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BBA - Molecular Cell Research (v.1823, #6)

Editorial Board (pp. i).

Tyrosine kinase signaling and the emergence of multicellularity by W. Todd Miller (pp. 1053-1057).

Tyrosine phosphorylation is an essential element of signal transduction in multicellular animals. Although tyrosine kinases were originally regarded as specific to the metazoan lineage, it is now clear that they evolved prior to the split between unicellular and multicellular eukaryotes (≈600million years ago). Genome analyses of choanoflagellates and other protists show an abundance of tyrosine kinases that rivals the most complex animals. Some of these kinases are orthologs of metazoan enzymes (e.g., Src), but others display unique domain compositions not seen in any metazoan. Biochemical experiments have highlighted similarities and differences between the unicellular and multicellular tyrosine kinases. In particular, it appears that the complex systems of kinase autoregulation may have evolved later in the metazoan lineage.► Tyrosine kinases (TKs) play critical signaling roles in multicellular organisms. ► Surprisingly, TKs have recently been discovered in single-celled eukaryotes. ► Primitive TKs display unique domain combinations. ► Biochemical experiments show lack of regulation in primitive Src kinases.

Keywords: Tyrosine kinase; Phosphorylation; Evolution; Choanoflagellate; SH2 domain; SH3 domain

The PDZ-binding motif of MCC is phosphorylated at position −1 and controls lamellipodia formation in colon epithelial cells by Laurent Pangon; Christa Van Kralingen; Melissa Abas; Roger J. Daly; Elizabeth A. Musgrove; Maija R.J. Kohonen-Corish (pp. 1058-1067).

In this study, we describe a new post-translational modification at position −1 of the PDZ-binding motif in the mutated in colorectal cancer (MCC) protein and its role in lamellipodia formation. Serine 828 at position −1 of this motif is phosphorylated, which is predicted to increase MCC binding affinity with the polarity protein Scrib. We show that endogenous MCC localizes at the active migratory edge of cells, where it interacts with Scrib and the non-muscle motor protein Myosin-IIB. Expression of MCC harboring a phosphomimetic mutation MCC-S828D strongly impaired lamellipodia formation and resulted in accumulation of Myosin-IIB in the membrane cortex fraction. We propose that MCC regulates lamellipodia formation by binding to Scrib and its downstream partner Myosin-IIB in a multiprotein complex. Importantly, we propose that the function of this complex is under the regulation of a newly described phosphorylation of the PDZ-binding motif at position −1.► First functional characterization of −1 phosphorylation of the PDZ binding motif. ► Mutation of the S828 phosphosite of MCC disrupts lamellipodia formation. ► Phosphorylation of MCC-S828 is predicted to increase MCC interaction with Scrib. ► MCC also interacts with the non-muscle motor protein Myosin-IIB. ► Mutation of MCC-S828 traps Myosin-IIB in the membrane cortex.

Keywords: Abbreviations; MCC; mutated in colorectal cancer; PDZ; post synaptic density protein (PSD95),; Drosophila; disc large tumor suppressor (DlgA), or Zonula occludens-1 protein (zo-1); PAK; p21 activated kinaseMCC; Lamellipodia; Scrib; PDZ; PDZ-binding motif; Myosin-IIB

Fluorescence correlation spectroscopy, combined with bimolecular fluorescence complementation, reveals the effects of β-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors by Laura E. Kilpatrick; Stephen J. Briddon; Nicholas D. Holliday (pp. 1068-1081).

Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis are powerful ways to study mobility and stoichiometry of G protein coupled receptor complexes, within microdomains of single living cells. However, relating these properties to molecular mechanisms can be challenging. We investigated the influence of β-arrestin adaptors and endocytosis mechanisms on plasma membrane diffusion and particle brightness of GFP-tagged neuropeptide Y (NPY) receptors. A novel GFP-based bimolecular fluorescence complementation (BiFC) system also identified Y1 receptor-β-arrestin complexes. Diffusion co-efficients ( D) for Y1 and Y2-GFP receptors in HEK293 cell plasma membranes were 2.22 and 2.15×10−⁹cm²s−¹ respectively. At a concentration which promoted only Y1 receptor endocytosis, NPY treatment reduced Y1-GFP motility ( D 1.48×10−⁹cm²s−¹), but did not alter diffusion characteristics of the Y2-GFP receptor. Agonist induced changes in Y1 receptor motility were inhibited by mutations (6A) which prevented β-arrestin recruitment and internalisation; conversely they became apparent in a Y2 receptor mutant with increased β-arrestin affinity. NPY treatment also increased Y1 receptor-GFP particle brightness, changes which indicated receptor clustering, and which were abolished by the 6A mutation. The importance of β-arrestin recruitment for these effects was illustrated by reduced lateral mobility ( D 1.20–1.33×10−⁹cm²s−¹) of Y1 receptor-β-arrestin BiFC complexes. Thus NPY-induced changes in Y receptor motility and brightness reflect early events surrounding arrestin dependent endocytosis at the plasma membrane, results supported by a novel combined BiFC/FCS approach to detect the underlying receptor-β-arrestin signalling complex.Display Omitted► First FCS / PCH analysis of the plasma membrane diffusion characteristics of NPY receptors. ► Demonstration that NPY changes in receptor diffusion and oligomeric state depend on early events in endocytosis. ► Design of a new GFP BiFC system for use in combination with FCS techniques. ► Isolation of the mobility and molecular brightness of defined receptor-arrestin complexes undergoing endocytosis.

Keywords: Abbreviations; BiFC; bimolecular fluorescence complementation; BIBO3304; (; R; )-; N; ₂; -(diphenylacetyl)-; N; -[(4-(aminocarbonylaminomethyl-)phenyl)methyl]-argininamide; BSA; bovine serum albumin; DMEM; Dulbecco's modified Eagle's medium; FCS; fluorescence correlation spectroscopy; FRAP; fluorescence recovery after photobleaching; Gc; sfGFP fragment 155–238; Gn; sfGFP fragment 2–173; GPCR; G protein coupled receptor; HBSS; HEPES buffered saline solution; NPY; neuropeptide Y; PCH; photon counting histogram; (sf)GFP; (superfolder) green fluorescent protein; YFP; yellow fluorescent proteinG protein coupled receptor; Neuropeptide Y; Arrestin; Fluorescence correlation spectroscopy; Bimolecular fluorescence complementation; Endocytosis

Overexpression of phospholipase D enhances Bcl-2 expression by activating STAT3 through independent activation of ERK and p38MAPK in HeLa cells by Hye-Jin Choi; Joong-Soo Han (pp. 1082-1091).

The purpose of this study was to identify the role of phospholipase D (PLD) isozymes in Bcl-2 expression. Overexpression of PLD1 or PLD2 increased Bcl-2 expression and phosphatidic acid (PA), the product of PLDs, also upregulated Bcl-2 expression. Treatment with PA activated the phospholipase A₂ (PLA₂)/G_i/ERK1/2, RhoA/Rho-associated kinase (ROCK)/p38 MAPK, and Rac1/p38 MAPK pathways. PA-induced phosphorylation of ERK1/2 was attenuated by a PLA₂ inhibitor (mepacrine) and, a G_i protein inhibitor (pertussis toxin, PTX). On the other hand, p38 MAPK phosphorylation was attenuated by a dominant negative Rac1 and a specific Rho-kinase inhibitor (Y-27632). These results suggest that PLA₂/G_i acts at the upstream of ERK1/2, while Rac1 and RhoA/ROCK act upstream of p38 MAPK. We next, tried to determine which transcription factor is involved in PLD-related Bcl-2 expression. When signal transducer and activator of transcription 3 (STAT3) activity was blocked by a STAT3 specific siRNA, PA-induced Bcl-2 expression was remarkably decreased, suggesting that STAT3 is an essential transcription factor linking PLD to Bcl-2 upregulation. Taken together, these findings indicate that PLD acts as an important regulator in Bcl-2 expression by activating STAT3 involving the phosphorylation of Ser727 through the PLA₂/G_i/ERK1/2, RhoA/ROCK/p38 MAPK, and Rac1/p38 MAPK pathways.► PLD and its catabolic product, PA, are involved in Bcl-2 expression in HeLa cells. ► ERK1/2 and p38 MAPK are responsible for PLD-induced Bcl-2 expression. ► ERK1/2 and p38 MAPK are activated through three separate pathways: PLA2/Gi/ERK1/2, RhoA/ROCK/p38 MAPK, and Rac1/p38 MAPK. ► PLD is also involved in the activation of STAT3, which is important to Bcl-2 expression as transcription factor in HeLa cells.

Keywords: Abbreviations; PLD; phospholipase D; PA; phosphatidic acid; Bcl-2; B-cell leukemia/lymphoma 2; PLA; ₂; phospholipases A; LPA; lysophosphatidic acid; DPPA; 1,2-dipalmitoyl-; sn; -glycero-3-phosphate; MAPK; mitogen-activated protein kinase; ERK; extracellular signal-regulated kinases; ROCK; Rho-associated kinase; GPCR; G protein-coupled receptor; STAT3; signal transducer and activator of transcription 3; DN; dominant negative; siRNA; small interfering RNA; PTX; pertussis toxin; RBD; Rho-binding domainPhospholipase D (PLD); Phosphatidic acid (PA); Bcl-2; MAPK; RhoA; STAT3 (ser727)

Characterization of the interaction of Aha1 with components of the Hsp90 chaperone machine and client proteins by Liang Sun; Thomas Prince; Jacob R. Manjarrez; Bradley T. Scroggins; Robert L. Matts (pp. 1092-1101).

The activator of Hsp90 ATPase, Aha1, is an Hsp90 co-chaperone that has been suggested to act as a general stimulator of Hsp90 function. In this report, we have characterized the interaction of Aha1 with Hsp90 and its co-chaperones in rabbit reticulocyte lysate (RRL) and in HeLa cell extracts. Complexes formed by Aha1 with Hsp90 in RRL were stabilized by molybdate and contained the co-chaperones FKBP52 and p23/Sba1, but lacked HOP/Sti1 and Cdc37. Aha1 complexes isolated from HeLa cell extracts also contained Hsp70 and DNAJA1. Over-expression of Aha1 has been reported to stimulate the activity of v-Src and steroid hormone receptors ectopically expressed in yeast, however, no interaction between Aha1 and nascent v-Src or the progesterone receptor could be detected in RRL. Contrary to expectations, over-expression of Aha1 also inhibited the rate of Hsp90-dependent refolding of denatured luciferase. A number of potential client proteins that specifically associated with Aha1 were identified by liquid chromatography/ tandem mass spectrometry (LC–MS/MS) and verified by Western blotting. The proteins identified suggest that Aha1 may play roles in modulating RNA splicing and DNA repair, in addition to other cellular processes.► Aha1 interacts with Hsp90, Hsp70, DNAJA1, p23 and FKBP52, but not with Cdc37 or HOP. ► Aha1 does not appear to modulate Hsp90-dependent folding of nascent polypeptides. ► Over-expression of Aha1 does not stimulate Hsp90's chaperone activity. ► Aha1 may function in regulation of proteins involved in cellular stress responses.

Keywords: Heat shock protein 90; Activator of Hsp90 ATPase; Non-receptor tyrosine kinase v-Src; Progesterone receptor; Aha1-interacting protein; Hsp90 inhibitor

Cap-independent Nrf2 translation is part of a lipoic acid-stimulated detoxification stress response by Kate Petersen Shay; Alexander J. Michels; Wenge Li; Ah-Ng Tony Kong; Tory M. Hagen (pp. 1102-1109).

Little is known about either the basal or stimulated homeostatic mechanisms regulating nuclear tenure of Nf-e2-related factor 2 (Nrf2), a transcription factor that mediates expression of over 200 detoxification genes. Our data show that stress-induced nuclear Nrf2 accumulation is largely from de novo protein synthesis, rather than translocation from a pre-existing cytoplasmic pool. HepG2 cells were used to monitor nuclear Nrf2 24h following treatment with the dithiol micronutrient ( R)-α-lipoic acid (LA; 50μM), or vehicle. LA caused a ≥2.5-fold increase in nuclear Nrf2 within 1h. However, pretreating cells with cycloheximide (50μg/ml) inhibited LA-induced Nrf2 nuclear accumulation by 94%. Providing cells with the mTOR inhibitor, rapamycin, decreased basal Nrf2 levels by 84% after 4h, but LA overcame this inhibition. LA-mediated de novo protein translation was confirmed using HepG2 cells transfected with a bicistronic construct containing an internal ribosome entry sequence (IRES) for Nrf2, with significant (P<0.05) increase in IRES use under LA treatment. These results suggest that a dithiol stimulus mediates Nrf2 nuclear tenure via cap-independent protein translation. Thus, translational control of Nrf2 synthesis, rather than reliance solely on pre-existing protein, may mediate the rapid burst of Nrf2 nuclear accumulation following stress stimuli.► ( R)-α-lipoic acid acts as a mild stressor to induce the transcription factor Nrf2. ► Stress-induced increase in nuclear Nrf2 doesn't draw solely from pre-existing pool. ► De novo translation of Nrf2 rapidly ensues after a lipoic acid stimulus. ► Lipoic acid induces Nrf2 protein expression via an IRES, even under mTOR inhibition.

Keywords: Nrf2; Lipoic acid; Mammalian target of rapamycin (mTOR); Cap-independent translation; Protein homeostasis; Cellular stress

Increase in claudin-2 expression by an EGFR/MEK/ERK/c-Fos pathway in lung adenocarcinoma A549 cells by Akira Ikari; Tomonari Sato; Ryo Watanabe; Yasuhiro Yamazaki; Junko Sugatani (pp. 1110-1118).

In human adenocarcinoma, claudin-2 expression is higher than that in normal lung tissue, but the regulatory mechanism of its expression has not been clarified. In human adenocarcinoma A549 cells, claudin-2 level time-dependently increased under the control conditions. In contrast, claudin-1 expression remained constant for 24h. The concentration of epidermal growth factor (EGF) in medium time-dependently increased, which was inhibited by matrix metalloproteinase (MMP) inhibitor II, an inhibitor of MMP-1, 3, 7, and 9. MMP inhibitor II decreased claudin-2 and phosphorylated ERK1/2 (p-ERK1/2) levels, which were recovered by EGF. Both claudin-2 and p-ERK1/2 levels were decreased by EGF neutralizing antibody, EGF receptor (EGFR) siRNA, AG1478, an inhibitor of EGFR, U0126, an inhibitor of MEK, and the exogenous expression of dominant negative-MEK. These results suggest that EGF is secreted from A549 cells by MMP and increases claudin-2 expression mediated via the activation of an EGFR/MEK/ERK pathway. The inhibition of the signaling pathway decreased phosphorylated c-Fos and nuclear c-Fos levels. The introduction of c-Fos siRNA decreased claudin-2 level without affecting claudin-1. The promoter activity of human claudin-2 was decreased by AG1478 and U0126. Furthermore, the activity was decreased by the deletion or mutation of the AP-1 binding site of claudin-2 promoter. Chromatin immunoprecipitation and avidin–biotin conjugated DNA assays showed that c-Fos binds to the AP-1 binding site. We suggest that a secreted EGF up-regulates the transcriptional activity of claudin-2 mediated by the activation of an EGFR/MEK/ERK/c-Fos pathway in A549 cells.► Claudin-2 is expressed in human lung adenocarcinoma. ► Secreted epidermal growth factor increases claudin-2 expression. ► Claudin-2 expression is regulated by an EGFR/MEK/ERK/c-Fos pathway. ► Transcriptional activity of claudin-2 is inhibited by c-Fos siRNA. ► c-Fos binds to promoter region of claudin-2.

Keywords: Abbreviations; ABCD; avidin–biotin conjugated DNA; CA-MEK; constitutively active-EMK; ChIP; chromatin immunoprecipitation; DN-MEK; dominant negative-MEK; EGF; epidermal growth factor; MMP; matrix metalloproteinase; NSCLC; non-small cell lung cancer; TJs; tight junctions; TGF; transforming growth factorClaudin-2; Lung; Matrix metalloproteinase; Epidermal growth factor; c-Fos

Cyclin D1 is a NF-κB corepressor by María F. Rubio; Pablo N. Larrosa Fernandez; Cecilia V. Alvarado; L.C. Panelo; Marina Ruiz Grecco; Georgina P. Colo; Martinez-Noel Giselle A. Martínez-Noel; Sabrina M. Micenmacher; Mónica A. Costas (pp. 1119-1131).

NF-κB regulates the expression of Cyclin D1 (CD1), while RAC3 is an NF-κB coactivator that enhances its transcriptional activity. In this work, we investigated the regulatory role of CD1 on NF-κB activity. We found that CD1 inhibits NF-κB transcriptional activity through a corepressor function that can be reverted by over-expressing RAC3. In both, tumoral and non-tumoral cells, the expression pattern of RAC3 and CD1 is regulated by the cell cycle, showing a gap between the maximal expression levels of each protein. The individual increase, by transfection, of either CD1 or RAC3 enhances cell proliferation. However the simultaneous and constitutive over-expression of both proteins has an inhibitory effect. Our results suggest that the relative amounts of CD1 and RAC3, and the timing of expression of these oncogenes could tilt the balance of tumor cell proliferation in response to external signals.► Cyclin D1 plays an inhibitory role over NF-κB transcriptional activity. ► The repression of NF-κB transactivation by CD1 involves a deacetylase activity. ► Cyclin D1 and the transcription factor NF-κB could be part of the same protein complex.

Keywords: Abbreviations; CD1; Cyclin D1; Cdk; Cyclin-dependent kinase; ER; Estrogen receptor; HAT; Histone acetylase; HDAC; Histone deacetylase; PMA; 12-O-tetraecanoylphorbol-13-acetate; SRC; Steroid receptor coactivator; RAC3; Retinoic acid coactivatorCyclin D1; NF-κB; RAC3; Transcriptional activity; Tumor development; Nuclear receptor coactivator

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BBA - Molecular Cell Research (v.1823, #6)