Protein Kinase C Signaling in Adenoviral Infection - Biochemistry

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Protein kinase C Signaling in Adenoviral Infection Mohammad Abu Yousuf, Ji Sun Lee, Xiaohong Zhou, Mirja Ramke, Jeong Yoon Lee, James Chodosh, and Jaya Rajaiya Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00858 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 10, 2016

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Biochemistry PKCα in Viral Infection bi-2016-00858e Yousuf, et al., page # 1

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Protein kinase C Signaling in Adenoviral Infection

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Funding: This work was supported by NIH grants EY013124, EY021558, and EY014104, a

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Senior Scientific Investigator Award grant (to JC) from Research to Prevent Blindness, Inc.,

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New York, NY, The Falk Foundation, and the Massachusetts Lions Eye Research Fund.

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Mohammad A. Yousuf, Ji Sun Lee, Xiaohong Zhou, Mirja Ramke, Jeong Yoon Lee, James

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Chodosh*, Jaya Rajaiya*

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Howe Laboratory, Mass Eye and Ear Infirmary, Department of Ophthalmology, Harvard

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Medical School, Boston, Massachusetts 02114

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Running title: PKCα in Viral Infection

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*Corresponding Authors: The Massachusetts Eye and Ear Infirmary, 243 Charles Street,

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Boston, MA 02114. Telephone: 617-573-6398; Fax: 617-573-4324; Emails:

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[email protected]; [email protected]

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ACS Paragon Plus Environment

Biochemistry

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Page 2 of 36 PKCα in Viral Infection bi-2016-00858e Yousuf, et al., page # 2

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ABBREVIATIONS

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CAR, coxsackie adenovirus receptor; DAG, diacylglycerol; IP3, 1,4,5-triphosphate; MOI,

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multiplicity of infection; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; RGD,

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arginine-glycine-aspartate.


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ABSTRACT

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Activation of protein kinase C (PKC), a serine/threonine protein kinase, ubiquitously influences

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cellular signal transduction, and has been shown to play a role in viral entry. In this study, we

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explored a role for PKC in human adenovirus type 37 infection of primary human corneal

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fibroblasts, a major target cell for infection. We sought evidence for an interaction between PKC

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activation and two potential downstream targets: cSrc kinase, shown previously to play a critical

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role in adenovirus signaling in these cells, and caveolin-1, reported earlier to be important to

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entry of adenovirus type 37. Infection of fibroblasts increased PKCα phosphorylation and

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translocation of PKCα from the cytosol to caveolin-1 containing vesicles. Virus-induced

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phosphorylation of both cSrc and AKT was abolished in cell lysates pretreated with calphostin

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C, a chemical inhibitor of PKC. Inhibition of PKC also reduced virus associated phosphorylation

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of caveolin-1, while inhibition of cSrc by the chemical inhibitor PP2 reduced only caveolin-1

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phosphorylation, but not PKCα phosphorylation, in lipid rafts. These results suggest a role for

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PKCα upstream to both cSrc and caveolin-1. Phosphorylated PKCα was found in the same

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endosomal fractions as phosphorylated cSrc, and PKCα was present to a greater degree in

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caveolin-1 pull downs from virus infected than mock infected cell lysates. Calphostin C also

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reduced early viral gene expression, indicating that PKCα activity may be required for viral

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entry. PKCα plays a central role in adenovirus infection of corneal fibroblasts and regulation of

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downstream molecules, including the important lipid raft component caveolin-1.

44 45 46 47


Biochemistry

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Protein kinase C (PKC) is a family of ~80 kDa phospholipid-dependent serine/threonine kinases

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with 10 isoforms.1 All share a common structure with an N-terminal regulatory domain, C-

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terminal catalytic domain, and a hinge region joining the two. Conventional PKC isoforms,

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including α, βI, βII and γ, are activated by diacylglycerol (DAG) requiring calcium, while the

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novel isoforms δ, ε, η, and θ are activated by DAG, and do not require calcium. Atypical

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isoforms include λ and ζ that require neither DAG nor calcium for activation. Interaction with

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endomembranes is a key aspect of conventional PKC function. Activation of phospholipase Cβ

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induces generation of DAG and inositol 1,4,5-triphosphate (IP3), with subsequent release of

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calcium from the endoplasmic reticulum. Together, DAG and calcium induce the movement of

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PKC from the cytosol to endomembranes where its kinase function is released. As a major

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upstream signaling molecule, PKC has been shown to play a critical role in the cellular entry of

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viruses, including rhabdovirus, alphavirus, poxvirus, herpesvirus,2 influenza virus,3 and

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respiratory syncytial virus.4 PKC isoforms play a central role in signaling events leading to

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alterations in the cell membrane and cytoskeleton, including the formation of caveolae,5 which

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have been implicated in the entry of filovirus,6 human enterovirus,7 echovirus,8 and human

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immunodeficiency virus.9,

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recently shown to infect corneal fibroblasts by use of caveolae.11

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Human adenovirus type 37, an important cause of keratitis, was

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Human adenoviruses utilize at least two distinct sets of cell surface receptors for

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attachment and internalization into target host cells. An initial interaction between the adenovirus

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capsid fiber knob with a cellular receptor including the coxsackie adenovirus receptor (CAR),12

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GD1a glycan,13 major histocompatibility class I,14 desmoglein,15 and CD46

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secondary interaction between arginine-glycine-aspartate (RGD) amino acid sequences in the

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adenoviral penton capsomer and target cell integrins αvβ3 and αvβ5.17,


18

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enables a

The secondary

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interaction induces activation of focal adhesion kinase, cSrc, and phosphoinositide 3-kinase

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(PI3K) that mediate endocytosis of the virus.19, 20 The same kinases act as key signaling nodes to

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prolong cell survival, thus enabling viral replication,21 and induce expression of cytokines by

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infected cells.22-27 In particular, cSrc is the kinase identified most upstream in the pathway

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initiated by adenovirus infection of corneal cells.

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Multiple lines of evidence point to functional interactions between PKC and cSrc,28-35

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and we sought to explore the possibility of upstream control of cSrc activation by PKC in the

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earliest stages of infection by adenovirus. We demonstrate herein that adenovirus infection

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results in activation of PKCα, which then translocates to caveolin-1 containing vesicles, also

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containing cSrc. Furthermore, inhibition of PKCα activity results in reduced cSrc

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phosphorylation and decreases adenovirus early gene expression. Our data are consistent with an

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endosomal signalosome induced by adenovirus infection, in which PKCα and cSrc act as master

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kinases for the control of a myriad of cellular responses to viral infection.

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EXPERIMENTAL PROCEDURES

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Antibodies and Reagents. Antibodies to total PKCα, phospho-cSrc, total AKT,

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phospho-AKT, phospho-PKCα, total caveolin-1, and isotype controls (anti-rabbit or anti-mouse)

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were obtained from Cell Signaling Technology (Beverly, MA) and Santa Cruz Biotechnology

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(Delaware, CA). Antibodies to total cSrc and phospho-caveolin-1 were purchased from EMD

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Millipore (Billerica, MA) and BD Bioscience (San Jose, CA). Antibody to actin was obtained

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from Thermo Scientific (Cambridge, MA). Inhibitors to PKC (calphostin C) and cSrc (PP2) were

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purchased from Calbiochem (La Jolla, CA). Horseradish peroxidase-conjugated goat anti-rabbit

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and goat anti-mouse IgG were obtained from Santa Cruz Biotechnology. Lipofectamine


Biochemistry

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RNAiMAX and OptiMEM cell culture medium were obtained from Invitrogen Life

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Technologies (Carlsbad, CA).

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Cell Culture and Viruses. Primary corneal fibroblasts were grown from human donor

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corneas as previously described.36 Cells from multiple donors were pooled and the cell

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monolayers used at passage two. The protocol for use of corneas from deceased human donors

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was approved by the Massachusetts Eye and Ear Infirmary Human Studies Committee, and is

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consistent with the principles expressed in the Declaration of Helsinki. In some experiments, cell

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cultures were pretreated with the PKC inhibitor calphostin C (1 µM), the cSrc inhibitor PP2 (10

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µM), or DMSO (control), for 3 hr prior to infection. Human adenovirus type 37 strain GW was

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obtained from American Type Culture Collection (ATCC, Manassas, VA), was grown in A549

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cells (CCL-185, ATCC), a human alveolar epithelial cell line, in Minimum Essential Media

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(MEM) with 2% FBS, penicillin G sodium, streptomycin sulfate, and amphotericin B. Virus was

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purified by cesium chloride gradient prior to use. In some instances, virus was conjugated to Cy3

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dye (GE Healthcare, Piscataway, NJ),37 to enable visualization of viral capsid for confocal

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microscopy, as previously described.38 One mg of Cy3 dye was reconstituted in 1 mL of 0.1 M

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sodium bicarbonate (pH 9.3), and conjugated to virus at a concentration approximately equal to

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1012 Ad particles/mL; reconstituted Cy3 dye was 20% of the final solution. The mixture was

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incubated for 30 min in the dark with gentle mixing, followed by dialysis overnight to remove

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the excess dye. Virus labeled with Cy3 grew to equal titer as unlabeled virus (data not shown).

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Immunoblot. Adenovirus type 37 and mock infected corneal cells were lysed with cell

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lysis buffer (Cell Signaling, Beverly, MA) containing protease inhibitor cocktail (Sigma, St.

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Louis, MO) and incubated at 4o C for 5 min. Cell lysates were centrifuged at 13,000 × g for 10

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min at 4o C. The protein concentration of each supernatant was measured by BCA analysis


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(Pierce, Rockford, IL) and equalized. Lysates were boiled in 1X sodium dodecyl sulfate (SDS)

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sample buffer and separated by 4-20% Tris-glycine gel (Invitrogen) and transferred onto

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nitrocellulose membranes (BioRad, Hercules, CA) and immunoblotted. Antibody reactivity was

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determined with enhanced chemiluminescent reagents (Amersham Bioscience, Piscataway, NJ)

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using horseradish peroxidase-coupled secondary antibodies. Densitometry was performed on

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images generated on a Kodak Image Station 4000R (Rochester, NY).

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Immunoprecipitation. Lysates obtained from adenovirus type 37 infected or mock

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infected primary keratocytes (200 µg) were precleared with protein A-agarose beads (Thermo-

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Fisher) for 60 min. Cleared supernatants were mixed with 3 µg of anti-caveolin-1 or isotype

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control (anti-rabbit) and 3 µg anti-PKCα or isotype control (anti-mouse) antibodies in buffer

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containing 50 mM Tris HCl (pH 8.5), 50 mM NaCl, 1% NP40, protease inhibitor cocktail, and

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rocked at 4o C overnight. Then 30 µl protein A agarose was added for an additional 2 hr at 4oC.

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Immunoprecipitates were washed three times with buffer containing 50 mM Tris HCl, 50 mM

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NaCl, 1% NP40, protease inhibitor cocktail, and proteins were eluted by the addition of sodium

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dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiled for 5

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min. Sample were run on 4-20% Tris-glycine gel and immunoblotted.

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Confocal Microscopy. Corneal fibroblasts were grown on slide chambers (Nunc,

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Rochester, NY) and infected with Cy3 labeled adenovirus type 37 for the indicated times, and

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then partially fixed in 0.05% paraformaldehyde for 10 min, washed in PBS containing 2% FBS,

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and permeabilized in 0.1% Triton X-100 for 5 min. After 30 min blocking in 3% FBS-PBS, the

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slides were incubated in 5 µg/ml of anti-PKCα, anti-caveolin-1, or isotype control antibodies for

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1 hr at RT, washed three times in 1X PBS containing 2% FBS. Slides were then incubated with

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Alexa Fluor 488 or 568 conjugated secondary antibody (Molecular Probes, Eugene, OR), washed


Biochemistry

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three times in 1X PBS containing 2% FBS, fixed in 2% paraformaldehyde, and mounted using

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vectashield (Vector labs, Burlingame, CA) mounting medium containing DAPI. Images were

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taken in a Leica SP5 confocal microscope using a 63x glycerol immersion objective. For

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inhibition experiments, cells were pretreated with calphostin C (1 µM) for 3 hr and then infected

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with Cy3 labeled adenovirus type 37 for 30 min, processed as above, and incubated in phalloidin

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conjugated with Alexa Fluor 488 for 30 min at RT, washed and mounted. Images were taken in a

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Leica SP5 confocal microscope using a 60x glycerol immersion objective. For quantitative

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analysis, ImageJ was used. Three frames from 3 separate experiments were chosen in a masked

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fashion to quantitate Cy3 labeled virus within cells.

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Non-detergent Isolation of Lipid Rafts. Cornea fibroblasts were treated with calphostin

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C or PP2, washed thoroughly, and infected with purified adenovirus type 37 at a multiplicity of

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infection (MOI) of 10 (for synchronous infection) or mock infected with virus-free dialysis

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buffer. After infection, lipid raft fractions were isolated using detergent free methods. Briefly,

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cells were washed once in ice cold 1X PBS, suspended in 500 mM Na2CO3 and lysed by 20

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strokes in a prechilled dounce homogenizer. Further disruption of cell membrane were achieved

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by passing the lysates through a 23 gauge needle five times, followed by sonication for 15 sec

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three times. The subsequent lysates were mixed with an equal volume of 90% sucrose-MBS (25

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mM MES (2-N-morpholinoethanesulfonic acid) plus 0.15 M NaCl (pH 6.5)), overlaid on

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equivalent volumes of 35% sucrose-MBS-Na2CO3 and 5% sucrose-MBS- Na2CO3, and

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centrifuged for 118 hr at 4o C. 500 µl fractions were collected and the protein concentrations

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measured and equalized. Lysates were subjected to SDS-PAGE and immunoblotted as above.

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Endosome Isolation. Flotation gradient fractionation of post nuclear supernatants were

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performed with 62%, (2.351 M) 35%, (1.177 M) and 25%, (0.806 M) sucrose gradients.39 One hr


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post viral infection or mock infection with dialysis buffer, cells were collected and post nuclear

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supernatants prepared with the NucBuster Protein Extraction Kit (Novagen, San Diego, CA).

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Post nuclear supernatants were then mixed 1:1 with 62% sucrose, layered over with 1.5 volume

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of 35% sucrose and 1 volume of 25% sucrose and centrifuged for 1 hr at 149,000 × g at 4oC.

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Fifty µl fractions were collected from the top of each gradient and subjected to Western blot

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analysis and used to determine cSrc kinase activity (as below).

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siRNA Transfection. The siRNA for PKCα was purchased from Ambion (Foster City,

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CA). The control scRNA (On Target PlusTM control pool) was purchased from Thermo

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Scientific. 100 picomole RNA was transfected into cells using Lipofectamine RNAiMAX

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(Invitrogen) following the manufacturer’s instructions. At 48 hr post transfection, cells were

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infected with adenovirus type 37 for 4 hr, and then analyzed for mRNA expression for PKCα and

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adenovirus type 37 E1A using real-time RT-PCR. For Western blot analysis, equal amounts of

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protein were separated in a 4-20% gradient gel (Invitrogen) and immunoblotted with antibodies

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against PKCα and actin.

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Polymerase Chain Reaction. For reverse transcriptase PCR, cells were infected with

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virus at an MOI of 10. Following 1 hr incubation, cells were washed twice with PBS and

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incubated an additional 4 hr with fresh media. Total RNA was isolated using TRIzol (Invitrogen)

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according to the manufacturer’s protocol. RNA concentrations and quality were analyzed on a

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Bio-Rad Smart-Spec Plus (Bio-Rad, Hercules, CA). To remove any genomic DNA

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contamination, RNA was treated with Turbo DNase (Ambion). For synthesis of cDNA, 2 µg of

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total RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase

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(Promega, Madison, WI) using an oligo(dT) 15 primer (Promega). A reaction without reverse

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transcription was run with each experiment to rule out the possibility of amplification of


Biochemistry

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contaminating genomic DNA in the PCR step. The cDNA product was amplified by PCR in a

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total volume of 25 µl of PCR mix, including 12.5 µl of 2 × PCR master mix (Promega), 8.5 µl

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ddH2O, and 1 µl of each primer (10 pmol). The PCR reaction was performed using the following

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cycling parameters: 95oC for 5 min for the initial denaturation step, 30 cycles of 95oC for 30 sec,

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54oC for 30 sec, 72oC for 30 sec, followed by the final extension at 72oC for 10 min and then

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kept at 4oC until analysis. The amplification products were analyzed by gel electrophoresis on

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1% agarose gel. PCR products were visualized after ethidium bromide staining using a Kodak

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Image Station 4000R.The primers used for PCR amplification in reverse transcriptase PCR were

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as

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5'ATCATCCCTGCCTCTACTGG3';

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adenovirus

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5'GCTGGGACCTACACTTCAGG3'; reverse: 5'TGTAACACAGAGCGCAGGAG3'.

follows:

for

type

GAPDH

37

E1A

(GenBank

13S

reverse:

Acc.

no.

NM

002046.3)

forward:

5'GTCAGGTCCACCACTGACAC3';

(GenBank

Acc.

no.

DQ900900)

for

forward:

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For real-time quantitative PCR, total RNA was extracted from treated cells using TRIzol

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(Invitrogen) according to the manufacturer’s instructions. Total RNA was then treated with

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DNase I (1 unit) (New England BioLabs, Ipswich, MA) at 37° C for 1 hr. Two µg of the DNase

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treated RNA was subjected to reverse transcription with M-MLV reverse transcriptase

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(Promega) and oligo dT15 primers (IDT). Primers for PKCα, caveolin-1 and E1A were designed

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using Primer3 plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi),

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and synthesized by IDT (Table 1). cDNAs were diluted 1:10 and 1 µl used for qRT-PCR with

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Fast SYBR Green master mix (Thermo Fisher) under the conditions: 40 cycles at 95° C (30 sec),

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60° C (1 min), 72° C (30 sec), and a final extension at 72° C (10 min). A non-template control

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and endogenous control (human GAPDH) were measured for relative quantification. The relative


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expression levels were calculated by the 2-∆∆CT method against untransfected/uninfected

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controls.40

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Src Kinase Assay. Src activity in endosomal fractions was assessed by the ProFluor®

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Src-Family Kinase Assay (Promega). Two µg of pooled endosomal fractions # 7-10 (peptide

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substrate) and 25 µl of 100 µM ATP was added to the reaction buffer and incubated for 60 min

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at room temperature, followed by the addition of protease reagent for another 60 min at RT. The

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reaction was then stopped by addition of stabilizer reagent. In the presence of active kinase, the

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peptide substrate is phosphorylated, effectively blocking the protease activity and resulting in

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low fluorescence of the substrate. When there is no kinase activity, the peptide substrate remains

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unphosphorylated, and the protease will remove all amino acids from the peptide substrate and

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liberate the highly fluorescent rhodamine 110. Therefore, kinase activity is inversely

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proportional to fluorescence. The resulting fluorescence was read with at excitation wavelength

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485 and emission wavelength of 530 nm.

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Statistical analysis. The results of at least 3 independent experiments were first checked

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for normality, and then subjected to analysis by ANOVA with α = .05. All analysis was

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performed in SAS (Cary, NC).

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RESULTS

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PKCα activation and translocation in adenovirus infection. As a first step toward the

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study of PKC in adenovirus infection of corneal cells, we subjected serum starved, primary,

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human corneal fibroblasts in culture to Western blot analysis for one PKC isoform of each

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activation group (conventional, novel, and atypical) (Figure 1A). PKCα was identified in

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fibroblasts at the expected 80 kDa molecular weight by use of an isoform specific antibody,


Biochemistry

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while bands for PKCθ and PKCζ were not seen. To determine whether PKCα was activated by

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infection with adenovirus, we infected the cells with 10 viral infectious units per cell

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(multiplicity of infection, MOI = 10) for one hr, or mock infected with virus-free dialysis buffer,

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and immunoblotted with antibody against phosphorylated PKCα. Other cells were pretreated

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with the PKC inhibitor, calphostin C

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increased phosphorylation of PKCα relative to total PKCα, and this effect was diminished by

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calphostin C pretreatment (Figure 1B). Densitometry analysis performed on three consecutive

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Western blots showed a significant increase in PKCα phosphorylation, and confirmed the effect

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of calphostin C on PKCα phosphorylation in adenovirus infection (Figure 1C; p

Protein Kinase C Signaling in Adenoviral Infection - Biochemistry

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