Kaposi's sarcoma-associated herpesvirus LANA protein downregulates nuclear glycogen synthase kinase 3 activity and consequently blocks differentiation

Abstract

The Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) protein interacts with glycogen synthase kinase 3 (GSK-3) and relocalizes GSK-3 in a manner that leads to stabilization of beta-catenin and upregulation of beta-catenin-responsive cell genes. The LANA-GSK-3 interaction was further examined to determine whether there were additional downstream consequences. In the present study, the nuclear GSK-3 bound to LANA in transfected cells and in BCBL1 primary effusion lymphoma cells was found to be enriched for the inactive serine 9-phosphorylated form of GSK-3. The mechanism of inactivation of nuclear GSK-3 involved LANA recruitment of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the ribosomal S6 kinase 1 (RSK1). ERK1/2 and RSK1 coprecipitated with LANA, and LANA was a substrate for ERK1 in vitro. A model is proposed for the overall inactivation of nuclear GSK-3 that incorporates the previously described GSK-3 phosphorylation of LANA itself. Functional inactivation of nuclear GSK-3 was demonstrated by the ability of LANA to limit phosphorylation of the known GSK-3 substrates C/EBPbeta and C/EBPalpha. The effect of LANA-mediated ablation of C/EBP phosphorylation on differentiation was modeled in the well-characterized 3T3L1 adipogenesis system. LANA-expressing 3T3L1 cells were impaired in their ability to undergo differentiation and adipogenesis. C/EBPbeta induction followed the same time course as that seen in vector-transduced cells, but there was delayed and reduced induction of C/EBPbeta transcriptional targets in LANA-expressing cells. We conclude that LANA inactivates nuclear GSK-3 and modifies the function of proteins that are GSK-3 substrates. In the case of C/EBPs, this translates into LANA-mediated inhibition of differentiation.

FIG. 1.

LANA bound GSK-3β is phosphorylated at Ser 9 and is inactive in an in vitro kinase assay. (A) Western blots probed with anti-Ser 9-GSK-3β (upper) or anti-GSK-3β (lower) antibodies to determine the phosphorylation status of HA-GSK-3β in Flag-LANA-cotransfected HeLa cells. Lane 1, transfected cell extract (Ext; 5% of that used for immunoprecipitation [IP]); lane 2, anti-HA precipitate; lane 3, anti-Flag precipitate; lane 4, control IgG precipitate. (B) Western blots probed with anti-Ser 9-GSK-3β (upper) or anti-GSK-3β (lower) antibodies to determine the phosphorylation status of GSK-3β in BCBL1 PEL cells. Lane 1, BCBL1 cell extract (5% of that used for immunoprecipitation); lane 2, anti-GSK-3β precipitate; lane 3, anti-LANA precipitate; lane 4, control IgG precipitate. (C) In vitro kinase assay using the BCBL1 samples from (B) and a primed GSK-3 peptide substrate. The peptide was subjected to gel electrophoresis and autoradiography to detect incorporation of [γ-³²P]ATP. Lane 1, BCBL1 cell extract; lane 2, anti-GSK-3β precipitate; lane 3, anti-LANA precipitate; lane 4, control IgG precipitate. Circled p, phospho.

FIG. 2.

LANA coprecipitates with phospho-ERK1/2. (A) Western blot probed with anti-Myc antibody showing coprecipitation of Myc-ERK2-MEK1 with Flag-LANA in cotransfected HeLa cells. Lane 1, anti-Flag coprecipitate; lane 2, anti-Myc direct precipitate; lane 3, control (ctrl) IgG precipitate. (B) Western blots probed with anti-ERK1/2 antibody (upper) and anti-phospho-ERK1/2 antibody (lower) to determine the phosphorylation status of endogenous LANA bound ERK1/2 in BCBL1 PEL cells. Lane 1, BCBL1 cell extract (Ext; 5% of that used for immunoprecipitation [IP]); lane 2, control IgG precipitate; lane 3, anti-LANA precipitate; lane 4, anti-ERK1/2 precipitate. (C) In vitro ERK1/2 kinase assay using the BCBL1 samples shown in panel B and a MAPK peptide substrate. The autoradiograph examines the incorporation of [γ-³²P]ATP in the presence of BCBL1 extract (lane 1), control IgG precipitate (lane 2), anti-LANA precipitate (lane 3), anti-ERK1/2 precipitate (lane 4), no added kinase (lane 5), purified ERK1 (lane 6), control peptide plus ERK1 kinase (lane 7), and substrate peptide plus ERK1 kinase (lane 8). Circled p, phospho. Lanes 7 and 8 were processed separately.

FIG. 3.

ERK1 phosphorylates LANA and can prime for GSK-3 phosphorylation in vitro. (A) In vitro ERK1 kinase assay. The autoradiograph examines the incorporation of [γ-³²P]ATP into control GST (lane 1), GST-LANA(C+N) (lane 2), GST-LANA-C (lane 3), GST-LANA-N (lane 4), and synthetic peptide LANA(246-258) (lane 5). (B) In vitro ERK1 priming and GSK-3 kinase assays. The autoradiograph examines the incorporation of [γ-³²P]ATP in a GSK-3 kinase assay performed without priming (top), a control ERK1 priming kinase assay to detect ERK background activity (middle), and an assay in which ERK1 primes for GSK-3 phosphorylation (bottom). Lane 1, control GST; lane 2, GST-LANA-N.

FIG. 4.

LANA-associated RSK1 phosphorylates GSK-3β. (A) Western blots probed with anti-LANA (upper) or anti-RSK1 (lower) antibodies showing coprecipitation of RSK1 and LANA from BCBL1 and BC3 PEL cell extracts. Lane 1, cell extract; lane 2, anti-LANA immunoprecipitate; lane 3, control IgG immunoprecipitate. The extract lane contained 5% of the protein used in immunoprecipitation. (B) In vitro phosphorylation assay showing Ser 9 inactivation of GSK-3β by ERK1 plus RSK1. GST-GSK-3β was incubated with kinase buffer containing [γ-³²P]ATP and control IgG immunoprecipitate from BCBL1 cells (lane 1), anti-LANA immunoprecipitate from BCBL1 cells (lane 2), or purified RSK1 and ERK1 (lane 3). Samples were separated by SDS-PAGE. A Western blot was probed with anti-Ser 9-GSK-3β antibody (upper). Samples were also subjected to autoradiography (middle) and Coomassie brilliant blue staining (lower). (C) Summary of LANA-associated ERK1/RSK-mediated serine 9 phosphorylation of nuclear GSK-3. Circled p, phospho.

FIG. 5.

GSK-3 phosphorylation of C/EBPα and C/EBPβ is inhibited by LANA. (A) HeLa cells transfected with Flag-C/EBPα, HA-GSK-3β, and GFP-LANA, as indicated, were metabolically labeled for 4 h with [³²P]orthophosphate 2 days posttransfection. Flag-C/EBPα was immunoprecipitated with anti-Flag M2 affinity beads and subjected to Western blotting to detect Flag-C/EBPα (lower) and to autoradiography to detect ³²P-labeled C/EBPα (upper). Detection of phosphorylated Flag-C/EBPα required cotransfection with HA-GSK-3β (lane 3), and this phosphorylation was blocked by cotransfected LANA (lane 5). (B) HeLa cells transfected with Flag-C/EBPβ, HA-GSK-3β, and GFP-LANA, as indicated, were metabolically labeled with [³²P]orthophosphate as described above. Flag-C/EBPβ was immunoprecipitated with anti-Flag M2 affinity beads and subjected to Western blotting to detect Flag-C/EBPβ (lower) and to autoradiography to detect ³²P-labeled C/EBPβ (upper). ³²P labeling of Flag-C/EBPβ increased in the presence of HA-GSK-3β (lane 3), and this increase was abolished by cotransfection of LANA (lane 7). LiCl is a GSK-3 inhibitor. Circled p, phospho.

FIG. 6.

Expression of LANA in transduced 3T3L1 preadipocytes. (A) Verification of LANA expression in retrovirus-transduced, puromycin-selected 3T3L1 cultures. Reverse transcriptase PCR detected LANA expression in 3T3L1-LANA but not in 3T3L1-vector transduced cultures. The LANA plasmid formed a positive PCR control. Ctrl, control. (B) Western blot probed with anti-LANA antibody comparing LANA expression with that in BCBL1 PEL cells. (C) Comparison of the growth rates of the parental 3T3L1 and transduced 3T3L1-vector and 3T3L1-LANA cultures. Cell growth was measured using CellTiter-Glo.

FIG. 7.

LANA inhibits differentiation of 3T3L1 preadipocytes. 3T3L1-vector and 3T3L1-LANA cultures were subjected to a standard 3T3L1 differentiation protocol. Cells were fixed and incubated with oil red O to stain accumulated fat droplets on days 0, 6, and 8 postinduction. Right panel, high-magnification photomicrograph of day 8 cultures (×200).

FIG. 8.

Expression of adipogenic mediators and markers in 3T3L1-vector and 3T3L1-LANA cultures. (A) Western blots examining expression of the differentiation regulators C/EBPβ, C/EBPα, PPARγ, β-catenin, and GSK-3β and the differentiation marker adiponectin in 3T3L1-vector and 3T3L1-LANA cultures subjected to a standard differentiation protocol. (B) Summary of the differentiation process showing the regulatory events blocked by LANA. Circled p, phospho.

FIG. 9.

Proposed mechanisms of LANA-mediated inhibition of nuclear GSK-3 activity and downstream modification of cell gene expression. LANA binds to GSK-3 entering the nucleus in S phase and prevents nuclear export, resulting in nuclear accumulation of GSK-3. GSK-3 phosphorylation of LANA is required for interaction between the two proteins. LANA also binds ERK1/2 and RSK1, which participate in Ser 9 inactivation of GSK-3. Thus, active nuclear GSK-3 is LANA bound, whereas inactivated GSK-3 is released from LANA, with the paradoxical outcome that LANA-mediated nuclear accumulation of GSK-3 is associated with a reduction in nuclear GSK-3 enzymatic activity and phosphorylation of downstream targets. Changes in transcription factor phosphorylation are likely to be reflected in a reprogramming of cell gene expression. Circled p, phospho.