scholarly journals The conserved Pkh–Ypk kinase cascade is required for endocytosis in yeast

2002 ◽  
Vol 156 (2) ◽  
pp. 241-248 ◽  
Author(s):  
Amy K.A. deHart ◽  
Joshua D. Schnell ◽  
Damian A. Allen ◽  
Linda Hicke
Keyword(s):  
Fluid Phase ◽  
Receptor Internalization ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Extracellular Signals ◽  
Fluid Phase Endocytosis ◽  
Endocytic Machinery ◽  
Kinase Cascade ◽  
Signaling Receptors ◽  
Mutant Cells

Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast α-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base–regulated serine–threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum– and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in α-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in α-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.

Ligand-stimulated beta 2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes

1995 ◽  
Vol 108 (9) ◽  
pp. 2983-2991 ◽  
Author(s):  
R.H. Moore ◽  
N. Sadovnikoff ◽  
S. Hoffenberg ◽  
S. Liu ◽  
P. Woodford ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Adrenergic Receptor ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Beta Agonist ◽  
Hek293 Cells ◽  
Fluid Phase Endocytosis ◽  
Beta 2

The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytosis. However, it is unknown whether rab5 regulates receptors whose internalization is stimulated by the binding of ligand, and whether such receptors change the underlying rate of the endocytic pathways they utilize. As a model for ligand-stimulated endocytosis, we used transfected HEK293 cells expressing high levels of an epitope-tagged human beta 2-adrenergic receptor. Nearly all receptors were on the cell surface in the absence of agonist, but within ten minutes of agonist addition > 50% of receptors internalized and colocalized extensively with rab5. Hypertonic sucrose blocked beta 2-adrenergic receptor internalization, as well as that of transferrin receptor, suggesting a clathrin-mediated process. In contrast, an inhibitor of potocytosis had little effect upon beta 2-adrenergic receptor internalization, suggesting that this process did not require active caveolae. Consistent with this finding, caveolin was not detectable in the 12 beta 6 line, as assessed by western blotting with a polyclonal anti-caveolin antibody. Stimulated receptor internalization did not affect the rate or capacity of the constitutive endocytic pathway since there was no detectable increase in fluid-phase endocytosis after addition of beta-agonist, nor was there a significant change in the amount of surface transferrin receptor. Altogether, these data suggest that beta 2-adrenergic receptors internalize by a clathrin-mediated and rab5-regulated constitutive endocytic pathway. Further, agonist-stimulated receptor internalization has no detectable effect upon the function of this pathway.

The Dictyostelium RasS protein is required for macropinocytosis, phagocytosis and the control of cell movement

2000 ◽  
Vol 113 (4) ◽  
pp. 709-719 ◽  
Author(s):  
J.R. Chubb ◽  
A. Wilkins ◽  
G.M. Thomas ◽  
R.H. Insall
Keyword(s):  
Cell Migration ◽  
Significant Proportion ◽  
Cell Movement ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Cell Cortex ◽  
Fluid Phase Endocytosis ◽  
Ras Family ◽  
Actin Protrusions ◽  
Mutant Cells

Endocytosis and cell migration both require transient localised remodelling of the cell cortex. Several lines of evidence suggest a key regulatory role in these activities for members of the Ras family of small GTPases. We have generated Dictyostelium cells lacking one member of this family, RasS, and the mutant cells are perturbed in endocytosis and cell migration. Mutant amoebae are defective in phagocytosis and fluid-phase endocytosis and are impaired in growth. Conversely, the rasS(-)cells show an enhanced rate of cell migration, moving three times faster than wild-type controls. The mutant cells display an aberrant morphology, are highly polarised, carry many elongated actin protrusions and show a concomitant decrease in formation of pinocytic crowns on the cell surface. These morphological aberrations are paralleled by changes in the actin cytoskeleton, with a significant proportion of the cortical F-actin relocalised to prominent pseudopodia. Rapid migration and endocytosis appear to be mutually incompatible and it is likely that RasS protein is required to maintain the normal balance between these two actin-dependent processes.

scholarly journals Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis

2004 ◽  
Vol 15 (2) ◽  
pp. 861-869 ◽  
Author(s):  
Yaya Lefkir ◽  
Marilyne Malbouyres ◽  
Daniel Gotthardt ◽  
Adrian Ozinsky ◽  
Sophie Cornillon ◽  
...  
Keyword(s):  
Early Stage ◽  
Fluid Phase ◽  
Small Particles ◽  
Medium Chain ◽  
Murine Macrophages ◽  
Fluid Phase Endocytosis ◽  
Phagocytic Cups ◽  
Golgi Network ◽  
Mutant Cells

The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1-cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1-cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1-cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation.

scholarly journals Mammalian Hippo signalling: a kinase network regulated by protein–protein interactions

2012 ◽  
Vol 40 (1) ◽  
pp. 124-128 ◽  
Author(s):  
Alexander Hergovich
Keyword(s):  
Protein Interactions ◽  
Tumour Suppressor ◽  
Protein Protein Interactions ◽  
Large Tumour ◽  
Serine Threonine Kinase ◽  
Ww Domains ◽  
Threonine Kinase ◽  
Signalling Molecules ◽  
Kinase Cascade ◽  
Hippo Signalling

The Hippo signal transduction cascade controls cell growth, proliferation and death, all of which are frequently deregulated in tumour cells. Since initial studies in Drosophila melanogaster were instrumental in defining Hippo signalling, the machinery was named after the central Ste20-like kinase Hippo. Moreover, given that loss of Hippo signalling components Hippo, Warts, and Mats resulted in uncontrolled tissue overgrowth, Hippo signalling was defined as a tumour-suppressor cascade. Significantly, all of the core factors of Hippo signalling have mammalian orthologues that functionally compensate for loss of their counterparts in Drosophila. Furthermore, studies in Drosophila and mammalian cell systems showed that Hippo signalling represents a kinase cascade that is tightly regulated by PPIs (protein–protein interactions). Several Hippo signalling molecules contain SARAH (Salvador/RASSF1A/Hippo) domains that mediate specific PPIs, thereby influencing the activities of MST1/2 (mammalian Ste20-like serine/threonine kinase 1/2) kinases, the human Hippo orthologues. Moreover, WW domains are present in several Hippo factors, and these domains also serve as interaction surfaces for regulatory PPIs in Hippo signalling. Finally, the kinase activities of LATS1/2 (large tumour-suppressor kinase 1/2), the human counterparts of Warts, are controlled by binding to hMOB1 (human Mps one binder protein 1), the human Mats. Therefore Hippo signalling is regulated by PPIs on several levels. In the present paper, I review the current understanding of how these regulatory PPIs are regulated and contribute to the functionality of Hippo signalling.

Assessing the role of the ASP56/CAP homologue of Dictyostelium discoideum and the requirements for subcellular localization

1999 ◽  
Vol 112 (19) ◽  
pp. 3195-3203 ◽  
Author(s):  
A.A. Noegel ◽  
F. Rivero ◽  
R. Albrecht ◽  
K.P. Janssen ◽  
J. Kohler ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Fluid Phase ◽  
Axenic Medium ◽  
Terminal Domain ◽  
Defect Growth ◽  
Fluid Phase Endocytosis ◽  
Altered Cell ◽  
Mutant Cells ◽  
Proline Rich Region

The CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP(2)-regulation of the sequestering activity. Here we have constructed GFP-fusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination. They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect. Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria. In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild-type cells. This was paralleled by a reduction in fluid phase endocytosis. Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected.

scholarly journals Betaglycan can act as a dual modulator of TGF-beta access to signaling receptors: mapping of ligand binding and GAG attachment sites

1994 ◽  
Vol 124 (4) ◽  
pp. 557-568 ◽  
Author(s):  
F López-Casillas ◽  
HM Payne ◽  
JL Andres ◽  
J Massagué
Keyword(s):  
Potent Inhibitor ◽  
Membrane Receptors ◽  
Deletion Mutants ◽  
Tgf Beta ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Terminal Domain ◽  
Extracellular Region ◽  
Attachment Sites ◽  
Signaling Receptors

Betaglycan, also known as the TGF-beta type III receptor, is a membrane-anchored proteoglycan that presents TGF-beta to the type II signaling receptor, a transmembrane serine/threonine kinase. The betaglycan extracellular region, which can be shed by cells into the medium, contains a NH2-terminal domain related to endoglin and a COOH-terminal domain related to uromodulin, sperm receptors Zp2 and 3, and pancreatic secretory granule GP-2 protein. We identified residues Ser535 and Ser546 in the uromodulin-related region as the glycosaminoglycan (GAG) attachment sites. Their mutation to alanine prevents GAG attachment but does not interfere with betaglycan stability or ability to bind and present TGF-beta to receptor II. Using a panel of deletion mutants, we found that TGF-beta binds to the NH2-terminal endoglin-related region of betaglycan. The remainder of the extracellular domain and the cytoplasmic domain are not required for presentation of TGF-beta to receptor II; however, membrane anchorage is required. Soluble betaglycan can bind TGF-beta but does not enhance binding to membrane receptors. In fact, recombinant soluble betaglycan acts as potent inhibitor of TGF-beta binding to membrane receptors and blocks TGF-beta action, this effect being particularly pronounced with the TGF-beta 2 isoform. The results suggest that release of betaglycan into the medium converts this enhancer of TGF-beta action into a TGF-beta antagonist.

scholarly journals The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo

1998 ◽  
Vol 142 (5) ◽  
pp. 1289-1299 ◽  
Author(s):  
Lisa D. Belmont ◽  
David G. Drubin
Keyword(s):  
Fluid Phase ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Cortical Actin ◽  
Fluid Phase Endocytosis ◽  
Synthetically Lethal ◽  
Actin Patch ◽  
Actin Cables ◽  
Mutant Cells

Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables.

scholarly journals Novel Proteins Linking the Actin Cytoskeleton to the Endocytic Machinery in Saccharomyces cerevisiae

2002 ◽  
Vol 13 (10) ◽  
pp. 3646-3661 ◽  
Author(s):  
H. Dewar ◽  
D. T. Warren ◽  
F. C. Gardiner ◽  
C. G. Gourlay ◽  
N. Satish ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Elevated Temperatures ◽  
Adaptor Protein ◽  
Fluid Phase ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Fluid Phase Endocytosis ◽  
Endocytic Machinery

The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.

Akt/PKB: one kinase, many modifications

2015 ◽  
Vol 468 (2) ◽  
pp. 203-214 ◽  
Author(s):  
Guillermo Risso ◽  
Matías Blaustein ◽  
Berta Pozzi ◽  
Pablo Mammi ◽  
Anabella Srebrow
Keyword(s):  
Large Body ◽  
Fine Tuning ◽  
Serine Threonine Kinase ◽  
Post Translational Modifications ◽  
Akt Phosphorylation ◽  
Akt Activation ◽  
Threonine Kinase ◽  
Cellular Processes ◽  
Extracellular Signals ◽  
Signalling Molecule

Akt/PKB, a serine/threonine kinase member of the AGC family of proteins, is involved in the regulation of a plethora of cellular processes triggered by a wide diversity of extracellular signals and is thus considered a key signalling molecule in higher eukaryotes. Deregulation of Akt signalling is associated with a variety of human diseases, revealing Akt-dependent pathways as an attractive target for therapeutic intervention. Since its discovery in the early 1990s, a large body of work has focused on Akt phosphorylation of two residues, Thr308 and Ser473, and modification of these two sites has been established as being equivalent to Akt activation. More recently, Akt has been identified as a substrate for many different post-translational modifications, including not only phosphorylation of other residues, but also acetylation, glycosylation, oxidation, ubiquitination and SUMOylation. These modifications could provide additional regulatory steps for fine-tuning Akt function, Akt trafficking within the cell and/or for determining the substrate specificity of this signalling molecule. In the present review, we provide an overview of these different post-translational modifications identified for Akt, focusing on their consequences for this kinase activity.

Sign in / Sign up

Export Citation Format

Share Document