As several downstream mediators of BCR-ABL are regulated by the proteasome degradation pathway, we also show that inhibition of this pathway, using bortezomib, causes regression of CML-like disease. Bortezomib treatment led to inhibition of BCR-ABL-induced suppression of FoxO proteins and their proapoptotic targets, tumor necrosis factor-related apoptosis-inducing ligand and BIM, thereby providing novel insights into the molecular effects of proteasome inhibitor therapy. We additionally show sensitivity of imatinib-resistant BCR-ABL T315I cells to bortezomib. Our data delineate the involvement of FoxO proteins in BCR-ABL-induced evasion of apoptosis and provide evidence that bortezomib is a candidate therapeutic in the treatment of BCR-ABL-induced leukemia.