Eri Asano

ARHGAP18, a GTPase activating protein for RhoA, controls cell shape, spreading and motility

Using a library of siRNAs, we found that ARHGAP18 was essential for the organization of actin stress fibers and focal adhesion. ARHGAP18 is one of the crucial factors for the regulation of RhoA in order to control cell motility and spreading.

ALX1 induces Snail expression to promote epithelial to mesenchymal transition and invasion of ovarian cancer cells

Ovarian cancer is a highly invasive and metastatic disease with a poor prognosis if diagnosed at an advanced stage, which is often the case. Recent studies argue that ovarian cancer cells that have undergone epithelial-to-mesenchymal transition (EMT) acquire aggressive malignant properties, but the relevant molecular mechanisms in this setting are not well-understood. Here, we report findings from an siRNA screen that identified the homeobox transcription factor ALX1 as a novel regulator of EMT. RNA interference-mediated attenuation of ALX1 expression restored E-cadherin expression and cell-cell junction formation in ovarian cancer cells, suppressing cell invasion, anchorage-independent growth, and tumor formation. Conversely, enforced expression of ALX1 in ovarian cancer cells or nontumorigenic epithelial cells induced EMT. We found that ALX1 upregulated expression of the key EMT regulator Snail (SNAI1) and that it mediated EMT activation and cell invasion by ALX1. Our results define the ALX1/Snail axis as a novel EMT pathway that mediates cancer invasion.

Misshapen-like kinase 1 (MINK1) Is a Novel Component of Striatin-interacting Phosphatase and Kinase (STRIPAK) and Is Required for the Completion of Cytokinesis

Background: Cytokinesis is regulated by phosphorylation and dephosphorylation of proteins. Results: MINK1 associated with STRN4, a regulatory subunit of PP2A, and depletion of either protein inhibited completion of cytokinesis. Conclusion: MINK1 and STRN4 are required for abscission, the final stage of cytokinesis. Significance: Our study reveals novel regulatory mechanisms for abscission. Cytokinesis is initiated by constriction of the cleavage furrow and terminated by abscission of the intercellular bridge that connects two separating daughter cells. The complicated processes of cytokinesis are coordinated by phosphorylation and dephosphorylation mediated by protein kinases and phosphatases. Mammalian Misshapen-like kinase 1 (MINK1) is a member of the germinal center kinases and is known to regulate cytoskeletal organization and oncogene-induced cell senescence. To search for novel regulators of cytokinesis, we performed a screen using a library of siRNAs and found that MINK1 was essential for cytokinesis. Time-lapse analysis revealed that MINK1-depleted cells were able to initiate furrowing but that abscission was disrupted. STRN4 (Zinedin) is a regulatory subunit of protein phosphatase 2A (PP2A) and was recently shown to be a component of a novel protein complex called striatin-interacting phosphatase and kinase (STRIPAK). Mass spectrometry analysis showed that MINK1 was a component of STRIPAK and that MINK1 directly interacted with STRN4. Similar to MINK1 depletion, STRN4-knockdown induced multinucleated cells and inhibited the completion of abscission. In addition, STRN4 reduced MINK1 activity in the presence of catalytic and structural subunits of PP2A. Our study identifies a novel regulatory network of protein kinases and phosphatases that regulate the completion of abscission.

Characterization of interaction between CLP36 and palladin

CLP36 is a member of the PDZ‐LIM family of proteins, which associates with α‐actinin and localizes to the actin cytoskeleton. CLP36 is involved in the formation of stress fibers and focal adhesions; however, the molecular mechanism of how CLP36 regulates stress fiber formation is still unknown. To investigate the physiological function of CLP36, we performed yeast two‐hybrid screening, and found that CLP36 interacts with palladin. Palladin is an important structural element of the actin cytoskeleton that is ubiquitously expressed and associates with α‐actinin. The interaction was dependent on the PDZ domain of CLP36 and the C‐terminus of palladin, and silencing of palladin suppressed localization of CLP36 to stress fibers. Overexpression of the PDZ domain of CLP36 also inhibited the localization of palladin to stress fibers, suggesting that the association of CLP36 and palladin is important for the localization of both proteins to stress fibers. Our experimental results indicate that α‐actinin, CLP36 and palladin form a protein complex and contribute to regulation of the actin cytoskeleton.

Role of Palladin Phosphorylation by Extracellular Signal-Regulated Kinase in Cell Migration

Phosphorylation of actin-binding proteins plays a pivotal role in the remodeling of the actin cytoskeleton to regulate cell migration. Palladin is an actin-binding protein that is phosphorylated by growth factor stimulation; however, the identity of the involved protein kinases remains elusive. In this study, we report that palladin is a novel substrate of extracellular signal-regulated kinase (ERK). Suppression of ERK activation by a chemical inhibitor reduced palladin phosphorylation, and expression of active MEK alone was sufficient for phosphorylation. In addition, an in vitro kinase assay demonstrated direct palladin phosphorylation by ERK. We found that Ser77 and Ser197 are essential residues for phosphorylation. Although the phosphorylation of these residues was not required for actin cytoskeletal organization, we found that expression of non-phosphorylated palladin enhanced cell migration. Finally, we show that phosphorylation inhibits the palladin association with Abl tyrosine kinase. Taken together, our results indicate that palladin phosphorylation by ERK has an anti-migratory function, possibly by modulating interactions with molecules that regulate cell migration.

The Roles of Two Distinct Regions of PINCH-1 in the Regulation of Cell Attachment and Spreading

PINCH-1, which comprises five LIM domains and the C-terminal region, is crucial for the regulation of cell–ECM adhesion. The LIM1 domain is essential for cell attachment, whereas C-terminal region is required for cell spreading by mediating the association with Rsu-1. PINCH-1–Rsu-1 pathway activates Rac to promote cell spreading.

The role of PLK1-phosphorylated SVIL in myosin II activation and cytokinetic furrowing.

Summary Polo-like kinase 1 (PLK1) is a widely conserved serine/threonine kinase that regulates progression of multiple stages of mitosis. Although extensive studies about PLK1 functions during cell division have been performed, it is still not known how PLK1 regulates myosin II activation at the equatorial cortex and ingression of the cleavage furrow. In this report, we show that an actin/myosin-II-binding protein, supervillin (SVIL), is a substrate of PLK1. PLK1 phosphorylates Ser238 of SVIL, which can promote the localization of SVIL to the central spindle and association with PRC1. Expression of a PLK1 phosphorylation site mutant, S238A-SVIL, inhibited myosin II activation at the equatorial cortex and induced aberrant furrowing. SVIL has both actin- and myosin-II-binding regions in the N-terminus. Expression of &Dgr;Myo-SVIL (deleted of the myosin-II-binding region), but not of &Dgr;Act-SVIL (deleted of actin-binding region), reduced myosin II activation and caused defects in furrowing. Our study indicates a possible role of phosphorylated SVIL as a molecular link between the central spindle and the contractile ring to coordinate the activation of myosin II for the ingression of the cleavage furrow.

UBE2S is associated with malignant characteristics of breast cancer cells

Ubiquitination is essential for various biological processes, such as signal transduction, intracellular trafficking, and protein degradation. Accumulating evidence has demonstrated that ubiquitination plays a crucial role in cancer development. In this report, we examine the expression and function of ubiquitin-conjugating enzyme E2S (UBE2S) in breast cancer. Immunohistochemical analysis revealed that UBE2S is highly expressed in breast cancer. The depletion of UBE2S by siRNA induced disruption of the actin cytoskeleton and focal adhesions. Interestingly, phosphorylation of FAK at Tyr397, which is important for the transduction of integrin-mediated signaling, was significantly reduced by UBE2S knockdown. We also show that UBE2S knockdown suppressed the malignant characteristics of breast cancer cells, such as migration, invasion, and anchorage-independent growth. Our results indicate that UBE2S could be a potential target for breast cancer treatment.

Silencing of STRN4 suppresses the malignant characteristics of cancer cells

The striatin family of proteins, comprising STRN, STRN3 and STRN4, are multidomain‐containing proteins that associate with additional proteins to form a large protein complex. We previously reported that STRN4 directly associated with protein kinases, such as MINK1, TNIK and MAP4K4, which are associated with tumor suppression or tumor progression. However, it remains unclear whether STRN4 is associated with tumor progression. In this report, we examined the role that STRN4 plays in cancer malignancy. We show that depletion of STRN4 suppresses proliferation, migration, invasion and the anchorage‐independent growth of cancer cells. In addition, STRN4 knockdown increases the sensitivity of pancreatic cancer cells to gemcitabine. Finally, we show that STRN4 knockdown suppresses the proliferation and metastasis of cancer cells in mice. Our results demonstrate a possible role of STRN4 in tumor progression.

The Aurora-B-mediated phosphorylation of SHCBP1 regulates cytokinetic furrow ingression.

Summary Centralspindlin, which is composed of MgcRacGAP and MKLP1, is essential for central spindle formation and cytokinetic furrow ingression. MgcRacGAP utilizes its GAP domain to inactivate Rac1 and induce furrow ingression in mammalian cells. In this report, we present a novel regulatory mechanism for furrowing that is mediated by the phosphorylation of SHC SH2-domain binding protein 1 (SHCBP1), a binding partner of centralspindlin, by Aurora B (AurB). AurB phosphorylates Ser634 of SHCBP1 during mitosis. We generated a phosphorylation site mutant, S634A-SHCBP1, which was prematurely recruited to the central spindle during anaphase and inhibited furrowing. An in vitro GAP assay demonstrated that SHCBP1 can suppress the MgcRacGAP-mediated inactivation of Rac1. In addition, the inhibition of Rac1 activity rescued the furrowing defect induced by S634A-SHCBP1 expression. Thus, AurB phosphorylates SHCBP1 to prevent the premature localization of SHCBP1 to the central spindle and ensures that MgcRacGAP inactivates Rac1 to promote the ingression of the cytokinetic furrow.