scholarly journals A Polybasic Domain in aPKC Mediates Par6-Dependent Control of Membrane Targeting and Kinase Activity

2019 ◽  
Author(s):  
Wei Dong ◽  
Juan Lu ◽  
Xuejing Zhang ◽  
Yan Wu ◽  
Kaela Lettieri ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Protein Interactions ◽  
Calcium Binding ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Allosteric Regulation ◽  
Cell Polarization ◽  
Physical Interaction ◽  
Binding Domains

SUMMARYMechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. Here we show that in both Drosophila and mammalian cells the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.eTOC SummaryDong et al. discover that the pseudo-substrate region (PSr) in aPKC is a polybasic domain capable of electrostatically targeting aPKC to plasma membrane. Allosteric regulation of PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of kinase activity.

scholarly journals A polybasic domain in aPKC mediates Par6-dependent control of membrane targeting and kinase activity

2020 ◽  
Vol 219 (7) ◽  
Author(s):  
Wei Dong ◽  
Juan Lu ◽  
Xuejing Zhang ◽  
Yan Wu ◽  
Kaela Lettieri ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Protein Interactions ◽  
Calcium Binding ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Cell Polarization ◽  
Physical Interaction ◽  
Protein Protein Interactions ◽  
Binding Domains ◽  
Electrostatic Binding

Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein–protein interactions. Here we show that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.

Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane

2001 ◽  
Vol 114 (10) ◽  
pp. 1893-1900 ◽  
Author(s):  
S. Lusa ◽  
T.S. Blom ◽  
E.L. Eskelinen ◽  
E. Kuismanen ◽  
J.E. Mansson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Ldl Cholesterol ◽  
Normal Cells ◽  
Recycling Endosomes ◽  
Regulate Protein ◽  
Niemann Pick ◽  
Accumulation Characteristic

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

scholarly journals aPKC: the Kinase that Phosphorylates Cell Polarity

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 903 ◽  
Author(s):  
Yang Hong
Keyword(s):  
Subcellular Localization ◽  
Cell Polarity ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Cell Polarization ◽  
Kinase C ◽  
Dividing Cells ◽  
Cell Embryo ◽  
Powerful Mechanism ◽  
Anterior Posterior

Establishing and maintaining cell polarity are dynamic processes that necessitate complicated but highly regulated protein interactions. Phosphorylation is a powerful mechanism for cells to control the function and subcellular localization of a target protein, and multiple kinases have played critical roles in cell polarity. Among them, atypical protein kinase C (aPKC) is likely the most studied kinase in cell polarity and has the largest number of downstream substrates characterized so far. More than half of the polarity proteins that are essential for regulating cell polarity have been identified as aPKC substrates. This review covers mainly studies of aPKC in regulating anterior-posterior polarity in the worm one-cell embryo and apical-basal polarity in epithelial cells and asymmetrically dividing cells (for example, Drosophila neuroblasts). We will go through aPKC target proteins in cell polarity and discuss various mechanisms by which aPKC phosphorylation controls their subcellular localizations and biological functions. We will also review the recent progress in determining the detailed molecular mechanisms in spatial and temporal control of aPKC subcellular localization and kinase activity during cell polarization.

scholarly journals Microtubules support a disk-like septin arrangement at the plasma membrane of mammalian cells

2011 ◽  
Vol 22 (23) ◽  
pp. 4588-4601 ◽  
Author(s):  
Mikael E. Sellin ◽  
Per Holmfeldt ◽  
Sonja Stenmark ◽  
Martin Gullberg
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell Model ◽  
Cell Types ◽  
Higher Order ◽  
Model Systems ◽  
Animal Cells ◽  
Endogenous Gene ◽  
Binding Domains ◽  
Substrate Adhesion

Septin family proteins oligomerize through guanosine 5′-triphosphate–binding domains into core heteromers, which in turn polymerize at the cleavage furrow of dividing fungal and animal cells. Septin assemblies during the interphase of animal cells remain poorly defined and are the topic of this report. In this study, we developed protocols for visualization of authentic higher-order assemblies using tagged septins to effectively replace the endogenous gene product within septin core heteromers in human cells. Our analysis revealed that septins assemble into microtubule-supported, disk-like structures at the plasma membrane. In the absence of cell substrate adhesion, this is the predominant higher-order arrangement in interphase cells and each of the seven to eight septin family members expressed by the two analyzed cell types appears equally represented. However, studies of myeloid and lymphoid cell model systems revealed cell type–specific alterations of higher-order septin arrangements in response to substrate adhesion. Live-cell observations suggested that all higher-order septin assemblies are mutually exclusive with plasma membrane regions undergoing remodeling. The combined data point to a mechanism by which densely arranged cortical microtubules, which are typical for nonadhered spherical cells, support plasma membrane–bound, disk-like septin assemblies.

The PtdIns3P phosphatase myotubularin is a cytoplasmic protein that also localizes to Rac1-inducible plasma membrane ruffles

2002 ◽  
Vol 115 (15) ◽  
pp. 3105-3117 ◽  
Author(s):  
Jocelyn Laporte ◽  
Francois Blondeau ◽  
Anne Gansmuller ◽  
Yves Lutz ◽  
Jean-Luc Vonesch ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Mammalian Cells ◽  
Transcriptional Regulators ◽  
Membrane Domains ◽  
Cytoplasmic Protein ◽  
Specific Antibodies ◽  
Myotubular Myopathy ◽  
Rac1 Gtpase ◽  
Aspartate Residue

Myotubularin, the phosphatase mutated in X-linked myotubular myopathy, was shown to dephosphorylate phosphatidylinositol 3-monophosphate(PtdIns3P) and was also reported to interact with nuclear transcriptional regulators from the trithorax family. We have characterized a panel of specific antibodies and investigated the subcellular localization of myotubularin. Myotubularin is not detected in the nucleus, and localizes mostly as a dense cytoplasmic network. Overexpression of myotubularin does not detectably affect vesicle trafficking in the mammalian cells investigated, in contrast to previous observations in yeast models. Both mutation of a key aspartate residue of myotubularin and dominant activation of Rac1 GTPase lead to the recruitment of myotubularin to specific plasma membrane domains. Localization to Rac1-induced ruffles is dependent on the presence of a domain highly conserved in the myotubularin family (that we named RID). We thus propose that myotubularin may dephosphorylate a subpool of PtdIns3P(or another related substrate) at the plasma membrane.

Lysophospholipid Acylation in RBC.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1575-1575
Author(s):  
Eric Soupene ◽  
Frans A. Kuypers
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Lysophosphatidic Acid ◽  
Calcium Binding ◽  
Mammalian Cells ◽  
Membrane Lipid ◽  
Polar Group ◽  
Acyl Transferase ◽  
Calcium Binding Site ◽  
E Coli

Abstract The formation, distribution and utilization of acyl-CoA plays a crucial role in plasma membrane phospholipid turnover in red blood cells (RBC). Upon de-acylation of glycero-phospholipids (PL) via the action of phospholipase, re-acylation of the lysophospholipids (LPL) requires activity of two enzymes of the Lands pathway. Long-chain acyl-CoA synthetases (ACSL) activate fatty acids to acyl-CoA which are subsequently ligated to LPL by LysoPhosphoLipid Acyl Transferase (LPLAT) a family of enzymes with exclusive specificity for the polar group of LPL (phosphatidic acid, choline, serine and ethanolamine). We recently identified ACSL6 as the enzyme responsible for the activation of fatty acid in RBC. None of the family members of LPLAT have been identified in RBC to date. LPC, either generated in the RBC or taken up from plasma, is rapidly acylated by RBC suggesting an important role for Lysohosphatidylcholine-acyl transferase (LPCAT) in RBC. We report the identification and characterization of LPCAT, the enzyme that generates PC from LPC and acylCoA. We identified the RNA expression of LPCAT, an approximately 60kD protein, in reticulocytes, confirming proteomic studies suggesting the presence of this protein in adult RBC membranes.). It is a modular protein containing an acyltransferase domain at the amino-terminus, three predicted membrane spanning domains, and a putative calcium binding site at the C-terminus, distinguishing it from the lysophosphatidic acid acyltransferease (LPAAT). The putative LPCAT was expressed in E. coli. It was found in the E. coli membrane fraction, and was able to use oleoyl-CoA and LPC as substrates to generate PC. Lysophosphatidic acid (LPA) was not acylated by this protein. In contrast the previously identified LPAAT (1) expressed in E. coli, utilized LPA but not LPC, indicating LPL specificity of these enzymes. Radioactive fatty acid added to RBC is also incorporated in phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS). Sequence analysis suggests that two other proteins present in the genome of mammals are homologues of LPCAT. We hypothesize that these putative acyltransferases are responsible for the acylation of lysophosphatidyl ethanolamine (LPE) and lysophosphatidyl serine (LPS). These proteins are essential to maintain a proper glycerophospholipid composition of the RBC membrane and thereby viability of the cells. A dysfunction of this system may underlie the observed differences in phospholipid molecular species composition in subpopulations of sickle cells contributing to sickle cell pathology. A complete description of these proteins involved in the maintenance of glycerophospholipid composition of RBC will aid to better understand the maintenance of plasma membrane lipid composition of all mammalian cells.

scholarly journals Interaction of the DNA modifying proteins VirD1 and VirD2 ofAgrobacterium tumefaciens: Analysis by subcellular localization in mammalian cells

1998 ◽  
Vol 95 (16) ◽  
pp. 9105-9110 ◽  
Author(s):  
Biserka Relić ◽  
Mirjana Andjelković ◽  
Luca Rossi ◽  
Yoshikuni Nagamine ◽  
Barbara Hohn
Keyword(s):  
Plant Cell ◽  
Nuclear Localization ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Bacterial Virulence ◽  
Plant Genome ◽  
Physical Interaction ◽  
Hek 293 ◽  
Human Embryo Kidney

Interaction betweenAgrobacterium tumefaciensand plants provides a unique example of interkingdom gene transfer.Agrobacterium, a plant pathogen, is capable to stably transform the plant cell with a segment of its own DNA called T-DNA (transferred DNA). This process depends, among others, on the specialized bacterial virulence proteins VirD1 and VirD2 that excise the T-DNA from its adjacent sequences. Subsequent to transfer to the plant cell, the virulence protein VirD2, through its nuclear localization signal (NLS), is believed to guide the T-DNA to the nucleus. The T-DNA then is integrated into the plant genome. Although both of these proteins are essential for bacterial virulence, physical interaction of them has not been analyzed so far. We studied associations between these proteins by expressing them in mammalian cells and by testing for intracellular localization and colocalization. When expressed in human cells [HeLa, human embryo kidney (HEK) 293], the VirD2 protein homogeneously distributed over the nucleoplasm. The presence of any of two NLSs, on the N and C termini of VirD2, was sufficient for its efficient nuclear localization whereas deletion of both NLSs rendered the protein cytoplasmic. However, this double NLS mutant was translocated to the nucleus in the presence of wild-type VirD2 protein, implying VirD2–VirD2 interaction. The VirD1 protein, by itself localized in the cytoplasm, moved to the nucleus when coexpressed with the VirD2 protein, suggesting VirD1–VirD2 interaction. This interaction was confirmed by coimmunoprecipitation tests. Of interest, both proteins coimported to the nucleus showed a similar, peculiar sublocalization. The data are discussed in terms of functions of the VirD proteins. In addition, coimport of proteins into nuclei is suggested as a useful system in studying individual protein–protein interactions.

scholarly journals Selective loss of substrate recognition induced by the tumour-associated D294G point mutation in protein kinase Cα

1998 ◽  
Vol 334 (2) ◽  
pp. 393-397 ◽  
Author(s):  
Corinne PRÉVOSTEL ◽  
Véronique ALVARO ◽  
Alice VALLENTIN ◽  
Annick MARTIN ◽  
Susan JAKEN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Protein Interactions ◽  
Kinase Activity ◽  
Substrate Recognition ◽  
Mutant Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Selective Loss ◽  
Protein Kinase Cα

The tumour-associated D294G mutant of protein kinase Cα (PKCα) was recently shown not to be translocated to the plasma membrane on stimulation with PMA, in contrast with the wild-type enzyme. Using recombinant wild-type and mutant PKCα, we establish here that, although the PKCα intrinsic lipid-dependent catalytic activity remains unaltered by the D294G mutation, the mutant enzyme exhibits a selective loss of substrate recognition. Indeed, whereas the mutant enzyme is still able to phosphorylate histone IIIS with comparable efficiency to that of the wild-type enzyme, it exhibits a lack of kinase activity towards the previously cloned 35F and 35H substrates for PKC. Overlay experiments demonstrate that this selective loss of kinase activity is correlated with a decrease in binding of D294G PKCα to the 35F and 35H proteins compared with that of the wild-type enzyme. Because the 35H and 35F proteins are predicted to be PKCα-anchoring proteins, these findings suggest a selective loss of PKCα–protein interactions that might fail to stabilize the location of the PKCα mutant at the plasma membrane.

scholarly journals Cell polarization is required for ricin sensitivity in a Caco-2 cell line selected for ricin resistance

1999 ◽  
Vol 341 (2) ◽  
pp. 323-327 ◽  
Author(s):  
Mark R. JACKMAN ◽  
Juliet A. ELLIS ◽  
Sally R. GRAY ◽  
Wenda SHURETY ◽  
J. Paul LUZIO
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Mammalian Cells ◽  
Cell Clone ◽  
Membrane Traffic ◽  
Retrograde Transport ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Cell Polarization ◽  
Apical Plasma Membrane

It has been proposed that killing of mammalian cells by ricin requires efficient endocytic delivery to the trans-Golgi network (TGN) prior to retrograde transport to the endoplasmic reticulum and entry to the cytosol. In polarized epithelial cells, an efficient membrane-traffic pathway to the TGN is present from the basolateral but not the apical plasma-membrane domain. Thus one can hypothesize that a ricin-resistant phenotype might be demonstrated by polarized cells that fail to differentiate and thus fail to develop an efficient membrane-traffic pathway from the basolateral plasma membrane to the TGN. We have isolated and studied a ricin-resistant Caco-2 cell clone (Caco-2-RCAr clone 2) which, when grown on plastic, was deficient in differentiation, measured by the development of polarized-cell-surface marker enzymes. The deficiency in differentiation was partially reversed, and ricin sensitivity was restored, when the cells were grown on filter supports. Our data provide the first evidence of a ricin-resistant cell line where resistance is due to the lack of development of polarized cell surfaces. The observed ricin resistance is consistent with the requirement that ricin is delivered to the TGN before its A chain enters the cytosol to mediate cell killing.

scholarly journals Plasma membrane organization and function: moving past lipid rafts

2013 ◽  
Vol 24 (18) ◽  
pp. 2765-2768 ◽  
Author(s):  
Mary L. Kraft
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Protein Interactions ◽  
Lipid Raft ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Imaging Techniques ◽  
Cellular Function ◽  
Membrane Organization ◽  
And Function

“Lipid raft” is the name given to the tiny, dynamic, and ordered domains of cholesterol and sphingolipids that are hypothesized to exist in the plasma membranes of eukaryotic cells. According to the lipid raft hypothesis, these cholesterol- and sphingolipid-enriched domains modulate the protein–protein interactions that are essential for cellular function. Indeed, many studies have shown that cellular levels of cholesterol and sphingolipids influence plasma membrane organization, cell signaling, and other important biological processes. Despite 15 years of research and the application of highly advanced imaging techniques, data that unambiguously demonstrate the existence of lipid rafts in mammalian cells are still lacking. This Perspective summarizes the results that challenge the lipid raft hypothesis and discusses alternative hypothetical models of plasma membrane organization and lipid-mediated cellular function.

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