1 The Rho target PRK2 regulates apical junction formation in 1 human bronchial epithelial cells 2 3

40 Pages·2010·3.41 MB·English

MCB Accepts, published online ahead of print on 25 October 2010
Mol. Cell. Biol. doi:10.1128/MCB.01001-10
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 The Rho target PRK2 regulates apical junction formation in
2 human bronchial epithelial cells
3
4 Sean W. Wallace, Ana Magalhaes1 & Alan Hall*
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6 Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, D
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7 New York, NY 10065 n
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15 1 Present address: Angiogenesis Lab, CIPM, Portuguese Institute of Oncology, Lisbon, n

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16 Portugal. r
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17 * Corresponding author: [email protected]; Tel: [phone]; Fax: [phone] ,
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18 9

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19 Word count for Materials and Methods: 919
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20 Combined word count for Introduction, Results and Discussion: 3203 s
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23 Running Title: PRK2 and epithelial apical junctions
24 Keywords: Rho, PRK2, epithelial morphogenesis, apical junction
1
1 ABSTRACT
2
3 Rho GTPases regulate multiple signaling pathways to control a number of cellular
4 processes during epithelial morphogenesis. To investigate the downstream pathways
5 through which Rho regulates epithelial apical junction formation, we screened an siRNA
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6 library targeting 28 known Rho target proteins in 16HBE human bronchial epithelial w
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7 cells. This led to the identification of the serine-threonine kinase PRK2 (protein kinase C
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8 related kinase 2, also called PKN2). Depletion of PRK2 does not block the initial d

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9 formation of primordial junctions at nascent cell-cell contacts, but prevents their m

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10 maturation into apical junctions. PRK2 is recruited to primordial junctions, and this tt
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11 localization depends on its C2-like domain. Rho binding is essential for PRK2 function m
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12 and also facilitates PRK2 recruitment to junctions. Kinase-dead PRK2 acts as a .a
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13 dominant-negative mutant and prevents apical junction formation. We conclude that .
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14 PRK2 is recruited to nascent cell-cell contacts through its C2-like domain and Rho- /
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15 binding domains, and promotes junctional maturation through a kinase-dependent A
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16 pathway. il 3
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1 INTRODUCTION
2
3 Apical junctions, including tight and adherens junctions, are important for epithelial cell-
4 cell adhesion, selective permeability and apical-basal polarity. The formation of apical
5 junctions is therefore essential for epithelia to regulate tissue integrity and homeostasis.
D
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6 Tight junctions and adherens junctions form at the apical margin of the lateral membrane w
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7 in vertebrate epithelial cells through the interactions of transmembrane junctional
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8 proteins. Tight junctions principally consist of the transmembrane proteins occludin and d

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9 the claudin family, while adherens junctions are principally composed of E-cadherin (2, m

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10 29). Additional transmembrane proteins including nectins, JAM (junctional adhesion tt
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11 molecule) and tricellulin also contribute to apical junctions. Junctional transmembrane m
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12 proteins associate via their cytoplasmic domains with a large number of adaptor and .a
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13 signaling proteins and with the actin cytoskeleton (21, 23). .
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14 /
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15 Epithelial apical junction formation is initiated by the trans-interaction of E-cadherin A
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16 molecules, which results in the stabilization of E-cadherin puncta at nascent cell-cell il 3
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17 contacts, referred to as spot-like or primordial junctions (1). Primordial junctions contain 0
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18 many of the proteins found in mature adherens junctions, including the catenins, as well b
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19 as the tight junction protein ZO-1 (4, 39). The formation of primordial junctions depends u
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20 on actin polymerization, and E-cadherin puncta are stabilized at cell-cell contacts by
21 interacting with actin filaments (10, 17). The formation of mature apical junctions,
22 consisting of distinct tight and adherens junctions, requires the recruitment of additional
23 tight junction proteins and the reorganization of the actin cytoskeleton to form the
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1 characteristic perijunctional actin belt, a process that requires actomyosin contractility
2 (17, 39, 47). Epithelial apical junctions can thus be regulated by a number of cellular
3 processes, including expression and trafficking of junctional proteins and the
4 organization of the actin cytoskeleton (13, 44). Many signaling pathways have been
5 implicated in the regulation of epithelial apical junctions, including those controlled by
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6 the Rho GTPase family members Rho, Rac and Cdc42 (15, 34). w
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7
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8 Rho plays a particularly important role in epithelial morphogenesis as one of its target d

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9 proteins, Rho kinase (ROCK), is a key regulator of myosin II-dependent actomyosin m

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10 contractility (32). ROCK activates myosin II, by inhibiting MLC (myosin light chain) tt
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11 phosphatase leading to increased MLC phosphorylation. During embryogenesis, apical m
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12 constriction of epithelial cells, as a result of apically localized myosin II activity, .a
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13 contributes to cell invagination events. In the Drosophila embryo, for example, localized .
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14 activation of Rho has been shown to control apical constriction during gastrulation and /
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15 spiracle cell invagination (19, 37). Another key morphogenetic event during A
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16 embryogenesis is the sealing of epithelial sheets and Rho, acting through myosin II, is il 3
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17 required for elongation of leading edge cells during Drosophila dorsal closure (12, 16). 0
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19 Evidence that Rho regulates apical junction formation in mammalian epithelial cells has u
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20 come from experimental manipulation of Rho activity, using bacterial C3 transferase, or
21 expression of mutant Rho proteins, in numerous cell types, including MDCK kidney
22 epithelial cells, keratinocytes, Eph4 mammary epithelial cells, T84 intestinal cells, MCF7
23 breast carcinoma cells and HCT116 colon carcinoma cells (6, 26, 33, 38, 40, 46).
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1 Investigation of the downstream signaling pathways through which Rho regulates apical
2 junctions has principally focused on ROCK. Inhibition of ROCK in T84 cells prevents
3 apical junction formation, and in MCF7 breast carcinoma cells results in reduced E-
4 cadherin accumulation at cell-cell contacts (36, 43). ROCK is believed to promote
5 reorganization of the characteristic perijunctional apical actin belt, which supports apical
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6 junction formation/stabilization in polarized epithelial cells, through actomyosin w
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7 contractility (17, 38, 47). However, ROCK inhibition has no effect on adherens junction
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8 formation in MDCK or HCT116 cells, suggesting alternative and/or redundant pathways d

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9 downstream of Rho are active in different cell types (33). m

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10 tt
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11 In addition to ROCK, more than 20 other Rho target proteins have been described. In the m
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12 present study we report a systematic analysis of Rho signaling pathways regulating apical .a
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13 junction formation in 16HBE human bronchial epithelial cells, an immortalized but non- .
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14 transformed cell-line derived from the epithelium of the lung airway (9). Understanding /
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15 the pathways that regulate the integrity of the lung epithelium is of great importance as A
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16 loss of epithelial integrity is a characteristic feature of lung diseases, including cancer and il 3
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17 chronic obstructive pulmonary disease (COPD) (45). In this study we identify the Rho 0
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18 target PRK2 (protein kinase C related kinase 2) as a regulator of apical junction b
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19 formation in human bronchial epithelial cells. u
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1 Materials and Methods
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3 Reagents and antibodies
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5 Unless stated otherwise all chemicals were from Sigma-Aldrich (St. Louis, MO). Primary
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6 antibodies: RhoA (clone 26C4), RhoA/C (rabbit polyclonal, sc-179) from Santa Cruz w
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7 Biotechnology (Santa Cruz, CA); Occludin (rabbit polyclonal), ZO-1 (clone 1A12), ZO-1
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8 (rabbit polyclonal), E-cadherin (clone ECCD-2) from Invitrogen (Carlsbad, CA); E- d

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9 cadherin (clone 34), PRK2 (clone 22) from BD Transduction (Lexington, KY); phospho- m

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10 PRK1 (Thr774)/PRK2 (Thr816) (rabbit polyclonal) from Cell Signaling (Beverly, MA); tt
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11 α-tubulin (clone YL1/2) from AbD Setotec (Raleigh, NC); β-actin (clone AC-74), FLAG m
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12 (clone M2) from Sigma-Aldrich; HA (clone 3F10) from Roche; myc (clone 9E10) from a
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13 Cancer Research UK (London, UK). Alexa488- and 568- conjugated secondary .o
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14 antibodies and Alexa488-conjugated phalloidin were from Invitrogen. AMCA-, FITC,
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15 and Cy3-conjugated secondary antibodies were from Jackson Immunoresearch (West A
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16 Grove, PA). il 3
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18 Cell culture and transfection b
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20 16HBE14o- cells were provided by Dr. Dieter Gruenert (California Pacific Medical
21 Center, San Francisco, CA) and were cultured in MEM + GlutaMAX (Invitrogen)
22 supplemented with 10% BenchMark FBS (Gemini Bio-Products, West Sacramento, CA)
23 and penicillin (100 U/ml)-streptomycin (100 µg/ml) (Invitrogen) at 37°C in 5% CO .
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1 Transfections were carried out by seeding cells at low density (1.5x104 cells/cm2, 10-20%
2 confluency) and allowing to adhere overnight. siRNA (50nM) was transfected in medium
3 without antibiotics, using 100pmol siRNA and 5µl lipofectamine LTX (Invitrogen) per
4 1.2x105 cells. For DNA transfection, 5µl lipofectamine LTX and 200ng plasmid DNA
5 were used per 1.2x105 cells. For retroviral infection, 16HBE cells were seeded as above,
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6 then incubated overnight in growth medium containing retroviral particles, produced in w
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7 HEK293T cells, supplemented with 8µg/ml polybrene (hexadimethrine bromide). 2 days a
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8 after infection, stable pools were selected using 1.5µg/ml puromycin (Invitrogen). For
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9 calcium-switch experiments, cells were washed extensively in PBS without calcium,

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10 incubated in low calcium medium for 4 hours, and then switched to normal growth p
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11 medium containing calcium. Low calcium medium was prepared using DMEM without c
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12 calcium chloride (Invitrogen) supplemented with 10% FBS pre-treated with Chelex 100 s
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13 resin (Bio-Rad, Hercules, CA). o
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15 HEK293T cells (ATCC, Manassas, VA) were cultured in DME HG + sodium pyruvate p
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16 supplemented with 10% FBS and penicillin (100U/ml)-streptomycin (100µg/ml) ,

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17 (Invitrogen) at 37 °C in 5% CO . For transfection, cells were seeded at 3x104 cells/cm2 1
2 9

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18 and allowed to adhere overnight. 1µg plasmid DNA per 3x105 cells was transfected using y
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19 5µl lipofectamine2000 (Invitrogen). For retroviral particle production, cells were triply- s
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20 transfected with VSV-G, GagPol and pBABE vector of interest, and 6 hours post-
21 transfection the medium was changed to 16HBE growth medium for 24 hours to collect
22 viral particles.
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1 siRNA reagents
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3 siRNAs were from Thermo Fisher Scientific (Lafayette, CO). RhoA SMARTpool M-
4 003860-03; RhoA duplex1 D-003860-01; RhoA duplex2 D-003860-02, RhoA duplex3
5 D-003860-03; RhoA duplex4 D-003860-04; RhoC SMARTpool M-008555-01; PRK2
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6 duplex1 D-004612-03; PRK2 duplex2 D-004612-10; siControl, custom sequence w
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7 GGAAAUUAUACAAGACCAA. Additional SMARTpool reagents used for screening
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8 are listed in Table 1. d

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9 m

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10 DNA constructs tt
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11 m
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12 Mouse RhoA and RhoC cDNAs were obtained from ATCC, and subcloned in to the .a
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13 pRK5myc expression vector. Mouse PRK2 (mPRK2) was obtained from RZPD .
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14 (Deutsches Resourcenzentrum für Genomforschung, Germany), and subloned in to /
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15 pBABE-HA and pRK5myc expression vectors. Note that the clone used (clone IRAV A
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16 p968C10112D6) contains a deletion of 11 amino acids (Gln 32 to Gln 42) compared to il 3
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17 NCBI reference sequence NM_178654.4. mPRK2(K685M) and mPRK2(D781A) were 0
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18 made by PCR amplification using primers containing the appropriate point mutations. b
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19 mPRK2(A66K,A155K) was made by carrying out 2 rounds of PCR amplification with u
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20 primers containing the appropriate point mutations. mPRK2∆C2 contains a deletion of
21 amino acids 381-462, and was made by overlap extension PCR using appropriate primers
22 to amplify residues 1-381 and 463-983. All primers were purchased from Sigma-
23 Genosys. All constructs were sequence verified.
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2 Immunoprecipitation and western blotting
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4 16HBE cell lysates were prepared by scraping cells in protein sample buffer (2% SDS,
5 100mM DTT, 50mM Tris-HCl pH6.8, 10% glycerol, 0.1% bromophenol blue) and
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6 boiling for 5 min at 100 °C. For immunoprecipitation, transfected HEK293T cells were w
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7 lysed in immunoprecipitation buffer (1% NP-40, 50mM Tris-HCl pH8.0, 150mM NaCl) a
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8 with 2mM PMSF and Complete protease inhibitor tablet (Roche) and cell debris was d

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9 pelleted by centrifugation at 13000 rpm 4°C for 10 min. The soluble fraction was m

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10 incubated at 4°C with primary antibody for 1 hour, followed by protein G sepharose tp
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11 beads (Sigma-Aldrich) for 1 hour. Beads were washed extensively with
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12 immunoprecipitation buffer and boiled in sample buffer. Proteins were resolved by SDS-
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13 PAGE, transferred to PVDF membrane (Millipore, Bedford, MA), and incubated with the o
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14 appropriate primary antibodies. Proteins were visualized using HRP-conjugated o
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15 secondary antibodies (Dako, Carpinteria, CA) and ECL detection reagents (GE p
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16 Healthcare, Waukesha, WI). 3
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18 Microscopy y

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20 16HBE cells grown on glass coverslips were fixed in 3.7% (v/v) formaldehyde for 15
21 min and permeabilized in 0.5% (v/v) Triton X-100 for 5 min. Primary and secondary
22 antibody incubations were carried out for 1 hour at RT. Coverslips were mounted with
23 fluorescent mounting medium (Dako) and visualized using a Zeiss AxioImager.A1
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1 fluorescence microscope with 40X NA0.75 and 63X NA1.4 objectives (Zeiss,
2 Thornwood, NY), using a Hammamatsu ORCA-ER 1394 C4742-80 digital camera
3 (Bridgewater, NJ) and AxioVision software (Zeiss).
4
5 Apical junction quantification
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6 w
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7 For each sample 12 random non-overlapping images were taken at 40X magnification (~
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8 400 cells) and apical junction formation quantified using the manual count function of d

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9 Metamorph image analysis software (Universal Imaging, West Chester, PA). Cells with a m

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10 continuous ring of occludin or ZO-1 at cell-cell contacts were scored as having intact tt
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11 apical junctions. Cells with punctate or discontinuous occludin or ZO-1 at cell-cell m
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12 contacts were scored as not having apical junctions. Results were analyzed using Prism .a
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13 (GraphPad Software, San Diego, CA). Error bars are SEM, and significance values have .
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14 been calculated using a two-tailed unpaired t-test at 95% confidence interval. /
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