Cofilin Phosphorylation and Actin Cytoskeletal Dynamics Regulated by Rho- and Cdc42-Activated Lim-Kinase 2

The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 and kinase-dead LIMK2 (LIMK2/KD) were respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylated cofilin and induced formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation was stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induced stress fibers and filopodia, whereas both Rho- induced stress fibers and Cdc42-induced filopodia were abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac did not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization.

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Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0555 ◽

2005 ◽

Vol 16 (2) ◽

pp. 649-664 ◽

Cited By ~ 240

Author(s):

Pirta Hotulainen ◽

Eija Paunola ◽

Maria K. Vartiainen ◽

Pekka Lappalainen

Keyword(s):

Cell Motility ◽

Actin Filament ◽

Actin Filaments ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Depolymerizing Factor ◽

Cytoskeletal Dynamics ◽

Nonmuscle Cells ◽

In Cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling “old” actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

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Annexin A2 Phosphorylation Mediates Cell Scattering and Branching Morphogenesis via Cofilin Activation

Molecular and Cellular Biology ◽

10.1128/mcb.01247-07 ◽

2007 ◽

Vol 28 (3) ◽

pp. 1029-1040 ◽

Cited By ~ 69

Author(s):

Marjo de Graauw ◽

Ine Tijdens ◽

Mirjam B. Smeets ◽

Paul J. Hensbergen ◽

André M. Deelder ◽

...

Keyword(s):

Mdck Cells ◽

Three Dimensional ◽

Branching Morphogenesis ◽

Annexin A2 ◽

Actin Binding ◽

Lim Kinase ◽

Cell Scattering ◽

Cytoskeletal Dynamics ◽

Upstream Kinase ◽

And Migration

ABSTRACT Dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration and can be regulated by tyrosine kinase activity. Phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein annexin A2 (AnxA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering. Expression of the phospho-mimicking mutant Y23E-AnxA2 itself was sufficient to induce actin reorganization and cell scattering in MDCK cells. While Y23E-AnxA2, but not Y23A-AnxA2, enhanced Src- or hepatocyte growth factor (HGF)-induced cell scattering, short hairpin RNA-mediated knockdown of AnxA2 inhibited both v-Src- and HGF-induced cell scattering. Three-dimensional branching morphogenesis was induced in wild-type-AnxA2-expressing cells only in the presence of HGF, while Y23E-AnxA2 induced HGF-independent branching morphogenesis. Knockdown of AnxA2 prevented lumen formation during cystogenesis. The Y23E-AnxA2-induced scattering was associated with dephosphorylation/activation of the actin-severing protein cofilin. Likewise, inactive S3E-cofilin and constitutively active LIM kinase, a direct upstream kinase of cofilin, inhibited Y23E-AnxA2-induced scattering. Together, our studies indicate an essential role for AnxA2 phosphorylation in regulating cofilin-dependent actin cytoskeletal dynamics in the context of cell scattering and branching morphogenesis.

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Role of Phosphoinositides in Cellular Signaling, Functions and Diseases

10.20944/preprints202009.0513.v1 ◽

2020 ◽

Author(s):

Kalpana Mandal

Keyword(s):

Cell Signaling ◽

Transmembrane Proteins ◽

Adaptor Proteins ◽

Membrane Curvature ◽

Membrane Dynamics ◽

Actin Binding ◽

Coated Pits ◽

Cytoskeletal Dynamics ◽

Actin Cytoskeletal Dynamics

In this review we summarize the recent development in understanding the role of PIP2 in cellular function and signaling. We first discuss the effect of PIP2 on actin binding proteins addressing the mechanism of the actin cytoskeletal dynamics such as polymerization or depolymerization of the filamentous network or the coupling to membrane to generate forces. Next, we outline the role of PIP2 in membrane dynamics. We summarized how the membrane organization depends upon PIP2 in the presence of ions or transmembrane proteins that are sensitive to membrane curvature. We discuss how clathrin coated pits interact with adaptor proteins during the endocytosis process, which is facilitated by PIP2. Finally, we discuss the role of PIP2 in cell signaling and diseases.

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Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics

Nature Cell Biology ◽

10.1038/12963 ◽

1999 ◽

Vol 1 (5) ◽

pp. 253-259 ◽

Cited By ~ 690

Author(s):

David C. Edwards ◽

Luraynne C. Sanders ◽

Gary M. Bokoch ◽

Gordon N. Gill

Keyword(s):

Lim Kinase ◽

Cytoskeletal Dynamics ◽

Actin Cytoskeletal Dynamics

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Cofilin Phosphorylation by Protein Kinase Testicular Protein Kinase 1 and Its Role in Integrin-mediated Actin Reorganization and Focal Adhesion Formation

Molecular Biology of the Cell ◽

10.1091/mbc.12.4.1131 ◽

2001 ◽

Vol 12 (4) ◽

pp. 1131-1145 ◽

Cited By ~ 188

Author(s):

Jiro Toshima ◽

Junko Y. Toshima ◽

Toru Amano ◽

Neng Yang ◽

Shuh Narumiya ◽

...

Keyword(s):

Protein Kinase ◽

Hela Cells ◽

Kinase Activity ◽

Adhesion Formation ◽

Focal Adhesions ◽

Specific Inhibitor ◽

Stress Fibers ◽

Lim Kinase ◽

Actin Reorganization ◽

Cofilin Phosphorylation

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of a kinase domain related to those of LIM-kinases and a unique C-terminal proline-rich domain. Like LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated the formation of actin stress fibers and focal adhesions. In contrast to LIM-kinases, the kinase activity of TESK1 was not enhanced by Rho-associated kinase (ROCK) or p21-activated kinase, indicating that TESK1 is not their downstream effector. Both the kinase activity of TESK1 and the level of cofilin phosphorylation increased by plating cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited LIM-kinase-induced cofilin phosphorylation but did not affect fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa cells. Expression of a kinase-negative TESK1 suppressed cofilin phosphorylation and formation of stress fibers and focal adhesions induced in cells plated on fibronectin. These results suggest that TESK1 functions downstream of integrins and plays a key role in integrin-mediated actin reorganization, presumably through phosphorylating and inactivating cofilin. We propose that TESK1 and LIM-kinases commonly phosphorylate cofilin but are regulated in different ways and play distinct roles in actin reorganization in living cells.

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Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648421 ◽

1992 ◽

Vol 67 (02) ◽

pp. 252-257 ◽

Cited By ~ 9

Author(s):

Anne M Aakhus ◽

J Michael Wilkinson ◽

Nils Olav Solum

Keyword(s):

Actin Filaments ◽

High Speed ◽

Binding Protein ◽

Protein A ◽

Actin Binding ◽

Platelet Glycoprotein ◽

Actin Binding Protein ◽

Crossed Immunoelectrophoresis ◽

Triton X ◽

Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

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