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

2010 ◽  
Vol 190 (5) ◽  
pp. 751-760 ◽  
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.

Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization

2001 ◽  
Vol 114 (9) ◽  
pp. 1787-1794 ◽  
Author(s):  
G.Z. Zhu ◽  
D.G. Myles ◽  
P. Primakoff
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Acrosome Reaction ◽  
Phosphorylation Site ◽  
Equatorial Region ◽  
Proteolytic Processing ◽  
Mature Sperm ◽  
Kinase C

Plasma membrane-anchored proteases have key roles in cell signaling, migration and refashioning the cell surface and its surroundings. We report the first example of a plasma membrane-anchored protease on mature sperm, testase 1 (ADAM 24). Unlike other studied sperm ADAMs (fertilin (α) and (β), cyritestin) whose metalloprotease domains are removed during sperm development, we found testase 1 retains an active metalloprotease domain, suggesting it acts as a protease on mature sperm. Testase 1 is a glycoprotein (molecular mass 88 kDa), localized to the equatorial region of the plasma membrane of cauda epididymal sperm. Typically, proteolytic removal of the pro-domain is an initial activation step for ADAM proteases. The pro-domain of the testase 1 precursor (108 kDa) is proteolytically removed as sperm transit the caput epididymis to produce processed (mature) testase 1 (88 kDa). Testase 1 is unique among all studied ADAMs in that its proteolytic processing occurs on the sperm plasma membrane instead of at an intracellular site (the Golgi). Using GST-fusion proteins and a synthetic testase 1 C-terminal peptide, we found that the cytoplasmic tail of testase 1 could be phosphorylated in vitro by protein kinase C (PKC). Thus testase 1 apparently has a cytoplasmic PKC phosphorylation site(s). Protein kinase C is known to stimulate other ADAMs' protease activity. Because events of the acrosome reaction include PKC activation, we speculate that testase 1 protease function could be important in sperm penetration of the zona pellucida after sperm PKC is activated during the acrosome reaction.

scholarly journals Distinct activities of Scrib module proteins organize epithelial polarity

2020 ◽  
Vol 117 (21) ◽  
pp. 11531-11540 ◽  
Author(s):  
Mark J. Khoury ◽  
David Bilder
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Epithelial Polarity ◽  
Protein Protein Interactions ◽  
Kinase C ◽  
Mutual Antagonism ◽  
Discs Large ◽  
Lethal Giant Larvae ◽  
Epithelial Structure ◽  
And Function

A polarized architecture is central to both epithelial structure and function. In many cells, polarity involves mutual antagonism between the Par complex and the Scribble (Scrib) module. While molecular mechanisms underlying Par-mediated apical determination are well-understood, how Scrib module proteins specify the basolateral domain remains unknown. Here, we demonstrate dependent and independent activities of Scrib, Discs-large (Dlg), and Lethal giant larvae (Lgl) using theDrosophilafollicle epithelium. Our data support a linear hierarchy for localization, but rule out previously proposed protein–protein interactions as essential for polarization. Cortical recruitment of Scrib does not require palmitoylation or polar phospholipid binding but instead an independent cortically stabilizing activity of Dlg. Scrib and Dlg do not directly antagonize atypical protein kinase C (aPKC), but may instead restrict aPKC localization by enabling the aPKC-inhibiting activity of Lgl. Importantly, while Scrib, Dlg, and Lgl are each required, all three together are not sufficient to antagonize the Par complex. Our data demonstrate previously unappreciated diversity of function within the Scrib module and begin to define the elusive molecular functions of Scrib and Dlg.

scholarly journals Phosphorylation of Zona Occludens-2 by Protein Kinase Cε Regulates Its Nuclear Exportation

2009 ◽  
Vol 20 (18) ◽  
pp. 4120-4129 ◽  
Author(s):  
David Chamorro ◽  
Lourdes Alarcón ◽  
Arturo Ponce ◽  
Rocio Tapia ◽  
Héctor González-Aguilar ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Nuclear Export ◽  
Phosphorylation Site ◽  
Putative Protein ◽  
Kinase C ◽  
Junction Protein ◽  
Zona Occludens ◽  
Intracellular Fate ◽  
Putative Protein Kinase

Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCε. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.

scholarly journals Phosphatidylinositol 4,5-Bisphosphate Mediates the Targeting of the Exocyst to the Plasma Membrane for Exocytosis in Mammalian Cells

2007 ◽  
Vol 18 (11) ◽  
pp. 4483-4492 ◽  
Author(s):  
Jianglan Liu ◽  
Xiaofeng Zuo ◽  
Peng Yue ◽  
Wei Guo
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Secretory Vesicles ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Conserved Residues ◽  
Vesicle Tethering ◽  
C Terminus ◽  
Evolutionarily Conserved ◽  
Vesicular Stomatitis

The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles at the plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the exocyst physically associates with the plasma membrane (PM). In this study, we report that the mammalian exocyst subunit Exo70 associates with the PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Furthermore, we have identified key conserved residues at the C-terminus of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P2. Disrupting Exo70-PI(4,5)P2 interaction abolished the membrane association of Exo70. We have also found that wild-type Exo70 but not the PI(4,5)P2-binding–deficient Exo70 mutant is capable of recruiting other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, we demonstrate that Exo70-PI(4,5)P2 interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM.

scholarly journals Confinement of β1- and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

2011 ◽  
Vol 22 (16) ◽  
pp. 2970-2982 ◽  
Author(s):  
Cathleen D. Valentine ◽  
Peter M. Haggie
Keyword(s):  
Plasma Membrane ◽  
Adrenergic Receptors ◽  
Protein Complexes ◽  
Pdz Domain ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Membrane Domains ◽  
Anchoring Proteins ◽  
Differential Signaling ◽  
Receptor Dynamics

The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β1- and β2AR, are structurally similar but mediate distinct signaling responses. Scaffold protein–mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β1- and β2AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)–domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β2AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β2AR confinement. For both β1- and β2AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β1- or β2AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes.

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

2015 ◽  
Vol 211 (2) ◽  
pp. 273-286 ◽  
Author(s):  
Wei Dong ◽  
Xuejing Zhang ◽  
Weijie Liu ◽  
Yi-jiun Chen ◽  
Juan Huang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Aurora Kinase ◽  
Cell Cortex ◽  
Membrane Targeting ◽  
Kinase C ◽  
Lethal Giant Larvae ◽  
Evolutionarily Conserved ◽  
Electrostatic Binding ◽  
Phosphatidylinositol Phosphates

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.

Explosion in the complexity of membrane protein recycling

2020 ◽  
Author(s):  
Fiona J McDonald
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Epithelial Polarity ◽  
Lipid Interactions

For decades, recycling of membrane proteins has been represented in figures by arrows between the 'endosome' and the plasma membrane, but recently there has been an explosion in the understanding of the mechanisms and protein complexes required to facilitate protein recycling. Here, some key discoveries will be introduced, including assigning function to a number of recently recognized protein complexes, and linking their function to protein recycling. Further, the importance of lipid interactions, and links to diseases and epithelial polarity will be summarized.

scholarly journals Autoregulation of the Raf-1 serine/threonine kinase

1998 ◽  
Vol 95 (16) ◽  
pp. 9214-9219 ◽  
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.

scholarly journals Cohesin: a critical chromatin organizer in mammalian gene regulation

2011 ◽  
Vol 89 (5) ◽  
pp. 445-458 ◽  
Author(s):  
Richard Chien ◽  
Weihua Zeng ◽  
Alexander R. Ball ◽  
Kyoko Yokomori
Keyword(s):  
Dna Repair ◽  
Gene Regulation ◽  
Cellular Differentiation ◽  
Developmental Disorders ◽  
Mitotic Chromosome ◽  
Protein Complexes ◽  
Chromatin Organization ◽  
Genome Integrity ◽  
Evolutionarily Conserved ◽  
Functional Hierarchy

Cohesins are evolutionarily conserved essential multi-protein complexes that are important for higher-order chromatin organization. They play pivotal roles in the maintenance of genome integrity through mitotic chromosome regulation, DNA repair and replication, as well as gene regulation critical for proper development and cellular differentiation. In this review, we will discuss the multifaceted functions of mammalian cohesins and their apparent functional hierarchy in the cell, with particular focus on their actions in gene regulation and their relevance to human developmental disorders.

scholarly journals Renal defects associated with improper polarization of the CRB and DLG polarity complexes in MALS-3 knockout mice

2007 ◽  
Vol 179 (1) ◽  
pp. 151-164 ◽  
Author(s):  
Olav Olsen ◽  
Lars Funke ◽  
Jia-fu Long ◽  
Masaki Fukata ◽  
Toshinari Kazuta ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Kidney Development ◽  
Protein Complexes ◽  
Large Cell ◽  
Cell Polarization ◽  
Renal Tubules ◽  
Metanephric Mesenchyme ◽  
Discs Large ◽  
Evolutionarily Conserved ◽  
Basolateral Complex

Kidney development and physiology require polarization of epithelia that line renal tubules. Genetic studies show that polarization of invertebrate epithelia requires the crumbs, partition-defective-3, and discs large complexes. These evolutionarily conserved protein complexes occur in mammalian kidney; however, their role in renal development remains poorly defined. Here, we find that mice lacking the small PDZ protein mammalian LIN-7c (MALS-3) have hypomorphic, cystic, and fibrotic kidneys. Proteomic analysis defines MALS-3 as the only known core component of both the crumbs and discs large cell polarity complexes. MALS-3 mediates stable assembly of the crumbs tight junction complex and the discs large basolateral complex, and these complexes are disrupted in renal epithelia from MALS-3 knockout mice. Interestingly, MALS-3 controls apico-basal polarity preferentially in epithelia derived from metanephric mesenchyme, and defects in kidney architecture owe solely to MALS expression in these epithelia. These studies demonstrate that defects in epithelial cell polarization can cause cystic and fibrotic renal disease.

Sign in / Sign up

Export Citation Format

Share Document