The C Terminus of Formin FMNL3 Accelerates Actin Polymerization and Contains a WH2 Domain-like Sequence That Binds Both Monomers and Filament Barbed Ends

Programmed cell death (PCD) is a physiological process in which infected or unnecessary cells due to their suicidal death capability can be selectively eliminated. Pro- and antiapoptotic proteins play an important role in the induction or inhibition of this process. Presented article shows property of Bax-1 (BI-1) inhibitor which is one of the conservative protein associated with the endoplasmic reticulum (ER) as well as its cytoprotective role in the regulation of cellular processes. It was shown that: 1) BI-1 is a small protein consisting of 237 amino acids (human protein - 36 kDa) and has 6 (in animals) and 7 (in plants) α-helical transmembrane domains, 2) BI-1 is expressed in all organisms and in most tissues, moreover its level depends on the functional condition of cells and it is involved in the development or reaction to biotic and abiotic stresses, 3) BI-1 forms a pH-dependent Ca2+ channel enabling release of these ions from the ER, 4) cytoprotective effects of BI-1 requires a whole, unchanged C-terminus, 5) BI-1 can interact directly with numerous other proteins, BI-1 protein affects numerous cellular processes, including: counteracting ER stress, oxidative stress, loss of cellular Ca2+ homeostasis as well as this protein influences on sphingolipid metabolism, autophagy, actin polymerization, lysosomal activity and cell proliferation. Studies of BI-1 functions will allow understanding the mechanisms of anticancer therapy or increases the knowledge of crop tolerance to environmental stresses.

Download Full-text

Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement

The Journal of Cell Biology ◽

10.1083/jcb.200705044 ◽

2007 ◽

Vol 179 (7) ◽

pp. 1539-1553 ◽

Cited By ~ 51

Author(s):

Rosa Ana Lacalle ◽

Rosa M. Peregil ◽

Juan Pablo Albar ◽

Ernesto Merino ◽

Carlos Martínez-A ◽

...

Keyword(s):

Actin Polymerization ◽

Cell Movement ◽

Leading Edge ◽

Cell Polarization ◽

Type I ◽

Lipid Kinase ◽

Erm Proteins ◽

Chemical Gradients ◽

C Terminus ◽

Phosphatidylinositol 4

Directional cell movement in response to external chemical gradients requires establishment of front–rear asymmetry, which distinguishes an up-gradient protrusive leading edge, where Rac-induced F-actin polymerization takes place, and a down-gradient retractile tail (uropod in leukocytes), where RhoA-mediated actomyosin contraction occurs. The signals that govern this spatial and functional asymmetry are not entirely understood. We show that the human type I phosphatidylinositol 4-phosphate 5-kinase isoform β (PIPKIβ) has a role in organizing signaling at the cell rear. We found that PIPKIβ polarized at the uropod of neutrophil-differentiated HL60 cells. PIPKIβ localization was independent of its lipid kinase activity, but required the 83 C-terminal amino acids, which are not homologous to other PIPKI isoforms. The PIPKIβ C terminus interacted with EBP50 (4.1-ezrin-radixin-moesin (ERM)-binding phosphoprotein 50), which enabled further interactions with ERM proteins and the Rho-GDP dissociation inhibitor (RhoGDI). Knockdown of PIPKIβ with siRNA inhibited cell polarization and impaired cell directionality during dHL60 chemotaxis, suggesting a role for PIPKIβ in these processes.

Download Full-text

Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0436 ◽

2007 ◽

Vol 18 (8) ◽

pp. 2893-2903 ◽

Cited By ~ 29

Author(s):

Sarah L. Barker ◽

Linda Lee ◽

B. Daniel Pierce ◽

Lymarie Maldonado-Báez ◽

David G. Drubin ◽

...

Keyword(s):

Late Stage ◽

Actin Polymerization ◽

Fluorescent Protein ◽

Temperature Sensitive ◽

Type I ◽

Reading Frame ◽

Rich Domain ◽

C Terminus

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

Download Full-text

The C-terminus SH3-binding domain of Kv1.3 is required for the actin-mediated immobilization of the channel via cortactin

Molecular Biology of the Cell ◽

10.1091/mbc.e14-07-1195 ◽

2015 ◽

Vol 26 (9) ◽

pp. 1640-1651 ◽

Cited By ~ 14

Author(s):

Peter Hajdu ◽

Geoffrey V. Martin ◽

Ameet A. Chimote ◽

Orsolya Szilagyi ◽

Koichi Takimoto ◽

...

Keyword(s):

T Lymphocytes ◽

Actin Polymerization ◽

Actin Dynamics ◽

Actin Network ◽

Immune Synapse ◽

Proximity Ligation ◽

C Terminus ◽

Kv1.3 Channels ◽

And Migration ◽

Channel Polarization

Kv1.3 channels play a pivotal role in the activation and migration of T-lymphocytes. These functions are accompanied by the channels' polarization, which is essential for associated downstream events. However, the mechanisms that govern the membrane movement of Kv1.3 channels remain unclear. F-actin polymerization occurs concomitantly to channel polarization, implicating the actin cytoskeleton in this process. Here we show that cortactin, a factor initiating the actin network, controls the membrane mobilization of Kv1.3 channels. FRAP with EGFP-tagged Kv1.3 channels demonstrates that knocking down cortactin decreases the actin-based immobilization of the channels. Using various deletion and mutation constructs, we show that the SH3 motif of Kv1.3 mediates the channel immobilization. Proximity ligation assays indicate that deletion or mutation of the SH3 motif also disrupts interaction of the channel with cortactin. In T-lymphocytes, the interaction between HS1 (the cortactin homologue) and Kv1.3 occurs at the immune synapse and requires the channel's C-terminal domain. These results show that actin dynamics regulates the membrane motility of Kv1.3 channels. They also provide evidence that the SH3 motif of the channel and cortactin plays key roles in this process.

Download Full-text

Withaferin A binds covalently to the N-terminal domain of annexin A2

Biological Chemistry ◽

10.1515/hsz-2012-0184 ◽

2012 ◽

Vol 393 (10) ◽

pp. 1151-1163 ◽

Cited By ~ 17

Author(s):

Gabriel Ozorowski ◽

Christopher M. Ryan ◽

Julian P. Whitelegge ◽

Hartmut Luecke

Keyword(s):

Actin Polymerization ◽

Inhibitory Effect ◽

Annexin A2 ◽

Actin Dynamics ◽

Withaferin A ◽

Core Domain ◽

Cancer Pathways ◽

Terminal Domain ◽

C Terminus

Abstract Annexin A2 (AnxA2), a 38-kDa member of the Ca2+-binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.

Download Full-text

Regulation of INF2-mediated actin polymerization through site-specific lysine acetylation of actin itself

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914072117 ◽

2019 ◽

Vol 117 (1) ◽

pp. 439-447 ◽

Cited By ~ 6

Author(s):

Mu A ◽

Tak Shun Fung ◽

Lisa M. Francomacaro ◽

Thao Huynh ◽

Tommi Kotila ◽

...

Keyword(s):

Actin Polymerization ◽

Full Length ◽

Common Mechanism ◽

Binding Studies ◽

Direct Binding ◽

Wh2 Domain ◽

Inhibitory Domain ◽

Low Levels ◽

U2os Cells ◽

Better Than

INF2 is a formin protein that accelerates actin polymerization. A common mechanism for formin regulation is autoinhibition, through interaction between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD). We recently showed that INF2 uses a variant of this mechanism that we term “facilitated autoinhibition,” whereby a complex consisting of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin) is required for INF2 inhibition, in a manner requiring INF2-DID. Deacetylation of actin in the CAP/KAc-actin complex activates INF2. Here we use lysine-to-glutamine mutations as acetylmimetics to map the relevant lysines on actin for INF2 regulation, focusing on K50, K61, and K328. Biochemically, K50Q- and K61Q-actin, when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. When not bound to CAP, these mutant actins polymerize similarly to WT-actin in the presence or absence of INF2, suggesting that the effect of the mutation is directly on INF2 regulation. In U2OS cells, K50Q- and K61Q-actin inhibit INF2-mediated actin polymerization when expressed at low levels. Direct-binding studies show that the CAP WH2 domain binds INF2-DID with submicromolar affinity but has weak affinity for actin monomers, while INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin. Actin in complex with full-length CAP2 is predominately ATP-bound. These interactions suggest an inhibition model whereby CAP/KAc-actin serves as a bridge between INF2 DID and DAD. In U2OS cells, INF2 is 90-fold and 5-fold less abundant than CAP1 and CAP2, respectively, suggesting that there is sufficient CAP for full INF2 inhibition.

Download Full-text

Arg interacts with cortactin to promote adhesion-dependent cell edge protrusion

The Journal of Cell Biology ◽

10.1083/jcb.200809085 ◽

2009 ◽

Vol 185 (3) ◽

pp. 503-519 ◽

Cited By ~ 57

Author(s):

Stefanie Lapetina ◽

Christopher C. Mader ◽

Kazuya Machida ◽

Bruce J. Mayer ◽

Anthony J. Koleske

Keyword(s):

Actin Polymerization ◽

Molecular Mechanisms ◽

Sh2 Domain ◽

Cell Edge ◽

Cell Matrix ◽

C Terminus ◽

Dependent Cell ◽

Src Homology ◽

Fibroblast Adhesion ◽

Cell Matrix Adhesion

The molecular mechanisms by which the Abelson (Abl) or Abl-related gene (Arg) kinases interface with the actin polymerization machinery to promote cell edge protrusions during cell–matrix adhesion are unclear. In this study, we show that interactions between Arg and the Arp2/3 complex regulator cortactin are essential to mediate actin-based cell edge protrusion during fibroblast adhesion to fibronectin. Arg-deficient and cortactin knockdown fibroblasts exhibit similar defects in adhesion-dependent cell edge protrusion, which can be restored via reexpression of Arg and cortactin. Arg interacts with cortactin via both binding and catalytic events. The cortactin Src homology (SH) 3 domain binds to a Pro-rich motif in the Arg C terminus. Arg mediates adhesion-dependent phosphorylation of cortactin, creating an additional binding site for the Arg SH2 domain. Mutation of residues that mediate Arg–cortactin interactions abrogate the abilities of both proteins to support protrusions, and the Nck adapter, which binds phosphocortactin, is also required. These results demonstrate that interactions between Arg, cortactin, and Nck1 are critical to promote adhesion-dependent cell edge protrusions.

Download Full-text

WASP and WAVE family proteins: key molecules for rapid rearrangement of cortical actin filaments and cell movement

Journal of Cell Science ◽

10.1242/jcs.114.10.1801 ◽

2001 ◽

Vol 114 (10) ◽

pp. 1801-1809 ◽

Cited By ~ 6

Author(s):

T. Takenawa ◽

H. Miki

Keyword(s):

Actin Filaments ◽

Actin Polymerization ◽

Cell Movement ◽

Activation Mechanism ◽

Cortical Actin ◽

C Terminus ◽

Inactive Form ◽

Extracellular Stimuli ◽

Proline Rich Region

Reorganization of cortical actin filaments plays critical roles in cell movement and pattern formation. Recently, the WASP and WAVE family proteins WASP and N-WASP, and WAVE1, WAVE2 and WAVE3 have been shown to regulate cortical actin filament reorganization in response to extracellular stimuli. These proteins each have a verprolin-homology (V) domain, cofilin-homology (C) domain and an acidic (A) region at the C-terminus, through which they activate the Arp2/3 complex, leading to rapid actin polymerization. N-WASP is usually present as an inactive form in which the VCA region is masked. Cooperative binding of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) exposes the VCA region, activating N-WASP. In addition to this activation mechanism, WISH also activates N-WASP independently of Cdc42 and PtdIns(4,5)P(2), by binding to the proline-rich region of N-WASP. N-WASP activation induces formation of filopodia in vivo. In contrast, the ubiquitously expressed form of WAVE2 is activated downstream of Rac, leading to formation of lamellipodia. In this case, IRSp53 transmits a signal from Rac to WAVE2 through formation of a ternary Rac-IRSp53-WAVE2 complex. Thus, N-WASP, which is activated downstream of Cdc42 or independently by WISH, induces formation of filopodia and WAVE2, which is activated via IRSp53 downstream of Rac, induces formation of lamellipodia.

Download Full-text

New players in actin polymerization – WH2-domain-containing actin nucleators

Trends in Cell Biology ◽

10.1016/j.tcb.2009.03.004 ◽

2009 ◽

Vol 19 (6) ◽

pp. 276-285 ◽

Cited By ~ 66

Author(s):

Britta Qualmann ◽

Michael M. Kessels

Keyword(s):

Actin Polymerization ◽

Wh2 Domain

Download Full-text

Characterization of a Rac1- and RhoGDI-Associated Lipid Kinase Signaling Complex

Molecular and Cellular Biology ◽

10.1128/mcb.18.2.762 ◽

1998 ◽

Vol 18 (2) ◽

pp. 762-770 ◽

Cited By ~ 112

Author(s):

Kimberley F. Tolias ◽

Anthony D. Couvillon ◽

Lewis C. Cantley ◽

Christopher L. Carpenter

Keyword(s):

Nadph Oxidase ◽

Rho Gtpases ◽

Actin Polymerization ◽

Cellular Proliferation ◽

Type I ◽

Lipid Kinase ◽

C Terminus ◽

Lipid Kinases

ABSTRACT Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.

Download Full-text