Masaru Mitsushima

Dual Role of Cdc42 in Spindle Orientation Control of Adherent Cells

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. βPix, 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-βPix-actin pathway for this mechanism.

Revolving movement of a dynamic cluster of actin filaments during mitosis

Arp2/3 actin filament nucleating complex drives circumnavigation of cortical actin clusters during mitosis.

Vinexin β Regulates the Anchorage Dependence of ERK2 Activation Stimulated by Epidermal Growth Factor

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 β, a novel focal adhesion protein, allows anchorage-independent ERK2 activation stimulated by epidermal growth factor. In contrast, expression of vinexin β had no effect on ERK2 activation in adherent cells, suggesting that vinexin β 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 β, but not the SH3 domains, is required for this function of vinexin β. 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 β. The dominant-negative mutant of vinexin β inhibited the anchorage-independent ERK2 activation induced by the PKA inhibitor. Together, these observations indicate that vinexin β plays a key role in regulating the anchorage dependence of ERK2 activation through PKA-PAK signaling.

Protein kinase A-dependent increase in WAVE2 expression induced by the focal adhesion protein vinexin

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 β 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 β with WAVE2 as well as WAVE1 and N‐WASP. Vinexin β interacts with the proline‐rich region of WAVE2 through the first and second SH3 domains of vinexin β. Mutations disrupting the interaction impaired the ability of vinexin β 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 β. Inhibition of protein kinase A (PKA) activity suppressed the vinexin‐induced increase in WAVE2 protein, while activation of PKA increased WAVE2 expression without vinexin β. These results suggest that vinexin β regulates the proteasome‐dependent degradation of WAVE2 in a PKA‐dependent manner.

Abl kinase interacts with and phosphorylates vinexin

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 α as well as β 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 α but not β was phosphorylated at tyrosine residue when c‐Abl or v‐Abl was co‐expressed. A mutational analysis identified tyrosine 127 on vinexin α as a major site of phosphorylation by c‐ or v‐Abl. These results suggest that vinexin α is a novel substrate for Abl.

Vinexin β regulates the phosphorylation of epidermal growth factor receptor on the cell surface

Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration and oncogenesis. In this study, we found that exogenous expression of vinexin β 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 β 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 β reduced the phosphorylation of EGFR on the cell surface in HeLa cells. The sustained phosphorylation of EGFR induced by vinexin β 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 β. We also found that E3 ubiquitin ligase c‐Cbl is a binding partner of vinexin β through the third SH3 domain. Expression of wild‐type vinexin β 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 β. These results suggest that vinexin β plays a role in maintaining the phosphorylation of EGFR on the plasma membrane through the regulation of c‐Cbl.