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Although the regulation of hemidesmosome dynamics during processes such as epithelial migration, wound healing, and carcinoma invasion is important, the mechanisms involved are poorly understood. The integrin α6β4 is an essential component of the hemidesmosome and a target of such regulation. Epidermal growth factor (EGF) can induce hemidesmosome disassembly by a mechanism that involves serine phosphorylation of the β4 integrin subunit. Using a combination of biochemical and mutational analyses, we demonstrate that EGF induces the phosphorylation of three specific serine residues (S1356, S1360, and S1364) located within the connecting segment of the β4 subunit and that phosphorylation on these residues accounts for the bulk of β4 phosphorylation stimulated by EGF. Importantly, phosphorylation of these serines is critical for the ability of EGF to disrupt hemidesmosomes. Using COS-7 cells, which assemble hemidesmosomes type II upon exogenous expression of the α6β4 integrin, we observed that expression of a β4 construct containing Ser-→Ala mutations of S1356, S1360, and S1364 reduced the ability of EGF to disrupt hemidesmosomes and that this effect appears to involve cooperation among these phosphorylation sites. Moreover, expression of Ser→Asp mutants that mimic constitutive phosphorylation reduced hemidesmosome formation. Protein kinase C-α (PKC-α) is the kinase responsible for phosphorylating at least two of these serines, based on in vitro kinase assays, peptide mapping, and mutational analysis. Together, these results highlight the importance of serine phosphorylation in regulating type II hemidesmosome disassembly, implicate a cluster of serine residues within the connecting segment of β4, and argue for a key role for PKC-α in regulating these structures. [ABSTRACT FROM AUTHOR]

The Ras-mitogen-activated protein (Ras.MAP) kinase pathway regulates various cellular processes, including gene expression, cell proliferation, and survival. Ribosomal S6 kinase (RSK), a key player in this pathway, modulates the activities of several cytoplasmic and nuclear proteins via phosphorylation. Here we report the characterization of the cytoskeletal protein filamin A (FLNa) as a membrane-associated RSK target. We show that the N-terminal kinase domain of RSK phosphorylates FLNa on Ser2152 in response to mitogens. Inhibition of MAP kinase signaling with UO126 or mutation of Ser2152 to Ala on FLNa prevents epidermal growth factor (EGF)-stimulated phosphorylation of FLNa in vivo. Furthermore, phosphorylation of FLNa on Ser2152 is significantly enhanced by the expression of wild-type RSK and antagonized by kinase-inactive RSK or specific reduction of endogenous RSK. Strikingly, EGF-induced, FLNa-dependent migration of human melanoma cells is significantly reduced by UO126 treatment. Together, these data provide substantial evidence that RSK phosphorylates FLNa on Ser2152 in vivo. Given that phosphorylation of FLNa on Ser2152 is required for Pak1-mediated membrane ruffling, our results suggest a novel role for RSK in the regulation of the actin cytoskeleton. [ABSTRACT FROM AUTHOR]

Blood vessel formation is a complex morphological process that is only beginning to be understood at the molecular level. In this study, we demonstrate a novel and critical role for the small GTPase, RhoB, in vascular development. RhoB null mice have retarded vascular development in the retina characterized by altered sprout morphology. Moreover, pharmaceutical means to deplete RhoB in neonatal rats is associated with apoptosis in the sprouting endothelial cells of newly forming vessels. Similarly, acute depletion of RhoB by antisense or dominant-negative strategies in primary endothelial cell culture models led to apoptosis and failures in tube formation. We identified a novel link between RhoB and the Akt survival signaling pathway to explain these changes. Confocal microscopy revealed that RhoB is highly localized to the nuclear margin with a small percentage found inside the nucleus. Similarly, total Akt is throughout the cell but has increased accumulation at the nuclear margin, and active phosphorylated Akt is found primarily inside the nucleoplasm, where it partially colocalizes with the RhoB therein. We show that this colocalization is functionally relevant, because when RhoB was depleted, Akt was excluded from the nucleus and total cellular Akt protein was decreased in a proteosome-dependent manner. Because the function of RhoB in vivo appears to only be rate limiting for endothelial cell sprouting, we propose that RhoB has a novel stage-specific function to regulate endothelial cell survival during vascular development. RhoB may offer a therapeutic target in diseases such as cancer, diabetic retinopathy, and macular degeneration, where the disruption of sprouting angiogenesis would be desirable. [ABSTRACT FROM AUTHOR]

Examines the activation of the alpha6beta4 integrin and its association with F-actin in cell protrusions during chemotactic migration of carcinoma cells. Activation of protein kinase C-alpha; Stimulation of A431 chemotaxis; Integrin mobilization from hemidesmosomes by indirect immunofluorescence microscopy.

Presents information pertaining to the integrin ...6B4 which is a receptor for the laminins. Reinforcement of the importance of the integrin in epithelial structure; Inhibition of the formation of lamellae in cells; Indication of results.

Here we provide the first evidence that tetraspanin CD151 can support de novo carcinogenesis. During two-stage mouse skin chemical carcinogenesis, CD151 reduces tumor lag time and increases incidence, multiplicity, size, and progression to malignant squamous cell carcinoma (SCC), while supporting both cell survival during tumor initiation and cell proliferation during the promotion phase. In human skin SCC, CD151 expression is selectively elevated compared to other skin cancer types. CD151 support of keratinocyte survival and proliferation may depend on activation of transcription factor STAT3, a regulator of cell proliferation and apoptosis. CD151 also supports PKCα-α6β4 integrin association and PKC-dependent β4 S1424 phosphorylation, while regulating α6β4 distribution. CD151-PKCα effects on integrin β4 phosphorylation and subcellular localization are consistent with epithelial disruption to a less polarized, more invasive state. CD151 ablation, while minimally affecting normal cell and normal mouse functions, markedly sensitized mouse skin and epidermoid cells to chemicals/drugs including DMBA (mutagen) and camptothecin (topoisomerase inhibitor), as well as to agents targeting EGFR, PKC, Jak2/Tyk2, and STAT3. Hence, CD151 ‘co-targeting’ may be therapeutically beneficial. These findings not only support CD151 as a potential tumor target, but also should apply to other cancers utilizing CD151-laminin-binding integrin complexes.