SH3 domain-containing proteins and the actin cytoskeleton in yeast

SH3 (Src homology-3) domains are involved in protein–protein interactions through proline-rich domains. Many SH3-containing proteins are implicated in actin cytoskeleton organization. The aim of our ongoing work is to study the functions of the SH3-containing proteins in actin cytoskeleton regulation. The yeast Saccharomyces cerevisiae proteome includes 29 SH3 domains distributed in 25 proteins. We have examined the direct involvement of these SH3 domains in actin polymerization using an in vitro polymerization assay on GST (glutathione S-transferase)–SH3-coated beads. As expected, not all SH3 domains show polymerization activity, and many recruit distinct partners as assessed by microscopy and pull-down experiments. One such partner, Las17p, the yeast homologue of WASP (Wiskott–Aldrich syndrome protein), was assayed because it stimulates actin nucleation via the Arp2/3 (actin-related protein 2/3) complex. Ultimately, proteins involved in specific biological processes, such as membrane trafficking, may also be recruited by some of these SH3 domains, shedding light on the SH3-containing proteins and actin cytoskeleton functions in these processes.

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Pan1p, Yeast eps15, Functions as a Multivalent Adaptor That Coordinates Protein–Protein Interactions Essential for Endocytosis

The Journal of Cell Biology ◽

10.1083/jcb.141.1.71 ◽

1998 ◽

Vol 141 (1) ◽

pp. 71-84 ◽

Cited By ~ 158

Author(s):

Beverly Wendland ◽

Scott D. Emr

Keyword(s):

Plasma Membrane ◽

Protein Interactions ◽

Membrane Trafficking ◽

Endocytic Pathway ◽

Binding Studies ◽

Dynamic Interactions ◽

Yeast Saccharomyces Cerevisiae ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization

A genetic screen for factors required for endocytosis in the budding yeast Saccharomyces cerevisiae previously identified PAN1. Pan1p is a homologue of the mammalian protein eps15, which has been implicated in endocytosis by virtue of its association with the plasma membrane clathrin adaptor complex AP-2. Pan1p contains two eps15 homology (EH) domains, a protein–protein interaction motif also present in other proteins that function in membrane trafficking. To address the role of Pan1p and EH domains in endocytosis, a yeast two-hybrid screen was performed using the EH domain–containing region of Pan1p. This screen identified yAP180A, one of two yeast homologues of a class of clathrin assembly proteins (AP180) that exhibit in vitro clathrin cage assembly activity. In vitro binding studies using GST fusion proteins and yeast extracts defined distinct binding sites on yAP180A for Pan1p and clathrin. yAP180 proteins and Pan1p, like actin, localize to peripheral patches along the plasma membrane. Mammalian synaptojanin, a phosphatidylinositol polyphosphate-5-phosphatase, also has been implicated in endocytosis recently, and three synaptojanin-like genes have been identified in yeast. We observed genetic interactions between the yeast SJL1 gene and PAN1, which suggest a role for phosphoinositide metabolites in Pan1p function. Together with other studies, these findings suggest that Pan1p coordinates regulatory interactions between proteins required for both endocytosis and actin-cytoskeleton organization; these proteins include the yAP180 proteins, clathrin, the ubiquitin–protein ligase Rsp5p, End3p, and synaptojanin. We suggest that Pan1p (and by extension eps15) serves as a multivalent adaptor around which dynamic interactions between structural and regulatory components of the endocytic pathway converge.

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Regulation of Yeast Actin Cytoskeleton-Regulatory Complex Pan1p/Sla1p/End3p by Serine/Threonine Kinase Prk1p

Molecular Biology of the Cell ◽

10.1091/mbc.12.12.3759 ◽

2001 ◽

Vol 12 (12) ◽

pp. 3759-3772 ◽

Cited By ~ 72

Author(s):

Guisheng Zeng ◽

Xianwen Yu ◽

Mingjie Cai

Keyword(s):

Actin Cytoskeleton ◽

Regulatory Protein ◽

Strong Support ◽

Stable Complex ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization

The serine/threonine kinase Prk1p is known to be involved in the regulation of the actin cytoskeleton organization in budding yeast. One possible function of Prk1p is the negative regulation of Pan1p, an actin patch regulatory protein that forms a complex in vivo with at least two other proteins, Sla1p and End3p. In this report, we identified Sla1p as another substrate for Prk1p. The phosphorylation of Sla1p by Prk1p was established in vitro with the use of immunoprecipitated Prk1p and in vivo with the use ofPRK1 overexpression, and was further supported by the finding that immunoprecipitated Sla1p contained PRK1- and ARK1-dependent kinase activities. Stable complex formation between Prk1p and Sla1p/Pan1p in vivo could be observed once the phosphorylation reaction was blocked by mutation in the catalytic site of Prk1p. Elevation of Prk1p activities in wild-type cells resulted in a number of deficiencies, including those in colocalization of Pan1p and Sla1p, endocytosis, and cell wall morphogenesis, likely attributable to a disintegration of the Pan1p/Sla1p/End3p complex. These results lend a strong support to the model that the phosphorylation of the Pan1p/Sla1p/End3p complex by Prk1p is one of the important mechanisms by which the organization and functions of the actin cytoskeleton are regulated.

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Protein-protein interactions in the synaptonemal complex.

Molecular Biology of the Cell ◽

10.1091/mbc.8.8.1405 ◽

1997 ◽

Vol 8 (8) ◽

pp. 1405-1414 ◽

Cited By ~ 42

Author(s):

M Tarsounas ◽

R E Pearlman ◽

P J Gasser ◽

M S Park ◽

P B Moens

Keyword(s):

Synaptonemal Complex ◽

Protein Interactions ◽

Coiled Coil ◽

Protein Protein Interactions ◽

Late Prophase ◽

Yeast Saccharomyces Cerevisiae ◽

Heterotypic Interactions ◽

Two Hybrid ◽

Homotypic Interactions

In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II.

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Regulation of the Actin Cytoskeleton Organization in Yeast by a Novel Serine/Threonine Kinase Prk1p

The Journal of Cell Biology ◽

10.1083/jcb.144.1.71 ◽

1999 ◽

Vol 144 (1) ◽

pp. 71-82 ◽

Cited By ~ 77

Author(s):

Guisheng Zeng ◽

Mingjie Cai

Keyword(s):

Actin Cytoskeleton ◽

Asymmetric Distribution ◽

Wild Type ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization ◽

Related Proteins ◽

Regulation Of Actin Cytoskeleton

Normal actin cytoskeleton organization in budding yeast requires the function of the Pan1p/ End3p complex. Mutations in PAN1 and END3 cause defects in the organization of actin cytoskeleton and endocytosis. By screening for mutations that can suppress the temperature sensitivity of a pan1 mutant (pan1-4), a novel serine/threonine kinase Prk1p is now identified as a new factor regulating the actin cytoskeleton organization in yeast. The suppression of pan1-4 by prk1 requires the presence of mutant Pan1p. Although viable, the prk1 mutant is unable to maintain an asymmetric distribution of the actin cytoskeleton at 37°C. Consistent with its role in the regulation of actin cytoskeleton, Prk1p localizes to the regions of cell growth and coincides with the polarized actin patches. Overexpression of the PRK1 gene in wild-type cells leads to lethality and actin cytoskeleton abnormalities similar to those exhibited by the pan1 and end3 mutants. In vitro phosphorylation assays demonstrate that Prk1p is able to phosphorylate regions of Pan1p containing the LxxQxTG repeats, including the region responsible for binding to End3p. Based on these findings, we propose that the Prk1 protein kinase regulates the actin cytoskeleton organization by modulating the activities of some actin cytoskeleton-related proteins such as Pan1p/End3p.

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A SAC phosphoinositide phosphatase controls rice development via hydrolyzing phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate

10.1101/740001 ◽

2019 ◽

Author(s):

Tao Guo ◽

Hua-Chang Chen ◽

Zi-Qi Lu ◽

Min Diao ◽

Ke Chen ◽

...

Keyword(s):

Actin Polymerization ◽

Membrane Lipids ◽

Dependent Manner ◽

Cytoskeleton Organization ◽

Cellular Processes ◽

Phosphoinositide Phosphatase ◽

Actin Cytoskeleton Organization ◽

Phosphatidylinositol 4 ◽

Related Proteins

AbstractPhosphoinositides (PIs) as regulatory membrane lipids play essential roles in multiple cellular processes. Although the exact molecular targets of PIs-dependent modulation remain largely elusive, the effects of disturbed PIs metabolism could be employed to propose regulatory modules associated with particular downstream targets of PIs. Here, we identified the role of GRAIN NUMBER AND PLANT HEIGHT 1 (GH1), which encodes a suppressor of actin (SAC) domain-containing phosphatase with unknown function in rice. Endoplasmic reticulum-localized GH1 specifically dephosphorylated and hydrolyzed phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Inactivation of GH1 resulted in massive accumulation of both PI4P and PI(4,5)P2, while excessive GH1 caused their depletion. Notably, superabundant PI4P and PI(4,5)P2 could both disrupt actin cytoskeleton organization and suppress cell elongation. Interestingly, both PI4P and PI(4,5)P2 inhibited actin-related proteins 2 and 3 (Arp2/3) complex-nucleated actin branching networks in vitro, whereas PI(4,5)P2 showed more dramatic effect in a dose-dependent manner. Overall, the overaccumulation of PI(4,5)P2 resulted from dysfunction of SAC phosphatase possibly perturbs Arp2/3 complex-mediated actin polymerization, thereby disordering the cell development. These findings imply that Arp2/3 complex might be the potential molecular target of PI(4,5)P2-dependent modulation in eukaryotes, thereby providing new insights into the relationship between PIs homeostasis and plants growth and development.

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The interaction of verprolin WIRE with the adapter proteins family intersectin

Visnik ukrains'kogo tovaristva genetikiv i selekcioneriv ◽

10.7124/visnyk.utgis.17.1.1195 ◽

2019 ◽

Vol 17 (1) ◽

pp. 3-7

Author(s):

S. V. Kropyvko ◽

A. V. Rynditch

Keyword(s):

Actin Cytoskeleton ◽

Protein Interactions ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Sh3 Domains ◽

Invasion And Migration ◽

The Family ◽

Cellular Signal ◽

And Migration ◽

Cytoskeleton Rearrangements

Aim. WIRE is a scafold protein that regulates actin cytoskeleton rearrangements and the formation of actin enriched membrane processes responsible for invasion and migration. ITSN1 and ITSN2 are representatives of the family of intersectins who participate in the reorganization of the actin cytoskeleton, as well as in other processes, such as endo/enzocytosis, cellular signal transduction, etc. As these proteins participate in the same processes, we checked their interaction with each other. Methods. Protein-protein interactions were identified using the GST pull-down method. Results. We showed that the SH3 domains of ITSN1 and ITSN2 interact with WIRE, and found that while WIRE is in a complex with endogenous actin. Conclusions. ITSN1 and ITSN2 interact with WIRE, which is located in a complex with endogenous actin. Keywords: WIRE, ITSN1 and ITSN2, actin.

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MiRNA-155 targets myosin light chain kinase and modulates actin cytoskeleton organization in endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00521.2013 ◽

2014 ◽

Vol 306 (8) ◽

pp. H1192-H1203 ◽

Cited By ~ 43

Author(s):

Martina Weber ◽

Sinae Kim ◽

Nicole Patterson ◽

Kimberly Rooney ◽

Charles D. Searles

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Actin Cytoskeleton ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization ◽

Mouse Aorta

Previously, we identified a microRNA (miRNA) signature for endothelial cells (ECs) subjected to unidirectional shear stress (USS). MiR-155, a multifunctional miRNA that has been implicated in atherosclerosis, was among the shear stress-responsive miRNAs. Here, we examined the role of miR-155 in modulating EC phenotype and function. In vitro, increased miR-155 levels in human ECs induced changes in morphology and filamentous (F)-actin organization. In addition, ECs transfected with miR-155 mimic were less migratory and less proliferative and had less apoptosis compared with control ECs. In mouse aorta, miR-155 expression was increased in the intima of thoracic aorta, where blood flow produces steady and unidirectional shear stress, compared with the intima of the lower curvature of the aortic arch, which is associated with oscillatory and low shear stress. These differences in miR-155 expression were associated with distinct changes in EC morphology and F-actin. The effects of miR-155 in vitro were mediated through suppression of two key regulators of the EC cytoskeleton organization: RhoA and myosin light chain kinase (MYLK). A novel direct interaction between miR-155 and the MYLK 3′UTR was verified by luciferase-MYLK 3′UTR reporter assays. Furthermore, the intensity of immunofluorescence staining for RhoA and MYLK in mouse aorta correlated inversely with miR-155 expression. In conclusion, a prominent effect of the multifunctional miR-155 in ECs is modulation of phenotype through alterations in RhoA, MYLK expression, and actin cytoskeleton organization.

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A Role for GEA1 and GEA2 in the Organization of the Actin Cytoskeleton in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/165.3.985 ◽

2003 ◽

Vol 165 (3) ◽

pp. 985-995

Author(s):

Ewa Zakrzewska ◽

Marjorie Perron ◽

André Laroche ◽

Dominick Pallotta

Keyword(s):

Saccharomyces Cerevisiae ◽

Actin Cytoskeleton ◽

Dependent Manner ◽

Yeast Saccharomyces Cerevisiae ◽

Cytoskeleton Organization ◽

Actin Cable ◽

Multicopy Suppressors ◽

Actin Cytoskeleton Organization ◽

Actin Cables ◽

Adp Ribosylation Factor

Abstract Profilin is an actin monomer-binding protein implicated in the polymerization of actin filaments. In the budding yeast Saccharomyces cerevisiae, the pfy1-111 rho2Δ double mutant has severe growth and actin cytoskeletal defects. The GEA1 and GEA2 genes, which code for paralog guanosine exchange factors for Arf proteins, were identified as multicopy suppressors of the mutant phenotype. These two genes restored the polarized distribution of actin cortical patches and produced visible actin cables in both the pfy1-111 rho2Δ and pfy1Δ cells. Thus, overexpression of GEA1 or GEA2 bypassed the requirement for profilin in actin cable formation. In addition, gea1 gea2 double mutants showed defects in budding and in actin cytoskeleton organization, while overexpression of GEA1 or GEA2 led to the formation of supernumerary actin cable-like structures in a Bni1p/Bnr1p-dependent manner. The ADP-ribosylation factor Arf3p may be a target of Gea1p/Gea2p, since overexpression of ARF3 partially suppressed the profilin-deficient phenotype and a deletion of ARF3 exacerbated the phenotype of a pfy1-111 mutant. Gea1p, Gea2p, Arf1p, and Arf2p but not Arf3p are known to function in vesicular transport between the endoplasmic reticulum and the Golgi. In this work, we demonstrate a role for Gea1p, Gea2p, and Arf3p in the organization of the actin cytoskeleton.

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Stability of actin cytoskeleton and PKC-δ binding to actin regulate NKCC1 function in airway epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00357.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. C487-C496 ◽

Cited By ~ 42

Author(s):

Carole M. Liedtke ◽

Melinda Hubbard ◽

Xiangyun Wang

Keyword(s):

Actin Cytoskeleton ◽

Protein Interactions ◽

Actin Polymerization ◽

Direct Interaction ◽

Airway Epithelial Cells ◽

Cell Periphery ◽

Airway Epithelial ◽

Protein Protein Interactions ◽

Signaling Mechanism ◽

Increased Activity

Activation of airway epithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activity of protein kinase C (PKC)-δ, which localizes predominantly to the actin cytoskeleton. Prompted by reports of a role for actin in NKCC1 function, we studied a signaling mechanism linking NKCC1 and PKC. Stabilization of actin polymerization with jasplakinolide increased activity of NKCC1, whereas inhibition of actin polymerization with latrunculin B prevented hormonal activation of NKCC1. Protein-protein interactions among NKCC1, actin, and PKC-δ were verified by Western blot analysis of immunoprecipitated proteins. PKC-δ was detected in immunoprecipitates of NKCC1 and vice versa. Actin was also detected in immunoprecipitates of NKCC1 and PKC-δ. Pulldown of endogenous actin revealed the presence of NKCC1 and PKC-δ. Binding of recombinant PKC-δ to NKCC1 was not detected in overlay assays. Rather, activated PKC-δ bound to actin, and this interaction was prevented by a peptide encoding δC2, a C2-like domain based on the amino acid sequence of PKC-δ. δC2 also blocked stimulation of NKCC1 function by methoxamine. Immunofluorescence and confocal microscopy revealed PKC-δ in the cytosol and cell periphery. Merged images of cells stained for actin and PKC-δ indicated colocalization of PKC-δ and actin at the cell periphery. The results indicate that actin is critical for the activation of NKCC1 through a direct interaction with PKC-δ.

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Mutational Analysis of STE5 in the Yeast Saccharomyces cerevisiae: Application of a Differential Interaction Trap Assay for Examining Protein-Protein Interactions

Genetics ◽

10.1093/genetics/147.2.479 ◽

1997 ◽

Vol 147 (2) ◽

pp. 479-492 ◽

Cited By ~ 12

Author(s):

Carla Inouye ◽

Namrita Dhillon ◽

Tim Durfee ◽

Patricia C Zambryski ◽

Jeremy Thorner

Keyword(s):

Protein Interactions ◽

Mutational Analysis ◽

Mapk Cascade ◽

Mitogen Activated Protein Kinase ◽

Missense Mutations ◽

Protein Protein Interactions ◽

Yeast Saccharomyces Cerevisiae ◽

Mitogen Activated Protein

Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold that organizes the components of the mitogen-activated protein kinase (MAPK) cascade. A new method was developed to isolate missense mutations in Ste5 that differentially affect the ability of Ste5 to interact with either of two MAPK cascade constituents, the MEKK (Ste11) and the MEK (Ste7). Mutations that affect association with Ste7 or with Ste11 delineate discrete regions of Ste5 that are critical for each interaction. Co-immunoprecipitation analysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G protein, β subunit), and Fus3 (MAPK), confirmed that each mutation specifically affects the interaction of Ste5 with only one protein. When expressed in a ste5Δ cell, mutant Ste5 proteins that are defective in their ability to interact with either Ste11 or Ste7 result in a markedly reduced mating proficiency. One mutation that clearly weakened (but did not eliminate) interaction of Ste5 with Ste7 permitted mating at wild-type efficiency, indicating that an efficacious signal is generated even when Ste5 associates with only a small fraction of (or only transiently with) Ste7. Ste5 mutants defective in association with Ste11 or Ste7 showed strong interallelic complementation when co-expressed, suggesting that the functional form of Ste5 in vivo is an oligomer.

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