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. 2011 Jan 20;409(2):299-307.
doi: 10.1016/j.virol.2010.10.022. Epub 2010 Nov 9.

The retroviral cyclin of walleye dermal sarcoma virus binds cyclin-dependent kinases 3 and 8

Connie D Brewster  1 , Claire H Birkenheuer, Megan B Vogt, Sandra L Quackenbush, Joel Rovnak
Affiliations

The retroviral cyclin of walleye dermal sarcoma virus binds cyclin-dependent kinases 3 and 8

Connie D Brewster et al. Virology. .

Abstract

Walleye dermal sarcoma virus encodes a retroviral cyclin (rv-cyclin) with a cyclin box fold and transcription activation domain (AD). Co-immune precipitation (co-IP) identified an association of rv-cyclin with cyclin-dependent kinase 8 (cdk8). Cdk8 is dependent upon cyclin C and regulates transcription with the Mediator complex, a co-activator of transcription. Mutation of cyclin residues, required for cdk binding, disrupts rv-cyclin-cdk8 co-IP. Mutation or removal of the AD has no effect on cdk8 interaction. Direct rv-cyclin-cdk8 binding is demonstrated by pulldown of active cdk8 and by GST-rv-cyclin binding to recombinant cdk8. Cdk3 is also activated by cyclin C and phosphorylates retinoblastoma protein to initiate entry into the cell division cycle. Co-IP and pulldowns demonstrate direct rv-cyclin binding to cdk3 as well. The rv-cyclin functions as a structural ortholog of cyclin C in spite of its limited amino acid sequence identity with C cyclins or with any known cyclins.

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Figures

Fig. 1
Fig. 1
A. Phyre alignment of WDSV rv-cyclin with cyclin A (D. melanogaster)(E-value=4e−43, identity 19.2%) and human cyclin C (E-value=5e−06, identity 14.9%). The alignment was generated with 998 additional cyclins. Black positions are identical; gray are conserved residues. Asterisks indicate conserved cdk contacts and correspond to residues K80 and E111 in the rv-cyclin. The unaligned carboxy terminal region containing the TAF9 binding motif, LDDV260MAVL (bold), extends beyond the cyclin box of all of the aligned cyclins. Small letters, ft in cyclin C and mt in cyclin A, indicate deletions introduced by the alignment software. B. HA-tagged cyclin C, rv-cyclin, rv-cyclin point mutants, K80A, E111A, double mutant (DM; K80A/E111A), and V260S, and a.a.1–255 were transiently co-expressed with FLAG-tagged cdk8 in HeLa cells and whole cell lysates were immune precipitated with anti-cdk8 antibody. Lysates represent 5% of the co-IP input protein (5 µg) (left panel). Expressed and precipitated HA-tagged cyclins were detected on western blots with anti-HA antibody (αHA). The bottom panel shows precipitated cdk8 in each reaction after reprobe with anti-cdk8 antibody (αcdk8).
Fig. 2
Fig. 2
A. GST-pulldowns of FLAG-cdk8 from cell lysates with GST, GST-rv-cyclin (rv-cyclin), and GST-cyclin C (cyclinC). GST-CTD served as in vitro substrate in kinase assays of bound cdk8 (top panel: autoradiograph of western blot of kinase reactions (32P-CTD)) followed by sequential antibody detection of input GST-CTD substrate (αCTD; anti-RNAPII CTD) and pulled down cdk8 (αcdk8). B. Ni Sepharose-His-cdk8 pulldown of soluble GST, GST-rv-cyclin (rv-cyclin), or GST-cyclin C (cyclinC) probed with anti-GST antibody (αGST). The blot was reprobed with anti-polyHis (αHis) to detect input His-cdk8. Aliquots of input GSTs were run separately (bottom panel). GST-rv-cyclin and GST-cyclin C are of similar molecular weight (64 KDa vs. 60 KDa); the GST panels show signal for proteins at 27 KDa (GST).
Fig. 3
Fig. 3
Co-IP of RNAPII with anti-cyclin C (αcyclinC), anti-cdk8 (αcdk8), and anti-HA (αHA) from nuclear extracts of cells without or with expressed, HA-tagged rv-cyclin. Co-precipitates were detected successively on western blots with antibodies specific for phosphorylated forms of the RNAPII CTD: H5 (phospho-Ser2; YSPTSPS), H14 (phospho-Ser5; YSPTSPS), and 8WG16 (un-phosphorylated RNAPII). The position of a 191 KDa marker is indicated to illustrate the size difference between un-phosphorylated RNAPII (predicted molecular weight 220 KDa) and the hyperphosphorylated forms (apparent molecular weight of 240 to 250 KDa).
Fig. 4
Fig. 4
A. Analysis of the levels of cellular transcripts from HeLa cells at 32 hours post-transfection with control vs. rv-cyclin expression vectors. 96 genes involved with cell cycle regulation were tested by hybridization with radiolabeled cDNA on membrane bound gene arrays (GEArray Q Human Cell Cycle Gene Array). The names of genes with greater than four-fold increases or greater than two-fold decreases are indicated. B. Quantitation of cyclin D and p19Ink4d transcripts at 10 hrs post-transfection with control vs. rv-cyclin expression vector.
Fig. 5
Fig. 5
Interaction of rv-cyclin and cdk3. A. Nuclear extracts from induced HeLa Tet-Off myc-rv-cyclin cells after 72 hours serum starvation followed by 3 hrs of serum restoration were subject to co-IP with the indicated IP antibodies, normal rabbit serum (NRS), anti-cdk3 (αcdk3) and anti-cdk8 (αcdk8). The western blot was probed first with anti-myc Ab (αmyc) to detect precipitation of myc-tagged rv-cyclin followed by anti-cdk3 Ab (αcdk3) to detect precipitation of endogenous cdk3. B. Over-expressed, HA-tagged cdk3 was subject to co-IP with anti-rv-cyclin (αrv-cyclin) from nuclear extracts of HeLa cells with or without induced expression of myc-tagged rv-cyclin. Blots were probed first with anti-HA (αHA) to detect HA-cdk3 followed by anti-myc (αmyc) to confirm myc-rv-cyclin expression. Co-IPs with anti-cdk3 (αcdk3) and normal rabbit sera (NRS) were from extracts of rv-cyclin-induced cells. C. Ni Sepharose-His-cdk3 pulldown of soluble GST, GST-rv-cyclin (rv-cyclin) or GST-cyclin C (cyclinC) probed with anti-GST antibody (αGST). The blot was reprobed with anti-polyHis (αHis) to detect input His-cdk3. Total unbound GSTs from each reaction were run separately (bottom panels). GST-rv-cyclin and GST-cyclin C are of similar molecular weight (64 KDa vs. 60 KDa); the GST panels show signal for proteins at 27 KDa (GST). D. GST-pulldowns of over-expressed cdk3 were tested in kinase assays for phosphorylation of an Rb fusion protein (MBP-Rb-C, a.a. 379–928). The top panel shows the autoradiograph (32P-Rb) followed by antibodies for phosphorylated residues S807 and S811 of Rb (αpRb, middle panel) and for input MBP-Rb-C substrate (αRb, bottom panel).
Fig. 6
Fig. 6
A. Un-induced and induced HeLa Tet-Off myc-rv-cyclin cells after 96 hrs of serum starvation. Arrows indicate cells undergoing cell division monitored over a 1 hr period and photographed at 0, 30 and 60 minutes. B. Un-induced HeLa Tet-Off myc-rv-cyclin cells were seeded in spinner flasks at 5×104 cells/ml under non-inducing (Tet+) or inducing conditions (Tet−) and viable cells monitored for cell concentration. rv-cyclin protein is detectable between 12 and 24 hrs post-induction (not shown).
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