PKC Regulation of an 5 Integrin-ZO-1 Complex Controls Lamellae Formation in Migrating Cancer Cells

Medical Biotechnology, VTT Technical Research Centre of Finland and University of Turku, FIN-20520 Turku, Finland.
Science Signaling (Impact Factor: 6.28). 02/2009; 2(77):ra32. DOI: 10.1126/scisignal.2000135
Source: PubMed


Disruption of intercellular adhesions, increased abundance of alpha(5)beta(1) integrin, and activation of protein kinase Cepsilon (PKCepsilon) correlate with invasion and unfavorable prognosis in lung cancer. However, it remains elusive how these distinct factors contribute to the invasive behavior of cancer cells. Persistent cell motility requires the formation of stable lamellae at the leading edge of a migrating cell. Here, we report that the tight junction protein zonula occludens-1 (ZO-1) preferentially interacts with alpha(5)beta(1) integrin at the lamellae of migrating cells. Disruption of ZO-1 binding to an internal PDZ-binding motif in the alpha(5) cytoplasmic tail prevented the polarized localization of ZO-1 and alpha(5) at the leading edge. Furthermore, silencing of alpha(5) integrin inhibited migration and invasion of lung cancer cells, and silencing of ZO-1 resulted in increased Rac activity and reduced directional cell motility. The formation of the alpha(5)-ZO-1 complex was dependent on PKCepsilon: Phosphorylation of ZO-1 at serine-168 regulated the subcellular localization of ZO-1 and thus controlled its association with alpha(5) integrin. In conclusion, PKCepsilon activation drives the formation of a spatially restricted, promigratory alpha(5)-ZO-1 complex at the leading edge of lung cancer cells.

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Available from: Tiina Öhman, Aug 05, 2015
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    • "This is in agreement with the reported occasional collapse back of the daughter cells in dividing PKCε null cells and indicates that PKCε may play a complex role in multiple different stages of mitosis [14]. We have shown earlier that the subcellular localization of ZO-1 is regulated, at least partly, by PKCε-dependent phosphorylation of serine 168 in ZO-1 [15]. To test the requirement for this site during cytokinesis, cells were transfected with Flag-tagged WT, S168A or S168D ZO-1 constructs and stained for tubulin, Flag and dapi. "
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    ABSTRACT: Recently, we demonstrated that integrin adhesion to the extracellular matrix at the cleavage furrow is essential for cytokinesis of adherent cells. Here, we report that tight junction protein ZO-1 (Zonula Occludens-1) is required for successful cytokinesis in NCI-H460 cells plated on fibronectin. This function of ZO-1 involves interaction with the cytoplasmic domain of α5-integrin to facilitate recruitment of active fibronectin-binding integrins to the base of the cleavage furrow. In the absence of ZO-1, or a functional ZO-1/α5β1-integrin complex, proper actin-dependent constriction between daughter cells is impaired and cells fail cytokinesis. Super-resolution microscopy reveals that in ZO-1 depleted cells the furrow becomes delocalized from the matrix. We also show that PKCε-dependent phosphorylation at Serine168 is required for ZO-1 localization to the furrow and successful cell division. Altogether, our results identify a novel regulatory pathway involving the interplay between ZO-1, α5-integrin and PKCε in the late stages of mammalian cell division.
    Full-text · Article · Aug 2013 · PLoS ONE
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    • "While a large body of evidence links PKC to the promotion of cancer cell invasion and metastatic dissemination, the specific roles of individual PKCs remain only partially understood. [17], [18], [19], [20], [21]. Most notably, phorbol esters promote actin cytoskeleton reorganization and stimulate cell motility in a number of cell lines [22], [23]. "
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    ABSTRACT: Protein kinase C (PKC) ε, a key signaling transducer implicated in mitogenesis, survival, and cancer progression, is overexpressed in human primary non-small cell lung cancer (NSCLC). The role of PKCε in lung cancer metastasis has not yet been established. Here we show that RNAi-mediated knockdown of PKCε in H358, H1299, H322, and A549 NSCLC impairs activation of the small GTPase Rac1 in response to phorbol 12-myristate 13-acetate (PMA), serum, or epidermal growth factor (EGF). PKCε depletion markedly impaired the ability of NSCLC cells to form membrane ruffles and migrate. Similar results were observed by pharmacological inhibition of PKCε with εV1-2, a specific PKCε inhibitor. PKCε was also required for invasiveness of NSCLC cells and modulated the secretion of extracellular matrix proteases and protease inhibitors. Finally, we found that PKCε-depleted NSCLC cells fail to disseminate to lungs in a mouse model of metastasis. Our results implicate PKCε as a key mediator of Rac signaling and motility of lung cancer cells, highlighting its potential as a therapeutic target.
    Full-text · Article · Feb 2012 · PLoS ONE
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    • "MAST2, by contrast, is a poorly studied microtubule-associated serine-threonine kinase. Interestingly, it contains a PDZ domain (Lumeng et al., 1999; Prehaud et al., 2010), which could interact with integrins and influence their function similarly to ZO-1 binding to a5 integrin through its PDZ domains (Tuomi et al., 2009). Furthermore, MAST2 has been shown to phosphorylate and stabilize the tumour suppressor PTEN, which could influence integrin activity by upregulation of the PI3K–AKT pathway (Valiente et al., 2005). "
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    ABSTRACT: β1 integrins constitute a large group of widely distributed adhesion receptors, which regulate the ability of cells to interact with their surroundings. This regulation of the expression and activity of integrins is crucial for tissue homeostasis and development and contributes to inflammation and cancer. We report an RNA interference screen to uncover genes involved in the regulation of β1-integrin activity using cell spot microarray technology in cancer cell lines. Altogether, ten cancer and two normal cell lines were used to identify regulators of β1 integrin activity. Cell biological analysis of the identified β1-integrin regulatory genes revealed that modulation of integrin activity can influence cell invasion in a three-dimensional matrix. We demonstrate with loss-of-function and rescue experiments that CD9 activates and MMP8 inactivates β1 integrins and that both proteins associate with β1 integrins in cells. Furthermore, CD9 and MMP8 regulate cancer cell extravasation in vivo. Our discovery of new regulators of β1-integrin activity highlight the complexity of integrin activity regulation and provide a set of new genes involved in regulation of integrin function.
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