Abstract
The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) protein is functionally pleiotropic. LANA contributes to KSHV-associated pathogenesis, in part, by increasing entry of cells into S phase through a process that is driven by LANA interaction with the serine-threonine kinase glycogen synthase kinase 3 (GSK-3) and stabilization of beta-catenin. We now show that LANA affects the activity of another protein involved in cell cycle regulation, c-Myc. Sequencing of c-Myc coding sequences revealed that c-Myc in KSHV-positive primary effusion lymphoma (PEL) cell lines is wild type in the N-terminal region that regulates c-Myc protein stability. Despite this, c-Myc in PEL cells is stabilized. In LANA-expressing cells, inactivation of nuclear GSK-3 reduced phosphorylation of c-Myc at Thr58 and contributed to c-Myc stabilization by decreasing c-Myc ubiquitination. Phosphorylation of c-Myc on Ser62 also affects c-Myc stability and function. We now show that LANA increases the level of phosphorylated extracellular signal-regulated kinase 1 (ERK1) and increases ERK phosphorylation of c-Myc on Ser62. LANA also interacted with c-Myc, and c-Myc amino acids 147 to 220 were required for this interaction. LANA (L1006P) retained the ability to bind to c-Myc and activate ERK1, indicating that these events did not require LANA interaction with GSK-3. Thus, LANA stabilizes c-Myc; prevents the phosphorylation of c-Myc at Thr58, an event that promotes Myc-induced apoptosis; and independently stimulates phosphorylation of c-Myc at Ser62, an event that transcriptionally activates c-Myc. LANA-mediated manipulation of c-Myc function is likely to contribute to KSHV-associated tumorigenesis through the induction of c-Myc regulated cellular genes, as well as by the stimulation of cell cycle progression.
Highlights
Kaposi’s sarcoma-associated herpesvirus (KSHV) was discovered in lesions of Kaposi’s sarcoma using differential display [12] and was subsequently recognized to be associated with primary effusion lymphoma and multicentric Castleman’s disease [10, 18, 52, 59]
Sequencing of c-Myc coding sequences revealed that c-Myc in KSHV-positive primary effusion lymphoma (PEL) cell lines is wild type in the N-terminal region that regulates c-Myc protein stability
We have recently shown that latency-associated nuclear antigen (LANA) is a substrate for extracellular signal-regulated kinase 1 (ERK1) and that ERK1 is present in anti-LANA immunoprecipitates generated using PEL cell extracts [41]
Summary
HeLa cells seeded at 106 per 10-cm dish were transfected by using calcium phosphate precipitation with HA– c-Myc (7 g), Flag-LANA (7 g), and/or S-GSK-3 (7 g). The supernatant was incubated with anti-HA monoclonal antibody (1 g) or anti-Flag M2 affinity gel for 2 h at 4°C, followed by incubation with protein G-Sepharose beads (50 l, swollen volume) for 2 h at 4°C. HeLa cells were rinsed with 1ϫ phosphate-buffered saline (pH 7.4), washed with phosphate-free DMEM, incubated in phosphate-free DMEM plus 10% dialyzed fetal bovine serum for 1 h, and incubated for 4 h with the same medium containing [32P]orthophosphate (carrier-free [Amersham; 4 mCi/10-cm dish, 250 Ci/ml), with or without 25 mM LiCl. Labeling was stopped by washing the the cells twice with ice-cold PBS. Labeled cells were lysed and subjected to immunoprecipitation with anti-c-Myc antibody as described above. Immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography and Western blotting
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