[show abstract][hide abstract] ABSTRACT: The actin cytoskeleton undergoes rapid changes in its architecture during mitosis. Here, we demonstrate novel actin assembly dynamics in M phase. An amorphous cluster of actin filaments appears during prometaphase, revolves horizontally along the cell cortex at a constant angular speed, and fuses into the contractile ring after three to four revolutions. Cdk1 activity is required for the formation of this mitotic actin cluster and its revolving movement. Rapid turnover of actin in the filaments takes place everywhere in the cluster and is also required for its cluster rotation during mitosis. Knockdown of Arp3, a component of the actin filament-nucleating Arp2/3 complex, inhibits the formation of the mitotic actin cluster without affecting other actin structures. These results identify Arp2/3 complex as a key factor in the generation of the dynamic actin cluster during mitosis.
The Journal of Cell Biology 10/2010; 191(3):453-62. · 10.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: The spindle orientation is regulated by the interaction of astral microtubules with the cell cortex. We have previously shown that spindles in nonpolarized adherent cells are oriented parallel to the substratum by an actin cytoskeleton- and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]-dependent mechanism. Here, we show that Cdc42, a Rho family of small GTPases, has an essential role in this mechanism of spindle orientation by regulating both the actin cytoskeleton and PtdIns(3,4,5)P3. Knockdown of Cdc42 suppresses PI(3)K activity in M phase and induces spindle misorientation. Moreover, knockdown of Cdc42 disrupts the cortical actin structures in metaphase cells. Our results show that p21-activated kinase 2 (PAK2), a target of Cdc42 and/or Rac1, plays a key role in regulating actin reorganization and spindle orientation downstream from Cdc42. Surprisingly, PAK2 regulates spindle orientation in a kinase activity-independent manner. BetaPix, a guanine nucleotide exchange factor for Rac1 and Cdc42, is shown to mediate this kinase-independent function of PAK2. This study thus demonstrates that spindle orientation in adherent cells is regulated by two distinct pathways downstream from Cdc42 and uncovers a novel role of the Cdc42-PAK2-betaPix-actin pathway for this mechanism.
Molecular and cellular biology 04/2009; 29(10):2816-27. · 6.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cultured adherent cells divide on the substratum, leading to formation of the cell monolayer. However, how the orientation of this anchorage-dependent cell division is regulated remains unknown. We have previously shown that integrin-dependent adhesion orients the spindle parallel to the substratum, which ensures this anchorage-dependent cell division. Here, we show that phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is essential for this spindle orientation control. In metaphase, PtdIns(3,4,5)P3 is accumulated in the midcortex in an integrin-dependent manner. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) reduces the accumulation of PtdIns(3,4,5)P3 and induces spindle misorientation. Introduction of PtdIns(3,4,5)P3 to these cells restores the midcortical accumulation of PtdIns(3,4,5)P3 and proper spindle orientation. PI(3)K inhibition causes dynein-dependent spindle rotations along the z-axis, resulting in spindle misorientation. Moreover, dynactin, a dynein-binding partner, is accumulated in the midcortex in a PtdIns(3,4,5)P3-dependent manner. We propose that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on spindles to the midcortex, and thereby orients the spindle parallel to the substratum.
[show abstract][hide abstract] ABSTRACT: Activation of extracellular signal-regulated kinase (ERK) is known to be regulated by cell adhesion, namely "anchorage dependence". Most studies on the anchorage-dependent regulation have focused on the upstream activating components. We previously reported that the focal adhesion protein vinexin beta can induce the anchorage-independent activation of ERK2. We show here that vinexin beta-induced anchorage-independent activation of ERK2 involves prevention of the dephosphorylation of ERK2, but not the promotion of MEK1 or Raf1 activity. Furthermore, knockdown of vinexin beta resulted in a faster dephosphorylation of ERK2 in A549 cells. Moreover, the coexpression of MKP3/rVH6, an ERK2 specific phosphatase, suppressed the anchorage-independent activation of ERK2 induced by vinexin beta. These results suggest that vinexin beta can prevent the dephosphorylation of ERK2 stimulated by cell detachment, leading to the anchorage-independent activation of ERK2. Furthermore, we found that phosphatase activity directed against activated ERK2 was higher in suspended cells than in adherent cells. In addition, orthovanadate efficiently induces anchorage-independent activation of ERK2 without marked activation of MEK1 in NIH3T3 cells. These observations suggest that the anchorage dependence of ERK1/2 activation is regulated not only by upstream kinases, Raf1 and MEK, but also by phosphatases acting against ERK1/2 and that vinexin beta can induce anchorage-independent activation of ERK by preventing the inactivation of ERK1/2.
Experimental Cell Research 06/2007; 313(9):1830-8. · 3.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration and oncogenesis. In this study, we found that exogenous expression of vinexin beta enhanced the phosphorylation of 180-kDa proteins in an EGF-dependent manner in Cos-7 cells. Western blot analysis using phospho-specific antibodies against EGFR identified EGFR as a phosphorylated 180-kDa protein. Vinexin beta did not stimulate the phosphorylation of EGFR but suppressed the dephosphorylation, resulting in a sustained phosphorylation. Mutational analyses revealed that both the first and third SH3 domains were required for a sustained phosphorylation of EGFR. Small interfering RNA-mediated knockdown of vinexin beta reduced the phosphorylation of EGFR on the cell surface in HeLa cells. The sustained phosphorylation of EGFR induced by vinexin beta was completely abolished by adding the EGFR-specific inhibitor AG1478 even after EGF stimulation, suggesting that the kinase activity of EGFR is required for the sustained phosphorylation induced by vinexin beta. We also found that E3 ubiquitin ligase c-Cbl is a binding partner of vinexin beta through the third SH3 domain. Expression of wild-type vinexin beta but not a mutant containing a mutation in the third SH3 domain decreased the cytosolic pool of c-Cbl and increased the amount of membrane-associated c-Cbl. Furthermore, over-expression of c-Cbl suppressed the sustained phosphorylation of EGFR induced by vinexin beta. These results suggest that vinexin beta plays a role in maintaining the phosphorylation of EGFR on the plasma membrane through the regulation of c-Cbl.
Genes to Cells 10/2006; 11(9):971-82. · 2.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Non-receptor tyrosine kinase Abl is a well known regulator of the actin-cytoskeleton, including the formation of stress fibers and membrane ruffles. Vinexin is an adapter protein consisting of three SH3 domains, and involved in signal transduction and the reorganization of actin cytoskeleton. In this study, we found that vinexin alpha as well as beta interacts with c-Abl mainly through the third SH3 domain, and that vinexin and c-Abl were colocalized at membrane ruffles in rat astrocytes. This interaction was reduced by latrunculin B, suggesting an F-actin-mediated regulatory mechanism. We also found that vinexin alpha but not beta was phosphorylated at tyrosine residue when c-Abl or v-Abl was co-expressed. A mutational analysis identified tyrosine 127 on vinexin alpha as a major site of phosphorylation by c- or v-Abl. These results suggest that vinexin alpha is a novel substrate for Abl.
[show abstract][hide abstract] ABSTRACT: The focal adhesion protein vinexin is a member of a family of adaptor proteins that are thought to participate in the regulation of cell adhesion, cytoskeletal reorganization, and growth factor signaling. Here, we show that vinexin beta increases the amount of and reduces the mobility on SDS-PAGE of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 protein, which is a key factor modulating actin polymerization in migrating cells. This mobility retardation disappeared after in vitro phosphatase treatment. Co-immunoprecipitation assays revealed the interaction of vinexin beta with WAVE2 as well as WAVE1 and N-WASP. Vinexin beta interacts with the proline-rich region of WAVE2 through the first and second SH3 domains of vinexin beta. Mutations disrupting the interaction impaired the ability of vinexin beta to increase the amount of WAVE2 protein. Treatments with proteasome inhibitors increased the amount of WAVE2, but did not have an additive effect with vinexin beta. Inhibition of protein kinase A (PKA) activity suppressed the vinexin-induced increase in WAVE2 protein, while activation of PKA increased WAVE2 expression without vinexin beta. These results suggest that vinexin beta regulates the proteasome-dependent degradation of WAVE2 in a PKA-dependent manner.
Genes to Cells 04/2006; 11(3):281-92. · 2.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although vinexin was originally identified as a protein binding to the proline-rich hinge region of vinculin, the functions and biochemical properties of the vinexin-vinculin interaction are not known. Here, we determined the affinity of the vinexin-vinculin interaction using surface plasmon resonance measurements and found that vinexin beta interacts with the C-terminal half of vinculin, which mimics an activated "open" form, with a threefold higher affinity than with the full-length "closed" vinculin. Coimmunoprecipitation experiments showed that cell adhesion on fibronectin enhances the vinexin-vinculin interaction. We also show that the interaction with vinculin is necessary for the efficient localization of vinexin alpha and beta at focal adhesions. These observations suggest a model that "activated" vinculin localized at focal adhesions recruits vinexins to focal adhesions.
Biochemical and Biophysical Research Communications 11/2005; 336(1):239-46. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Extracellular signal-regulated kinase 1/2 (ERK1/2) is activated by various extracellular stimuli including growth factors and cytokines and plays a pivotal role in regulating cell proliferation and differentiation by phosphorylating nuclear transcription factors. Recently, it was reported that activated ERK1/2 also concentrates at adhesion sites and regulates cell spreading and migration. Vinexin is a focal adhesion protein regulating both cell spreading and growth factor signaling. We show here that vinexin was directly phosphorylated by ERK1/2 upon stimulation with growth factors. ERK1/2 phosphorylated the linker region of vinexin between the second and third SH3 domains. Site-directed mutagenesis revealed that ERK2 mainly phosphorylated the serine 189 residue of vinexin beta. Furthermore, vinexin beta interacted with ERK1/2 both in vitro and in vivo. Vinexin interacted with the active but not inactive form of ERK1/2. A putative DEF (docking for ERK FXFP) domain located in the linker region of vinexin was required for the interaction with ERK1/2 and efficient phosphorylation of vinexin beta by ERK2. Finally, we showed that cell adhesion to fibronectin also induced the association of vinexin beta with ERK2 and the phosphorylation of vinexin beta. Furthermore, vinexin and ERK were co-localized to the periphery of cells during cell spreading on fibronectin. Together, these results suggest that vinexin is a novel substrate of ERK2 and may play roles in ERK-dependent cell regulation during cell spreading as well as in growth factor-induced responses.
Journal of Biological Chemistry 09/2004; 279(33):34570-7. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vinexin is a recently identified cytoskeletal protein and plays a key role in the regulation of cytoskeletal organization and signal transduction. Vinexin localizes at sites of cell-extracellular matrix adhesion in NIH3T3 fibroblasts and at sites of cell-cell contact in epithelial LLC-PK1 cells. Expression of vinexin promotes the formation of actin stress fiber, but the role of vinexin at sites of cell-cell contact is unclear. Here we identified lp-dlg/KIAA0583 as a novel binding partner for vinexin by using yeast two-hybrid screening. lp-dlg/KIAA0583 has a NH2-terminal coiled-coil-like domain, in addition to four PDZ domains, an Src homology (SH) 3 domain, and a guanylate kinase domain, which are conserved structures in membrane-associated guanylate kinase family proteins. The third SH3 domain of vinexin bound to the region between the second and third PDZ domain of lp-dlg, which contains a proline-rich sequence. lp-dlg colocalized with vinexin at sites of cell-cell contact in LLC-PK1 cells. Furthermore, lp-dlg colocalized with beta-catenin, a major adherens junction protein, in LLC-PK1 cells. Co-immunoprecipitation experiments revealed that both endogenous and epitope-tagged deletion mutants of lp-dlg/KIAA0583 associated with beta-catenin. We also showed that these three proteins could form a ternary complex. Together these findings suggest that lp-dlg/KIAA0583 is a novel scaffolding protein that can link the vinexin-vinculin complex and beta-catenin at sites of cell-cell contact.
Journal of Biological Chemistry 07/2003; 278(24):21709-14. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: ERK is activated by soluble growth factors in adherent cells. However, activation of ERK is barely detectable and not sufficient for cell proliferation in non-adherent cells. Here, we show that exogenous expression of vinexin beta, a novel focal adhesion protein, allows anchorage-independent ERK2 activation stimulated by epidermal growth factor. In contrast, expression of vinexin beta had no effect on ERK2 activation in adherent cells, suggesting that vinexin beta regulates the anchorage dependence of ERK2 activation. Analyses using deletion mutants demonstrated that a linker region between the second and third SH3 domains of vinexin beta, but not the SH3 domains, is required for this function of vinexin beta. To evaluate the pathway regulating the anchorage dependence of ERK2 activation, we used a dominant-negative mutant of p21-activated kinase (PAK) and a specific inhibitor (H89) of cAMP-dependent protein kinase (PKA) because PAK and PKA are known to regulate the anchorage dependence of ERK2 activation. The dominant-negative mutant of PAK suppressed the anchorage-independent ERK2 activation induced by expression of vinexin beta. The dominant-negative mutant of vinexin beta inhibited the anchorage-independent ERK2 activation induced by the PKA inhibitor. Together, these observations indicate that vinexin beta plays a key role in regulating the anchorage dependence of ERK2 activation through PKA-PAK signaling.
Journal of Biological Chemistry 05/2002; 277(15):13053-8. · 4.65 Impact Factor