scholarly journals The intracellular fate of zonula occludens 2 is regulated by the phosphorylation of SR repeats and the phosphorylation/O-GlcNAcylation of S257

2013 ◽  
Vol 24 (16) ◽  
pp. 2528-2543 ◽  
Author(s):  
Miguel Quiros ◽  
Lourdes Alarcón ◽  
Arturo Ponce ◽  
Thomas Giannakouros ◽  
Lorenza González-Mariscal
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Posttranslational Modifications ◽  
Proliferating Cells ◽  
Akt Activation ◽  
Dual Localization ◽  
Epidermal Growth ◽  
Proteosomal Degradation ◽  
Zona Occludens ◽  
Intracellular Fate

Zona occludens 2 (ZO-2) has a dual localization. In confluent epithelia, ZO-2 is present at tight junctions (TJs), whereas in sparse proliferating cells it is also found at the nucleus. Previously we demonstrated that in sparse cultures, newly synthesized ZO-2 travels to the nucleus before reaching the plasma membrane. Now we find that in confluent cultures newly synthesized ZO-2 goes directly to the plasma membrane. Epidermal growth factor induces through AKT activation the phosphorylation of the kinase for SR repeats, serine arginine protein kinase 1, which in turn phosphorylates ZO-2, which contains 16 SR repeats. This phosphorylation induces ZO-2 entry into the nucleus and accumulation in speckles. ZO-2 departure from the nucleus requires intact S257, and stabilizing the β-O-linked N-acetylglucosylation (O-GlcNAc) of S257 with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate, an inhibitor of O-GlcNAcase, triggers nuclear exportation and proteosomal degradation of ZO-2. At the plasma membrane ZO-2 is not O-GlcNAc, and instead, as TJs mature, it becomes phosphorylated at S257 by protein kinase Cζ. This late phosphorylation of S257 is required for the correct cytoarchitecture to develop, as cells transfected with ZO-2 mutant S257A or S257E form aberrant cysts with multiple lumens. These results reveal novel posttranslational modifications of ZO-2 that regulate the intracellular fate of this protein.

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 Phosphoinositide-Dependent Phosphorylation of PDK1 Regulates Nuclear Translocation

2005 ◽  
Vol 25 (6) ◽  
pp. 2347-2363 ◽  
Author(s):  
Michael P. Scheid ◽  
Michael Parsons ◽  
James R. Woodgett
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Posttranslational Modifications ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Pleckstrin Homology Domain ◽  
Subcellular Trafficking ◽  
Kinase C ◽  
Ribosomal S6 Kinase ◽  
Nuclear Shuttling

ABSTRACT 3-Phosphoinositide-dependent kinase 1 (PDK1) phosphorylates the activation loop of a number of protein serine/threonine kinases of the AGC kinase superfamily, including protein kinase B (PKB; also called Akt), serum and glucocorticoid-induced kinase, protein kinase C isoforms, and the p70 ribosomal S6 kinase. PDK1 contains a carboxyl-terminal pleckstrin homology domain, which targets phosphoinositide lipids at the plasma membrane and is central to the activation of PKB. However, PDK1 subcellular trafficking to other compartments is not well understood. We monitored the posttranslational modifications of PDK1 following insulin-like growth factor 1 stimulation. PDK1 underwent rapid and transient phosphorylation on S396, which was dependent upon plasma membrane localization. Phosphorylation of S396 was necessary for nuclear shuttling of PDK1, possibly through its influence on an adjacent nuclear export sequence. Thus, mitogen-stimulated phosphorylation of PDK1 provides a means for directed PDK1 subcellular trafficking, with potential implications for PDK1 signaling.

scholarly journals Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

2007 ◽  
Vol 18 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Hisayoshi Yoshizaki ◽  
Naoki Mochizuki ◽  
Yukiko Gotoh ◽  
Michiyuki Matsuda
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Time Course ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Akt Inhibitor ◽  
Epidermal Growth ◽  
Ral Gtpase

We studied the spatiotemporal regulation of Akt (also called protein kinase B), phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] by using probes based on the principle of fluorescence resonance energy transfer. On epidermal growth factor (EGF) stimulation, the amount of PtdIns(3,4,5)P3 was increased diffusely in the plasma membrane, whereas that of PtdIns(3,4)P2 was increased more in the nascent lamellipodia than in the plasma membrane of the central region. The distribution and time course of Akt activation were similar to that of increased PtdIns(3,4)P2 levels, which were most prominent in the nascent lamellipodia. Moreover, we found that upon EGF stimulation 3-phosphoinositide–dependent protein kinase-1 (PDK1) was also recruited to nascent lamellipodia in an Akt-dependent manner. Because PDK1 is known to activate Ral GTPase and because Ral is required for EGF-induced lamellipodial protrusion, we speculated that the PDK1–Akt complex may be indispensable for the induction of lamellipodia. In agreement with this idea, EGF-induced lamellipodia formation was promoted by the overexpression of Akt and inhibited by an Akt inhibitor or a Ral-binding domain of Sec5. These results identified the Akt–PDK1 complex as an upstream positive regulator of Ral GTPase in the induction of lamellipodial protrusion.

The presence of an unusual PKC isozyme profile in rat liver cells

1998 ◽  
Vol 76 (1) ◽  
pp. 73-82 ◽  
Author(s):  
Hayfa A Al-Mazidi ◽  
Leonard P Kleine ◽  
Douglas J Franks
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Mitogenic Response ◽  
Kinase C ◽  
Epidermal Growth ◽  
Pkc Β ◽  
Pkc Ζ

We have previously shown that protein kinase C (PKC) is involved in the mitogenic response of T51B cells to epidermal growth factor. In fact, epidermal growth factor was an excellent mitogen, even after prolonged pretreatment of cells with TPA, suggesting that the PKC isoform implicated in proliferation is not down-regulated by 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have now determined that the PKC isozymes -α, -βI, -δ, -ε, and -ζ are present in T51B cells. All five isoforms are associated with the plasma membrane and the cytoplasm and are either in or around the nucleus. PKC-βI has a slightly different subcellular profile from that of the other isoforms in that it is clearly and strongly associated with the nuclear membrane. Also, a unique and novel pattern is obtained from immunoblots with anti-PKC-βI. PKC-βI is detected as a single band of 70 kDa in the cytosolic fraction and as a doublet of 65 and 77 kDa in the membrane fraction. PKC-α, -δ, and -ε were down-regulated by pretreatment of cells with TPA, while PKC-ζ was unaffected. Of particular interest was the fact that TPA did not down-regulate PKC-βI. In fact, the amount of this isoform associated with the plasma membrane increased. These findings indicate that it is probably PKC-βI that is involved in the mitogenic response of T51B cells to epidermal growth factor. Since PKC-ζ is also not down-regulated by TPA, the possible involvement of this isoform needs to be resolved.Key words: protein kinase C, intracellular localization, cell proliferation, liver.

scholarly journals Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase

1984 ◽  
Vol 220 (3) ◽  
pp. 677-683 ◽  
Author(s):  
J E Kudlow ◽  
Y Leung
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Egf Receptor ◽  
Light Exposure ◽  
Atp Binding ◽  
Receptor Kinase ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Dependent Protein ◽  
Epidermal Growth

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3′-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5′-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5′-[beta gamma-imido]triphosphate or 20 mM-guanosine 5′-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.

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