The calcium/calcineurin pathway promotes hemidesmosome stability through inhibition of ß4 integrin phosphorylation.

Cell migration depends on cells being able to create and disassemble adhesive contacts. Hemidesmosomes are multiprotein structures that attach epithelia to basal lamina and disassemble during migration and carcinoma invasion. Phosphorylation of the ß4 integrin, a hemidesmosome component, induces disassembly. Although kinases involved in ß4 phosphorylation have been identified, little is known about phosphatases countering kinase action. Here we report that calcineurin, a serine-threonine protein phosphatase, regulates ß4 phosphorylation. Calcineurin inhibitor cyclosporin A (CsA) and calcineurin-siRNA increase ß4 phosphorylation, induce hemidesmosome disassembly, and increase migration in HaCat keratinocytes, suggesting that calcineurin negatively regulates ß4 phosphorylation. We found no direct dephosphorylation of ß4 by calcineurin or association between ß4 and calcineurin, suggesting indirect regulation of ß4 phosphorylation. We therefore assessed calcineurin influence on MAPK and PKC, known to phosphorylate ß4. CsA increased MAPK activity, whereas MAPK inhibitors reduced CsA-induced ß4 phosphorylation, suggesting that calcineurin restricts ß4 phosphorylation by MAPK. Calcineurin is activated by calcium. Increased [Ca(2+)](i) reduces ß4 phosphorylation and stabilizes hemidesmosomes, effects that are reversed by CsA, indicating that calcineurin mediates calcium effects on ß4. However, MAPK activation is increased when [Ca(2+)](i) is increased, suggesting that calcineurin activates an additional mechanism that counteracts MAPK-induced ß4 phosphorylation. Interestingly, in some squamous cell carcinoma cells, which have reduced hemidesmosomes and increased ß4 phosphorylation, an increase in [Ca(2+)](i) using thapsigargin, bradykinin, or acetylcholine can increase hemidesmosomes and reduce ß4 phosphorylation in a calcineurin-dependent manner. These findings have implications in calcineurin-inhibitor induced carcinoma, a complication of immunosuppressive therapy.

Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase.

CD36 plays a critical role in the inhibition of angiogenesis through binding to the type 1 repeats of thrombospondin-1 (TSP-1) and activating Fyn tyrosine kinase and MAPK pathways. Here, we reveal a novel association of CD36 with VEGFR-2 and spleen tyrosine kinase (Syk). We also address the correlation between the expression of CD36 and Syk by demonstrating that overexpression of CD36 in HUVECs up-regulates endogenous Syk expression. We also define a new role for TSP-1 and CD36 in the activation of the VEGFR-2 signaling pathway that requires Syk. Our findings also identify a role for Syk as a stimulator of VEGF-A-induced angiogenesis by increasing phosphorylation of Y1175 in VEGFR-2, which is a major tyrosine for promoting VEGF-A-induced endothelial cell migration. Together, these studies introduce a new signaling pathway for TSP-1, CD36, and Syk, and address the role of these proteins in regulating the angiogenic switch.

Palmitoylation by DHHC3 is critical for the function, expression, and stability of integrin α6ß4.

The laminin-binding integrin α6ß4 plays key roles in both normal epithelial and endothelial cells and during tumor cell progression, metastasis, and angiogenesis. Previous cysteine mutagenesis studies have suggested that palmitoylation of α6ß4 protein supports a few integrin-dependent functions and molecular associations. Here we took another approach and obtained strikingly different results. We used overexpression and RNAi knockdown in multiple cell types to identify protein acyl transferase DHHC3 as the enzyme responsible for integrin ß4 and α6 palmitoylation. Ablation of DHHC3 markedly diminished integrin-dependent cellular cable formation on Matrigel, integrin signaling through Src, and ß4 phosphorylation on key diagnostic amino acids (S1356 and 1424). However, unexpectedly, and in sharp contrast to prior α6ß4 mutagenesis results, knockdown of DHHC3 accelerated the degradation of α6ß4, likely due to an increase in endosomal exposure to cathepsin D. When proteolytic degradation was inhibited (by Pepstatin A), rescued α6ß4 accumulated intracellularly, but was unable to reach the cell surface. DHHC3 ablation effects were strongly selective for α6ß4. Cell-surface levels of ~10 other proteins (including α3ß1) were not diminished, and the appearance of hundreds of other palmitoylated proteins was not altered. Results obtained here demonstrate a new substrate for the DHHC3 enzyme and provide novel opportunities for modulating α6ß4 expression, distribution, and function.

Role of ß4 integrin phosphorylation in human invasive squamous cell carcinoma: regulation of hemidesmosome stability modulates cell migration.

Hemidesmosomes (HDs) are multiprotein structures that anchor epithelia to the basement membrane. During squamous cell carcinoma (SCC) invasion, there is a reduction in the number of HDs, which may facilitate dissemination. Mechanisms of HD disassembly are incompletely understood. Previous work has shown that epidermal growth factor (EGF)-induced phosphorylation of the ß4 integrin on three of its serines, S(1356)S(1360)S(1364), can induce HD disassembly in normal cells. Here, we examine the role of ß4 integrin serine phosphorylation in SCC. We have found that around 60% of invasive cutaneous SCC show increased ß4 phosphorylation on S(1356) when compared with carcinoma in situ or normal tissue. To assess the mechanisms by which SCC increases ß4 phosphorylation, we performed in vitro analyses. Compared with keratinocytes, SCC cells showed increased levels of S(1356) phosphorylation in the absence of EGF, correlating with reduced HD-like structures. In addition, phospho-S(1356) signal was largely segregated from other HD components. Epidermal growth factor receptor and PKC inhibitors inhibited basal levels of S(1356) phosphorylation in SCC, suggesting that cells use intrinsic mechanisms to activate the EGF signaling pathway to induce ß4 phosphorylation. Moreover, these inhibitors stabilized HD-like structures in SCC cells and reduced their migratory ability. Mutation of S(1356)S(1360)S(1364) in SCC cells to non-phosphorylatable alanines stabilized HD-like structures and substantially reduced migration, while mutation into phosphorylation mimicking aspartate reduced HD-like structures but had no effect on migration, suggesting that serine phosphorylation function is releasing anchorage rather than promoting migration. Altogether these results suggest that ß4 serine phosphorylation may have an important role during SCC invasion by destabilizing HDs and facilitating migration.

Protein kinase C-alpha phosphorylation of specific serines in the connecting segment of the beta 4 integrin regulates the dynamics of type II hemidesmosomes.

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 alpha 6 beta 4 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 beta 4 integrin subunit. Using a combination of biochemical and mutational analyses, we demonstrate that EGF induces the phosphorylation of three specific serine residues (S(1356), S(1360), and S(1364)) located within the connecting segment of the beta 4 subunit and that phosphorylation on these residues accounts for the bulk of beta 4 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 alpha 6 beta 4 integrin, we observed that expression of a beta 4 construct containing Ser-->Ala mutations of S(1356), S(1360), and S(1364) 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-alpha (PKC-alpha) 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 beta 4, and argue for a key role for PKC-alpha in regulating these structures.

Ribosomal S6 kinase (RSK) regulates phosphorylation of filamin A on an important regulatory site.

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 Ser(2152) in response to mitogens. Inhibition of MAP kinase signaling with UO126 or mutation of Ser(2152) to Ala on FLNa prevents epidermal growth factor (EGF)-stimulated phosphorylation of FLNa in vivo. Furthermore, phosphorylation of FLNa on Ser(2152) 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 Ser(2152) in vivo. Given that phosphorylation of FLNa on Ser(2152) is required for Pak1-mediated membrane ruffling, our results suggest a novel role for RSK in the regulation of the actin cytoskeleton.

Use of RNA interference to inhibit integrin (alpha6beta4)-mediated invasion and migration of breast carcinoma cells.

The application of small interfering RNA (siRNA) oligonucleotides to silence gene expression has profound implications for the intervention of human diseases including cancer. Using this technique, we explored the possibility that the alpha6beta4 integrin, a laminin adhesion receptor with a recognized role in the invasive phenotype of many carcinomas, represents a potential therapeutic target to inhibit the migration and invasion of carcinoma cells. We found that siRNA oligonucleotides targeted to either subunit of the alpha6beta4 integrin reduced cell surface expression of this integrin and resulted in decreased invasion of MDA-MB-231 breast carcinoma cells. Interestingly, reduced alpha6beta4 expression also promoted decreased migration on non-laminin substrata indicating that this integrin can function in a ligand-independent manner. In addition, the absence of beta4 expression in these cells augmented the formation of alpha6beta1 heterodimers and increased adhesion to laminin-1. Taken together, these results substantiate the importance of the alpha6beta4 integrin in invasion and migration that has been demonstrated previously by expression of the beta4 subunit in beta4-deficient cell lines and by function blocking antibodies. Furthermore, these data suggest that the utilization of siRNA oligonucleotides to reduce the expression of the alpha6beta4 integrin may be a useful approach to prevent carcinoma cell progression.

Integrin-associated CD151 drives ErbB2-evoked mammary tumor onset and metastasis.

ErbB2+ human breast cancer is a major clinical problem. Prior results have suggested that tetraspanin CD151 might contribute to ErbB2-driven breast cancer growth, survival, and metastasis. In other cancer types, CD151 sometimes supports tumor growth and metastasis. However, a definitive test of CD151 effects on de novo breast cancer initiation, growth, and metastasis has not previously been done. We used CD151 gene-deleted mice expressing the MMTV-ErbB2 transgene to show that CD151 strongly supports ErbB2+ mammary tumor initiation and metastasis. Delayed tumor onset (by 70-100 days) in the absence of CD151 was accompanied by reduced survival of mammary epithelial cells and impaired activation of FAK- and MAPK-dependent pathways. Both primary tumors and metastatic nodules showed smooth, regular borders, consistent with a less invasive phenotype. Furthermore, consistent with impaired oncogenesis and decreased metastasis, CD151-targeted MCF-10A/ErbB2 cells showed substantial decreases in three-dimensional colony formation, EGF-stimulated tumor cell motility, invasion, and transendothelial migration. These CD151-dependent functions were largely mediated through α6ß4 integrin. Moreover, CD151 ablation substantially prevented PKC- and EGFR/ERK-dependent α6ß4 integrin phosphorylation, consistent with retention of epithelial cell polarity and intermediate filament cytoskeletal connections, which helps to explain diminished metastasis. Finally, clinical data analyses revealed a strong correlation between CD151 and ErbB2 expression and metastasis-free survival of breast cancer patients. In conclusion, we provide strong evidence that CD151 collaborates with LB integrins (particularly α6ß4 and ErbB2 (and EGFR) receptors to regulate multiple signaling pathways, thereby driving mammary tumor onset, survival, and metastasis. Consequently, CD151 is a useful therapeutic target in malignant ErbB2+ breast cancer.

Protein phosphatase 2A reactivates FOXO3a through a dynamic interplay with 14-3-3 and AKT.

Forkhead box transcription factor FOXO3a, a key regulator of cell survival, is regulated by reversible phosphorylation and subcellular localization. Although the kinases regulating FOXO3a activity have been characterized, the role of protein phosphatases (PP) in the control of FOXO3a subcellular localization and function is unknown. In this study, we detected a robust interaction between FOXO3a and PP2A. We further demonstrate that 14-3-3, while not impeding the interaction between PP2A and FOXO3a, restrains its activity toward AKT phosphorylation sites T32/S253. Disruption of PP2A function revealed that after AKT inhibition, PP2A-mediated dephosphorylation of T32/S253 is required for dissociation of 14-3-3, nuclear translocation, and transcriptional activation of FOXO3a. Our findings reveal that distinct phosphatases dephosphorylate conserved AKT motifs within the FOXO family and that PP2A is entwined in a dynamic interplay with AKT and 14-3-3 to directly regulate FOXO3a subcellular localization and transcriptional activation.

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.

Akt blocks breast cancer cell motility and invasion through the transcription factor NFAT.

The phosphoinositide 3-kinase (PI 3-K) signaling axis is intimately associated with deregulated cancer cell growth, primarily by promoting increased survival through Akt/PKB (protein kinase B). However, there is relatively little information on the role of Akt in cancer cell motility, a key phenotype of invasive carcinomas. Here we report that activation of Akt inhibits carcinoma migration and invasion of breast cancer cells. Conversely, downregulation of Akt using RNA interference increased migration and invasion. Akt blunts invasion by inhibiting the transcriptional activity of NFAT (nuclear factor of activated T cells). Specifically, signaling through Akt reduces NFAT expression levels due to ubiquitination and proteasomal degradation, mediated by the E3 ubiquitin ligase HDM2. These results indicate that while Akt can promote tumor progression through increased cell survival mechanisms, it can block breast cancer cell motility and invasion by a mechanism that depends, at least in part, on the NFAT transcription factor.

RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development.

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.