scholarly journals TheSaccharomyces cerevisiaeHomologue of Human Wiskott–Aldrich Syndrome Protein Las17p Interacts with the Arp2/3 Complex

1999 ◽  
Vol 10 (10) ◽  
pp. 3521-3538 ◽  
Author(s):  
Ammar Madania ◽  
Pascal Dumoulin ◽  
Sandrine Grava ◽  
Hiroko Kitamoto ◽  
Claudia Schärer-Brodbeck ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Temperature Sensitive ◽  
Multicopy Suppressor ◽  
Wiskott Aldrich Syndrome ◽  
Regulatory Cascades ◽  
Src Homology ◽  
Synthetically Lethal ◽  
Actin Patch ◽  
Syndrome Protein

Yeast Las17 protein is homologous to the Wiskott–Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least oneARP3 ts mutant are synthetically lethal withlas17Δ ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Δcells showed that receptor-mediated internalization of α factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton.

scholarly journals Abelson Interactor 1 (Abi1) and Its Interaction with Wiskott-Aldrich Syndrome Protein (Wasp) Are Critical for Proper Eye Formation in Xenopus Embryos

2013 ◽  
Vol 288 (20) ◽  
pp. 14135-14146 ◽  
Author(s):  
Arvinder Singh ◽  
Emily F. Winterbottom ◽  
Yon Ju Ji ◽  
Yoo-Seok Hwang ◽  
Ira O. Daar
Keyword(s):  
Progenitor Cells ◽  
Cultured Cells ◽  
Scaffold Protein ◽  
Wiskott Aldrich Syndrome ◽  
Src Homology 3 ◽  
Eye Field ◽  
Src Homology 3 Domain ◽  
Src Homology ◽  
Syndrome Protein

Abl interactor 1 (Abi1) is a scaffold protein that plays a central role in the regulation of actin cytoskeleton dynamics as a constituent of several key protein complexes, and homozygous loss of this protein leads to embryonic lethality in mice. Because this scaffold protein has been shown in cultured cells to be a critical component of pathways controlling cell migration and actin regulation at cell-cell contacts, we were interested to investigate the in vivo role of Abi1 in morphogenesis during the development of Xenopus embryos. Using morpholino-mediated translation inhibition, we demonstrate that knockdown of Abi1 in the whole embryo, or specifically in eye field progenitor cells, leads to disruption of eye morphogenesis. Moreover, signaling through the Src homology 3 domain of Abi1 is critical for proper movement of retinal progenitor cells into the eye field and their appropriate differentiation, and this process is dependent upon an interaction with the nucleation-promoting factor Wasp (Wiskott-Aldrich syndrome protein). Collectively, our data demonstrate that the Abi1 scaffold protein is an essential regulator of cell movement processes required for normal eye development in Xenopus embryos and specifically requires an Src homology 3 domain-dependent interaction with Wasp to regulate this complex morphogenetic process.

scholarly journals A Novel Neural Wiskott-Aldrich Syndrome Protein (N-Wasp) Binding Protein, Wish, Induces Arp2/3 Complex Activation Independent of Cdc42

2001 ◽  
Vol 152 (3) ◽  
pp. 471-482 ◽  
Author(s):  
Maiko Fukuoka ◽  
Shiro Suetsugu ◽  
Hiroaki Miki ◽  
Kiyoko Fukami ◽  
Takeshi Endo ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Leucine Zipper ◽  
Adaptor Protein ◽  
Sh3 Domain ◽  
Brain Extract ◽  
Wiskott Aldrich Syndrome ◽  
Neural Tissues ◽  
Src Homology ◽  
Syndrome Protein

We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP–induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract–induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP–depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP–dependent and –independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.

scholarly journals The Novel Adaptor Protein, Mti1p, and Vrp1p, a Homolog of Wiskott-Aldrich Syndrome Protein-Interacting Protein (WIP), May Antagonistically Regulate Type I Myosins in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (3) ◽  
pp. 923-934
Author(s):  
Junko Mochida ◽  
Takaharu Yamamoto ◽  
Konomi Fujimura-Kamada ◽  
Kazuma Tanaka
Keyword(s):  
Adaptor Protein ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Null Mutation ◽  
Type I ◽  
Cortical Actin ◽  
Tail Region ◽  
Interacting Protein ◽  
Wiskott Aldrich Syndrome ◽  
Syndrome Protein

Abstract Type I myosins in yeast, Myo3p and Myo5p (Myo3/5p), are involved in the reorganization of the actin cytoskeleton. The SH3 domain of Myo5p regulates the polymerization of actin through interactions with both Las17p, a homolog of mammalian Wiskott-Aldrich syndrome protein (WASP), and Vrp1p, a homolog of WASP-interacting protein (WIP). Vrp1p is required for both the localization of Myo5p to cortical patch-like structures and the ATP-independent interaction between the Myo5p tail region and actin filaments. We have identified and characterized a new adaptor protein, Mti1p (Myosin tail region-interacting protein), which interacts with the SH3 domains of Myo3/5p. Mti1p co-immunoprecipitated with Myo5p and Mti1p-GFP co-localized with cortical actin patches. A null mutation of MTI1 exhibited synthetic lethal phenotypes with mutations in SAC6 and SLA2, which encode actin-bundling and cortical actin-binding proteins, respectively. Although the mti1 null mutation alone did not display any obvious phenotype, it suppressed vrp1 mutation phenotypes, including temperature-sensitive growth, abnormally large cell morphology, defects in endocytosis and salt-sensitive growth. These results suggest that Mti1p and Vrp1p antagonistically regulate type I myosin functions.

scholarly journals Fyn and PTP-PEST–mediated Regulation of Wiskott-Aldrich Syndrome Protein (WASp) Tyrosine Phosphorylation Is Required for Coupling T Cell Antigen Receptor Engagement to WASp Effector Function and T Cell Activation

2004 ◽  
Vol 199 (1) ◽  
pp. 99-112 ◽  
Author(s):  
Karen Badour ◽  
Jinyi Zhang ◽  
Fabio Shi ◽  
Yan Leng ◽  
Michael Collins ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Actin Polymerization ◽  
Cell Activation ◽  
Synapse Formation ◽  
Tyrosine Phosphatase ◽  
Structural Constraints ◽  
Wiskott Aldrich Syndrome ◽  
Syndrome Protein

Involvement of the Wiskott-Aldrich syndrome protein (WASp) in promoting cell activation requires its release from autoinhibitory structural constraints and has been attributed to WASp association with activated cdc42. Here, however, we show that T cell development and T cell receptor (TCR)-induced proliferation and actin polymerization proceed normally in WASp−/− mice expressing a WASp transgene lacking the cdc42 binding domain. By contrast, mutation of tyrosine residue Y291, identified here as the major site of TCR-induced WASp tyrosine phosphorylation, abrogated induction of WASp tyrosine phosphorylation and its effector activities, including nuclear factor of activated T cell transcriptional activity, actin polymerization, and immunological synapse formation. TCR-induced WASp tyrosine phosphorylation was also disrupted in T cells lacking Fyn, a kinase shown here to bind, colocalize with, and phosphorylate WASp. By contrast, WASp was tyrosine dephosphorylated by protein tyrosine phosphatase (PTP)-PEST, a tyrosine phosphatase shown here to interact with WASp via proline, serine, threonine phosphatase interacting protein (PSTPIP)1 binding. Although Fyn enhanced WASp-mediated Arp2/3 activation and was required for synapse formation, PTP-PEST combined with PSTPIP1 inhibited WASp-driven actin polymerization and synapse formation. These observations identify key roles for Fyn and PTP-PEST in regulating WASp and imply that inducible WASp tyrosine phosphorylation can occur independently of cdc42 binding, but unlike the cdc42 interaction, is absolutely required for WASp contributions to T cell activation.

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

2007 ◽  
Vol 18 (8) ◽  
pp. 2893-2903 ◽  
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.

scholarly journals A Fourth Component of the Fission Yeast γ-Tubulin Complex, Alp16, Is Required for Cytoplasmic Microtubule Integrity and Becomes Indispensable When γ-Tubulin Function Is Compromised

2002 ◽  
Vol 13 (7) ◽  
pp. 2360-2373 ◽  
Author(s):  
Akiko Fujita ◽  
Leah Vardy ◽  
Miguel Angel Garcia ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Microtubule ◽  
Multicopy Suppressor ◽  
Large Complex ◽  
Synthetically Lethal ◽  
And Function

γ-Tubulin functions as a multiprotein complex, called the γ-tubulin complex (γ-TuC), and composes the microtubule organizing center (MTOC). Fission yeast Alp4 and Alp6 are homologues of two conserved γ-TuC proteins, hGCP2 and hGCP3, respectively. We isolated a novel gene, alp16 + , as a multicopy suppressor of temperature-sensitive alp6-719mutants. alp16 + encodes a 759-amino-acid protein with two conserved regions found in all other members of γ-TuC components. In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210. Gene disruption shows that alp16 + is not essential for cell viability. However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip. Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225,alp4-1891, or alp6-719 mutants. Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6. Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase. Alp16 coimmunoprecipitates with γ-tubulin and cosediments with the γ-TuC in a large complex (>20 S). Alp16 is, however, not required for the formation of this large complex. We discuss evolutional conservation and divergence of structure and function of the γ-TuC between yeast and higher eukaryotes.

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