A conserved polybasic domain mediates plasma membrane targeting of Lgl and its regulation by hypoxia

Lethal giant larvae (Lgl) plays essential and conserved functions in regulating both cell polarity and tumorigenesis in Drosophila melanogaster and vertebrates. It is well recognized that plasma membrane (PM) or cell cortex localization is crucial for Lgl function in vivo, but its membrane-targeting mechanisms remain poorly understood. Here, we discovered that hypoxia acutely and reversibly inhibits Lgl PM targeting through a posttranslational mechanism that is independent of the well-characterized atypical protein kinase C (aPKC) or Aurora kinase–mediated phosphorylations. Instead, we identified an evolutionarily conserved polybasic (PB) domain that targets Lgl to the PM via electrostatic binding to membrane phosphatidylinositol phosphates. Such PB domain–mediated PM targeting is inhibited by hypoxia, which reduces inositol phospholipid levels on the PM through adenosine triphosphate depletion. Moreover, Lgl PB domain contains all the identified phosphorylation sites of aPKC and Aurora kinases, providing a molecular mechanism by which phosphorylations neutralize the positive charges on the PB domain to inhibit Lgl PM targeting.

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Analysis of the roles of phosphatidylinositol-4,5-bisphosphate and individual subunits in assembly, localization, and function of Saccharomyces cerevisiae target of rapamycin complex 2

Molecular Biology of the Cell ◽

10.1091/mbc.e18-10-0682 ◽

2019 ◽

Vol 30 (12) ◽

pp. 1555-1574 ◽

Cited By ~ 2

Author(s):

Maria Nieves Martinez Marshall ◽

Anita Emmerstorfer-Augustin ◽

Kristin L. Leskoske ◽

Lydia H. Zhang ◽

Biyun Li ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Lipid Metabolism ◽

Eukaryotic Cell ◽

Target Of Rapamycin ◽

Multiprotein Complex ◽

Ph Domain ◽

Evolutionarily Conserved ◽

And Function

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2-associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5- bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or cause disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.

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Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

Molecular Biology of the Cell ◽

10.1091/mbc.e10-03-0256 ◽

2011 ◽

Vol 22 (2) ◽

pp. 189-201 ◽

Cited By ~ 40

Author(s):

Roman Gorelik ◽

Changsong Yang ◽

Vasumathi Kameswaran ◽

Roberto Dominguez ◽

Tatyana Svitkina

Keyword(s):

Plasma Membrane ◽

Electrostatic Interactions ◽

Coiled Coil ◽

Small Gtpases ◽

Coincidence Detection ◽

Membrane Targeting ◽

Amino Terminal ◽

N Terminus

The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1–FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.

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Formation of a Bazooka–Stardust complex is essential for plasma membrane polarity in epithelia

The Journal of Cell Biology ◽

10.1083/jcb.201006029 ◽

2010 ◽

Vol 190 (5) ◽

pp. 751-760 ◽

Cited By ~ 65

Author(s):

Michael P. Krahn ◽

Johanna Bückers ◽

Lars Kastrup ◽

Andreas Wodarz

Keyword(s):

Plasma Membrane ◽

Protein Complexes ◽

Pdz Domain ◽

Phosphorylation Site ◽

Direct Interaction ◽

Epithelial Polarity ◽

Kinase C ◽

Discs Large ◽

Evolutionarily Conserved ◽

Functional Hierarchy

Apical–basal polarity in Drosophila melanogaster epithelia depends on several evolutionarily conserved proteins that have been assigned to two distinct protein complexes: the Bazooka (Baz)–PAR-6 (partitioning defective 6)–atypical protein kinase C (aPKC) complex and the Crumbs (Crb)–Stardust (Sdt) complex. These proteins operate in a functional hierarchy, in which Baz is required for the proper subcellular localization of all other proteins. We investigated how these proteins interact and how this interaction is regulated. We show that Baz recruits Sdt to the plasma membrane by direct interaction between the Postsynaptic density 95/Discs large/Zonula occludens 1 (PDZ) domain of Sdt and a region of Baz that contains a phosphorylation site for aPKC. Phosphorylation of Baz causes the dissociation of the Baz–Sdt complex. Overexpression of a nonphosphorylatable version of Baz blocks the dissociation of Sdt from Baz, causing phenotypes very similar to those of crb and sdt mutations. Our findings provide a molecular mechanism for the phosphorylation-dependent interaction between the Baz–PAR-3 and Crb complexes during the establishment of epithelial polarity.

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Immunocytochemical evidence for translocation of protein kinase C in human megakaryoblastic leukemic cells: synergistic effects of Ca2+ and activators of protein kinase C on the plasma membrane association.

The Journal of Cell Biology ◽

10.1083/jcb.107.3.929 ◽

1988 ◽

Vol 107 (3) ◽

pp. 929-937 ◽

Cited By ~ 42

Author(s):

T Ito ◽

T Tanaka ◽

T Yoshida ◽

K Onoda ◽

H Ohta ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Synergistic Effects ◽

Leukemic Cells ◽

Immunofluorescent Staining ◽

Protein Kinase Activity ◽

Intact Cells ◽

Kinase C

Immunological analysis using monoclonal antibodies against subspecies of protein kinase C revealed the predominant expression of the isozyme, type II, in human megakaryoblastic leukemic cells. We investigated the effects of phorbol diester 12-O-tetradecanoyl phorbol-13-acetate (TPA), the Ca2+ ionophore ionomycin and synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) on the immunocytochemical localization of protein kinase C in these cells. Indirect immunofluorescence techniques revealed the enzyme to be located in a diffuse cytosolic pattern, in the intact cells. When the cells were exposed to 100 nM TPA, the immunofluorescent staining was translocated from the cytoplasm to the plasma membrane. The translocation was protracted and staining on the membrane decreased in parallel with the Ca2+, phospholipid-dependent protein kinase activity. Treatment of the cells with 500 nM ionomycin caused an apparent translocation comparable with that seen with TPA, however, this translocation was transient and most of the cytosolic staining was within 60 min. We also found that 30 micrograms/ml OAG did not have significant effects on distribution of the staining, but rather acted synergistically on the translocation with the suboptimal concentration of 100 nM ionomycin. A similar synergism was also observed with 10 nM TPA and 100 nM ionomycin. These results obtained in situ provide evidence that intracellular Ca2+ and diacylglycerol regulate membrane binding of the enzyme in vivo.

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Human NDR Kinases Are Rapidly Activated by MOB Proteins through Recruitment to the Plasma Membrane and Phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.25.18.8259-8272.2005 ◽

2005 ◽

Vol 25 (18) ◽

pp. 8259-8272 ◽

Cited By ~ 77

Author(s):

Alexander Hergovich ◽

Samuel J. Bichsel ◽

Brian A. Hemmings

Keyword(s):

Plasma Membrane ◽

Regulatory Mechanism ◽

Membrane Targeting ◽

Membrane Translocation ◽

Cancer Types ◽

Membrane Bound ◽

Insight Into ◽

Constitutively Active

ABSTRACT Human nuclear Dbf2-related kinases (NDRs) are up-regulated in certain cancer types, yet their precise function(s) and regulatory mechanism(s) still remain to be defined. Here, we show that active (phosphorylated on Thr444) and inactive human NDRs are both mainly cytoplasmic. Moreover, NDR kinases colocalize at the plasma membrane with human MOBs (hMOBs), which are recently described coactivators of human NDR in vitro. Strikingly, membrane targeting of NDR results in a constitutively active kinase due to phosphorylation on Ser281 and Thr444 that is further activated upon coexpression of hMOBs. Membrane-targeted hMOBs also robustly promoted activation of NDR. We further demonstrate that the in vivo activation of human NDR by membrane-bound hMOBs is dependent on their interaction and occurs solely at the membrane. By using a chimeric molecule of hMOB, which allows inducible membrane translocation, we found that NDR phosphorylation and activation at the membrane occur a few minutes after association of hMOB with membranous structures. We provide insight into a potential in vivo mechanism of NDR activation through rapid recruitment to the plasma membrane mediated by hMOBs.

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Lgl cortical dynamics are independent of binding to the Scrib-Dlg complex but require Dlg-dependent restriction of aPKC

10.1101/867929 ◽

2019 ◽

Cited By ~ 2

Author(s):

Guilherme Ventura ◽

Sofia Moreira ◽

André Barros-Carvalho ◽

Mariana Osswald ◽

Eurico Morais-de-Sá

Keyword(s):

Plasma Membrane ◽

Dynamic Behavior ◽

Binding Sites ◽

Fluorescence Recovery After Photobleaching ◽

Follicle Cells ◽

Cortical Dynamics ◽

Discs Large ◽

Lethal Giant Larvae ◽

Epithelial Barriers

AbstractApical-basal polarity underpins the formation of specialized epithelial barriers that are critical for metazoan physiology. Although apical-basal polarity is long known to require the basolateral determinants Lethal Giant Larvae (Lgl), Discs Large (Dlg) and Scribble (Scrib), mechanistic understanding of their function is limited. Lgl plays a role as an aPKC inhibitor, but it remains unclear whether Lgl also forms a complex with Dlg or Scrib. Using fluorescence recovery after photobleaching, we show that Lgl does not form immobile complexes at the lateral domain of Drosophila follicle cells. Optogenetic depletion of plasma membrane phosphatidylinositol 4,5-biphosphate (PIP2) or Dlg removal accelerate Lgl cortical dynamics. However, whereas Lgl turnover relies on PIP2 binding, Dlg and Scrib are only required for Lgl localization and dynamic behavior in the presence of aPKC function. Furthermore, light-induced oligomerization of basolateral proteins indicate that Lgl is not part of the Scrib-Dlg complex in vivo. Thus, Scrib-Dlg are necessary to repress aPKC activity in the lateral domain but do not provide cortical binding sites for Lgl. Our work therefore highlights that Lgl does not act in a complex but in parallel with Scrib-Dlg to antagonize apical determinants.

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Dlg1 controls planar spindle orientation in the neuroepithelium through direct interaction with LGN

The Journal of Cell Biology ◽

10.1083/jcb.201405060 ◽

2014 ◽

Vol 206 (6) ◽

pp. 707-717 ◽

Cited By ~ 42

Author(s):

Mehdi Saadaoui ◽

Mickaël Machicoane ◽

Florencia di Pietro ◽

Fred Etoc ◽

Arnaud Echard ◽

...

Keyword(s):

Mitotic Spindle ◽

Direct Interaction ◽

Human Cells ◽

Spindle Orientation ◽

Cell Cortex ◽

Precise Localization ◽

Cell Divisions ◽

Evolutionarily Conserved ◽

Cortical Localization

Oriented cell divisions are necessary for the development of epithelial structures. Mitotic spindle orientation requires the precise localization of force generators at the cell cortex via the evolutionarily conserved LGN complex. However, polarity cues acting upstream of this complex in vivo in the vertebrate epithelia remain unknown. In this paper, we show that Dlg1 is localized at the basolateral cell cortex during mitosis and is necessary for planar spindle orientation in the chick neuroepithelium. Live imaging revealed that Dlg1 is required for directed spindle movements during metaphase. Mechanistically, we show that direct interaction between Dlg1 and LGN promotes cortical localization of the LGN complex. Furthermore, in human cells dividing on adhesive micropatterns, homogenously localized Dlg1 recruited LGN to the mitotic cortex and was also necessary for proper spindle orientation. We propose that Dlg1 acts primarily to recruit LGN to the cortex and that Dlg1 localization may additionally provide instructive cues for spindle orientation.

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Autoregulation of the Raf-1 serine/threonine kinase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.16.9214 ◽

1998 ◽

Vol 95 (16) ◽

pp. 9214-9219 ◽

Cited By ~ 97

Author(s):

Richard E. Cutler ◽

Robert M. Stephens ◽

Misty R. Saracino ◽

Deborah K. Morrison

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Regulatory Region ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Rich Domain ◽

Kinase C ◽

Evolutionarily Conserved ◽

Phosphorylation Event ◽

Cysteine Rich Domain

The Raf-1 serine/threonine kinase is a key protein involved in the transmission of many growth and developmental signals. In this report, we show that autoinhibition mediated by the noncatalytic, N-terminal regulatory region of Raf-1 is an important mechanism regulating Raf-1 function. The inhibition of the regulatory region occurs, at least in part, through binding interactions involving the cysteine-rich domain. Events that disrupt this autoinhibition, such as mutation of the cysteine-rich domain or a mutation mimicking an activating phosphorylation event (Y340D), alleviate the repression of the regulatory region and increase Raf-1 activity. Based on the striking similarites between the autoregulation of the serine/threonine kinases protein kinase C, Byr2, and Raf-1, we propose that relief of autorepression and activation at the plasma membrane is an evolutionarily conserved mechanism of kinase regulation.

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Dictyostelium Dock180-related RacGEFs Regulate the Actin Cytoskeleton during Cell Motility

Molecular Biology of the Cell ◽

10.1091/mbc.e08-09-0899 ◽

2009 ◽

Vol 20 (2) ◽

pp. 699-707 ◽

Cited By ~ 20

Author(s):

Alessia Para ◽

Miriam Krischke ◽

Sylvain Merlot ◽

Zhouxin Shen ◽

Michael Oberholzer ◽

...

Keyword(s):

Cell Motility ◽

Protein Function ◽

Actin Polymerization ◽

Cell Movement ◽

Leading Edge ◽

Cell Cortex ◽

Proteomic Approach ◽

Evolutionarily Conserved ◽

Membrane Protrusions

Cell motility of amoeboid cells is mediated by localized F-actin polymerization that drives the extension of membrane protrusions to promote forward movements. We show that deletion of either of two members of the Dictyostelium Dock180 family of RacGEFs, DockA and DockD, causes decreased speed of chemotaxing cells. The phenotype is enhanced in the double mutant and expression of DockA or DockD complements the reduced speed of randomly moving DockD null cells' phenotype, suggesting that DockA and DockD are likely to act redundantly and to have similar functions in regulating cell movement. In this regard, we find that overexpressing DockD causes increased cell speed by enhancing F-actin polymerization at the sites of pseudopod extension. DockD localizes to the cell cortex upon chemoattractant stimulation and at the leading edge of migrating cells and this localization is dependent on PI3K activity, suggesting that DockD might be part of the pathway that links PtdIns(3,4,5)P3 production to F-actin polymerization. Using a proteomic approach, we found that DdELMO1 is associated with DockD and that Rac1A and RacC are possible in vivo DockD substrates. In conclusion, our work provides a further understanding of how cell motility is controlled and provides evidence that the molecular mechanism underlying Dock180-related protein function is evolutionarily conserved.

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Rapid clearance of malaria circumsporozoite protein (CS) by hepatocytes.

Journal of Experimental Medicine ◽

10.1084/jem.179.2.695 ◽

1994 ◽

Vol 179 (2) ◽

pp. 695-701 ◽

Cited By ~ 40

Author(s):

C Cerami ◽

U Frevert ◽

P Sinnis ◽

B Takacs ◽

V Nussenzweig

Keyword(s):

Plasma Membrane ◽

Circumsporozoite Protein ◽

Amino Acid Sequence Homology ◽

Evolutionarily Conserved ◽

A Cell ◽

Rapid Clearance ◽

Region Ii ◽

Striking Parallelism

The circumsporozoite protein (CS) covers uniformly the plasma membrane of malaria sporozoites. In vitro, CS multimers bind specifically to regions of the hepatocyte plasma membrane that are exposed to circulating blood in the Disse space. The ligand is in the region II-plus of CS, an evolutionarily conserved stretch of the protein that has amino acid sequence homology to a cell adhesive motif of thrombospondin. We have now found that intravenously injected CS constructs bind rapidly to the basolateral surface of hepatocytes, provided that the recombinant proteins contain region II-plus, and that they are aggregated. Significant amounts of CS were not retained in any other organ. The striking parallelism between these in vitro and in vivo findings with the target specificity of malaria sporozoites, reinforces the hypothesis that the attachment of the parasites to hepatocytes is via region II-plus of CS.

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