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. 2008 Jun 5;375(2):550-60.
doi: 10.1016/j.virol.2008.01.034. Epub 2008 Mar 17.

Walleye dermal sarcoma virus Orf B functions through receptor for activated C kinase (RACK1) and protein kinase C

Candelaria C Daniels  1 , Joel Rovnak, Sandra L Quackenbush
Affiliations

Walleye dermal sarcoma virus Orf B functions through receptor for activated C kinase (RACK1) and protein kinase C

Candelaria C Daniels et al. Virology. .

Abstract

Walleye dermal sarcoma virus is a complex retrovirus that is associated with walleye dermal sarcomas that are seasonal in nature. Fall developing tumors contain low levels of spliced accessory gene transcripts A and B, suggesting a role for the encoded proteins, Orf A and Orf B, in oncogenesis. In explanted tumor cells the 35 kDa Orf B accessory protein is localized to the cell periphery in structures similar to focal adhesions and along actin stress fibers. Similar localization was observed in mammalian cells. The cellular protein, receptor for activated C kinase 1 (RACK1), bound Orf B in yeast two-hybrid assays and in cell culture. Sequence analysis of walleye RACK1 demonstrated high conservation to other known RACK1 sequences. RACK1 binds to activated protein kinase C (PKC). Orf B associates with PKCalpha, which is constitutively activated and localized at the membrane. Activated PKC promoted cell survival, proliferation, and increased cell viability in Orf B-expressing cells.

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Figures

Fig. 1
Fig. 1
Expression of WDSV Orf B in explanted tumor cells and NIH3T3 cells. (Upper panels) Explanted spring tumor cells were labeled consecutively with rabbit anti-Orf B sera and FITC-conjugated goat anti-rabbit IgG (green). 400 X magnification. (Lower panel) NIH3T3 (left) and NIH3T3-Orf B (right) cells were labeled with anti-HA mAb (HA.11) and FITC-conjugated anti-mouse IgG (green). 400 X magnification. Nuclei were stained with DAPI (blue).
Fig. 2
Fig. 2
WDSV Orf B interacts with RACK1. Lysates from NIH3T3 cells and 3T3 cells stably expressing Orf B (NIH3T3-Orf B) were immunoprecipitated with mouse anti-RACK1 antibody or normal mouse serum (NMS). HA-tagged Orf B was detected with anti-HA antibody (12CA5). Lysate represents 4% of the total amount used for immunoprecipitation.
Fig. 3
Fig. 3
Amino acid sequence alignment of walleye RACK1 with human, mouse, and zebrafish RACK1. Identical residues are darkly shaded, similar residues are lightly shaded, and non-identical residues are unshaded. Positions of the seven WD repeats (WD1-7) are indicated with arrows.
Fig. 4
Fig. 4
A. Co-immunoprecipitation of Orf B and walleye RACK1 from HeLa cell lysates. Lysates from HeLa cells co-transfected with HA-tagged Orf-B (pKH3-Orf B) and FLAG-tagged walleye RACK1 (pFLAG-wRACK1) or empty vectors pKH3 and pFLAG were immunoprecipitated with anti-HA or anti-FLAG antibodies. HA-tagged Orf B was detected with anti-HA MAb (12CA5) and walleye RACK1 was detected with anti-FLAG MAb (M2). HA-tagged Orf B and FLAG-tagged walleye RACK1 run at approximately 39 kDa and 37 kDa, respectively. Lysates represent 4% of the total amount used for immunoprecipitation. B. Immunoprecipitation of Orf B with endogenous walleye RACK1. Lysates from walleye cells (W12) transfected with pKH3-Orf B or pKH3 empty vector were immunoprecipitated with MAb to human RACK1. Orf B was detected with anti-HA (12CA5).
Fig. 5
Fig. 5
WDSV Orf B interacts with PKCα. Lysates from NIH3T3 cells stably expressing Orf B (NIH3T3-Orf B) and NIH3T3 control cells (NIH3T3) were immunoprecipitated with rabbit anti-PKCα antibody or with rabbit anti-PKCδ antibody. HA-tagged Orf B was detected with anti-HA (12CA5) MAb. Lysate represents 4% of total amount used for immunoprecipitation.
Fig. 6
Fig. 6
WDSV Orf B expressing cells remain viable after treatment with staurosporine. NIH3T3 and NIH3T3-Orf B cells were treated with 25, 50, or 100 nM staurosporine, and cell viability was measured by MTS assay after 16 hrs of incubation. The mean ± standard deviation of OD490 readings from replicates of six wells was determined and normalized to % viability. The data represent one of three independent experiments. Statistically significant differences (P< 0.004).
Fig. 7
Fig. 7
PKCα is constitutively activated in Orf B-expressing cells. NIH3T3 and NIH3T3-Orf B cells were grown without serum for 24 hours and then treated with dimethyl sulfoxide (DMSO), bisindolymaleimide I (Bim), or phorbol ester (PMA). Cell lysates were harvested, separated into membrane (A) and cytosolic (B) fractions by ultracentrifugation, and analyzed by western blot with the indicated antibodies; anti-pPKCα detects PKCα when phosphorylated at threonine 638, anti-PKCα detects total PKCα anti-RACK1 detects endogenous RACK1, and anti-HA (12CA5) detects HA-tagged Orf B. β-actin and cadherin serve as loading controls. The data presented in this figure is representative of four independent experiments.
Fig. 8
Fig. 8
Orf B-expressing cells proliferate and survive in the absence of serum. A. NIH3T3 cells and NIH3T3-Orf B cells were grown under serum-free conditions and viable cells measured each day for 7 days by MTS assay. The mean of six replicate wells was determined for each time point. Viable cells at Day 0 were normalized to 100% and ODs from subsequent days were adjusted accordingly. The data presented in this figure is representative of three independent experiments. B. Phase contrast images of NIH3T3 and NIH3T3-Orf B cells illustrate cell morphology at day 0 and day 3 of culture without serum. Magnification was 200X.
Fig. 9
Fig. 9
Activated PKC drives proliferation and survival of Orf B-expressing cells. A. NIH3T3-Orf B cells were grown without serum in the presence of 5 µM Bim in DMSO (NIH3T3-Orf B no serum+Bim) or DMSO only (NIH3T3-Orf B no serum). NIH3T3-Orf B cells were also cultured with serum and 5µM Bim (NIH3T3-Orf B serum+Bim). Viable cells were measured each day for 7 days by MTS assay. The mean of six replicate wells was determined for each time point. Viable cells at Day 0 were normalized to 100% viability and the ODs from subsequent days were adjusted accordingly. The data presented in this figure is representative of three independent experiments. B. Phase contrast images illustrate the morphology NIH3T3-Orf B cells cultured with serum and treated with 5 µM Bim (left), cells without serum and treated with DMSO (middle), and cells without serum and with 5 µM Bim (right) at day 0 and day 3. Magnification was 200X.
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