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. 2012 May;86(10):5742-51.
doi: 10.1128/JVI.07006-11. Epub 2012 Feb 29.

Retroviral cyclin enhances cyclin-dependent kinase-8 activity

Joel Rovnak  1 , Connie D Brewster, Sandra L Quackenbush
Affiliations

Retroviral cyclin enhances cyclin-dependent kinase-8 activity

Joel Rovnak et al. J Virol. 2012 May.

Abstract

Alterations in the functional levels of cyclin-dependent kinase-8 (CDK8) or its partner, cyclin C, have been clearly associated with cancers, including colon cancer, melanoma, and osteosarcoma. Walleye dermal sarcoma virus encodes a retroviral cyclin (RV-cyclin) that localizes to interchromatin granule clusters and binds CDK8. It also binds to the Aα subunit (PR65) of protein phosphatase 2A (PP2A). Binding to the Aα subunit excludes the regulatory B subunit, but not the catalytic C subunit, in a manner similar to that of T antigens of the small DNA tumor viruses. The expression of the RV-cyclin enhances the activity of immune affinity-purified CDK8 in vitro for RNA polymerase II carboxy-terminal domain (CTD) and histone H3 substrates. PP2A also enhances CDK8 kinase activity in vitro for the CTD but not for histone H3. The PP2A enhancement of CDK8 is independent of RV-cyclin expression and likely plays a role in the normal regulation of CDK8. The manipulation of endogenous PP2A activity by inhibition, amendment, or depletion confirmed its role in CDK8 activation by triggering CDK8 autophosphorylation. Although RV-cyclin and PP2A both enhance CDK8 activity, their actions are uncoupled and additive in kinase reactions. PP2A may be recruited to CDK8 in the Mediator complex by a specific PP2A B subunit or additionally by the RV-cyclin in infected cells, but the RV-cyclin appears to activate CDK8 directly and in a manner independent of its physical association with PP2A.

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Figures

Fig 1
Fig 1
RV-cyclin enhancement of CDK8 activity. Anti-CDK8 IPs from nuclear extracts of matched uninduced (−RV-cyclin) and induced (+RV-cyclin) HeLa cells were assayed in triplicate reactions. Equal aliquots of washed CDK8 Ab-protein G magnetic beads were suspended in a common reaction mix. After incubation, GST fusion substrate was captured with added glutathione magnetic beads. IP and substrate were washed and separated by polyacrylamide gel electrophoresis and blotted onto nylon for the imaging of labeled GST-CTD substrate (32P-CTD). The blot was subsequently probed with specific antibodies for GST-CTD (74 to 76 kDa; αCTD), CDK8 (53 kDa; αCDK8), and MED23 (140 kDa; αMED23). The chart presents means and standard deviations of signal (relative band volume) from the phosphorimage of the CTD band. Asterisks indicate the statistical significance of the increase associated with RV-cyclin expression (P < 0.001).
Fig 2
Fig 2
WDSV RV-cyclin interacts with PP2A. (A) Lysates of HeLa cells transfected with constructs expressing myc-tagged PR65 (aa 212 to 568; 40 kDa; myc-PR65) and HA-tagged RV-cyclin (37 kDa; HA-RV-cyclin) were subject to IP with anti-HA (αHA) and anti-myc (αmyc) antibodies, and Western blots were probed with the reciprocal antibodies. Lysate lanes contain 10 μg cell lysate. (B) Co-IP of expressed HA-RV-cyclin with endogenous PR65. Lysate from HeLa cells transfected with control vector or HA-RV-cyclin expression vector were subjected to IP with monoclonal antibodies that distinguish free PR65 Aα and Aβ subunits of PP2A (65 kDa; 6G3; A), the PP2A core enzyme (5H4; A+C), or the PP2A holoenzyme (6F9; A+B+C) and with polyclonal anti-PR65 Aα/β that precipitates all forms (αPR65). The input lane contains 10 μg lysate.
Fig 3
Fig 3
Okadaic acid (OA) inhibits CDK8 kinase activity. (A) Anti-CDK8 IPs from nuclear extracts of matched uninduced (−RV-cyclin) and induced (+RV-cyclin) HeLa cells were assayed in triplicate reactions. Equal aliquots of washed CDK8 Ab-protein G magnetic beads were suspended in a common reaction mix with or without added OA (10 nM). After incubation, GST fusion substrate was captured with added glutathione magnetic beads. IP and substrate were washed and separated by polyacrylamide gel electrophoresis and blotted onto nylon for the imaging of labeled GST-CTD substrate (32P-CTD). The blot was subsequently probed with specific antibodies for the GST-CTD (74 to 76 kDa; αCTD), CDK8 (53 kDa; αCDK8), and MED23 (140 kDa; αMED23). The chart presents means and standard deviations of signal (relative band volume) from the phosphorimage of the CTD band. Statistical significance is represented by asterisks (*, P < 0.01; **, P < 0.001). These data are representative of three independent experiments. (B) Assay of anti-CDK8 IPs from nuclear extracts of walleye cells with and without the inclusion of OA (10 nM) in the reaction mix. (C) Titration of OA in kinase assay of anti-CDK8 IPs from RV-cyclin-positive HeLa cell nuclear extract. Equal aliquots of washed CDK8 Ab-protein G magnetic beads were suspended in a common reaction mix with or without added OA as indicated. (D) Assay of anti-CDK8 and combined anti-CDK8/anti-PP2A IPs from RV-cyclin-positive HeLa cell nuclear extract [NE (+RV-cyclin)] or the same extract immune depleted of PP2A (−PP2A NE) in a common reaction mix with or without added OA (10 nM). (E) Assay of anti-CDK8 IPs from RV-cyclin-positive HeLa cell nuclear extracts previously immune depleted of PP2A (−PP2A NE) or PP1 (−PP1 NE) with or without added OA (10 nM). Charts present signal (relative band volume) from the phosphorimage of the CTD band in each experiment. (F) Western blot evaluation of depleted (Depl.) nuclear extracts. Ten μg total protein was probed for PR65 (65 kDa; αPR65) and PP1 (34 kDa; αPP1) before and after immune depletion.
Fig 4
Fig 4
RV-cyclin only enhances CDK8 activity. (A) Anti-CDK7, anti-CDK8, and anti-CDK9 IPs from nuclear extracts of matched uninduced and induced (RV-cyclin +) HeLa cells were assayed for kinase activity with the GST-CTD substrate. Equal aliquots of washed Ab-protein G magnetic beads were suspended in a common reaction mix with or without added OA (10 nM). After incubation, GST fusion substrate was captured with added glutathione magnetic beads. IP and substrate were washed and separated by polyacrylamide gel electrophoresis and blotted onto nylon for the imaging of labeled GST-CTD substrate (32P-CTD). Blots were subsequently probed with specific antibodies for the CTD (74 to 76 kDa; αCTD) and the corresponding IP Ab (first panel, αCDK7, 40 to 42 kDa; second panel, αCDK8, 53 kDa; third panel, αCDK9, 42 kDa). Image exposures were 30 min for CDK7 IPs and 3 h for CDK8 and CDK9 IPs. The charts present signal (relative band volume) from the phosphorimage of the CTD band. (B) Coprecipitation of CDKs. Western blots of CDK IPs (αCDK7, αCDK8, or αCDK9) and 10 μg HeLa nuclear extract (NE) were probed for CDK8 (αCDK8; upper panels) and for CDK7 and CDK9 (αCDK7 or αCDK9; lower panels).
Fig 5
Fig 5
Exogenous FLAG-CDK8 kinase activity is regulated by PP2A and RV-cyclin. HeLa cell nuclear extracts with transiently expressed FLAG-CDK8, FLAG-CDK8 with RV-cyclin (RV-cyclin), FLAG-CDK8 with cyclin C (cyclin C), or kinase-deficient FLAG-CDK8 (FLAG-CDK8 KD) were immune precipitated with anti-FLAG antibody or with combined anti-FLAG and anti-PP2A (αPP2A) antibodies for kinase assay with GST-CTD as the substrate. Equal aliquots of washed Ab-protein G magnetic beads were suspended in a common reaction mix with or without added OA (10 nM). After incubation, GST fusion substrate was captured with added glutathione magnetic beads. IP and substrate were washed and separated by polyacrylamide gel electrophoresis and blotted onto nylon for the imaging of labeled GST-CTD substrate (32P-CTD). The blot was subsequently probed with specific antibodies for the CTD (74 to 76 kDa; αCTD) and FLAG-CDK8 (56 kDa; αFLAG). The chart presents signal (relative band volume) from the phosphorimage of the CTD band.
Fig 6
Fig 6
CDK8 autophosphorylation is regulated by PP2A. (A) HeLa cell nuclear extracts with transiently expressed FLAG-CDK8 or kinase-deficient FLAG-CDK8 (FLAG-CDK8 KD) were immune precipitated with anti-FLAG antibody or with combined anti-FLAG and anti-PP2A (αPP2A) antibodies for kinase assay with no added substrate. Equal aliquots of washed Ab-protein G magnetic beads were suspended in a common reaction mix with or without added OA (10 nM). After incubation, IPs were washed and separated by polyacrylamide gel electrophoresis and blotted onto nylon for the imaging of labeled IP proteins (56 kDa; 32P-FLAG-CDK8). The single radiolabeled band was identified subsequently with anti-CDK8 Ab (αCDK8). The chart presents signal (relative band volume) from the phosphorimage of the FLAG-CDK8 band. (B) Anti-CDK8 IPs from HeLa cell nuclear extracts were assayed in triplicate reactions. Equal aliquots of washed CDK8 Ab-protein G magnetic beads were preincubated in a common reaction mix with or without added OA (20 nM) for 50 min prior to the addition of a single mix with GST-CTD substrate, [γ32P]ATP, and excess OA (100 nM) to both reactions. After further incubation, GST-CTD was captured with added glutathione magnetic beads. IP and substrate were washed and separated by polyacrylamide gel electrophoresis and blotted onto nylon for the imaging of labeled GST-CTD substrate (32P-CTD). Blots were subsequently probed with specific antibodies for the CTD (74 to 76 kDa; αCTD), CDK8 (53 kDa; αCDK8), and MED23 (140 kDa; αMED23). The chart presents signal (relative band volume) from the phosphorimage of the CTD band. The asterisk indicates statistical significance associated with preincubation with OA (P < 0.01).
Fig 7
Fig 7
RV-cyclin enhances, but okadaic acid (OA) does not inhibit, histone H3 phosphorylation. Anti-CDK8 IPs from nuclear extracts of matched uninduced (−RV-cyclin) and induced (+RV-cyclin) HeLa cells were assayed in triplicate reactions. Equal aliquots of washed CDK8 Ab-protein G magnetic beads were suspended in a common reaction mix containing recombinant mouse histone H3 and GST-CTD as substrates and with or without added OA (10 nM). After incubation, histone H3 substrate was captured with specific antibody and additional protein G magnetic beads and GST-CTD with glutathione magnetic beads. IP and substrates were washed and separated by polyacrylamide gel electrophoresis and blotted onto nylon for the imaging of labeled histone H3 substrate (32P-H3) (Fig. 3A, CTD). The blot was subsequently probed with specific antibody for histone H3 (17 kDa; αH3). The chart presents means and standard deviations of signal (relative band volume) from the phosphorimage of the H3 band.
Fig 8
Fig 8
Proposed model of RV-cyclin regulation of CDK8 activity. RV-cyclin displaces cyclin C from CDK8 in the submodule and in CDK8-Mediator complexes associated with RNA Pol II and transcription complexes. CDK8 has greater enzymatic activity with RV-cyclin than cyclin C binding. CDK8-Mediator is also subject to dephosphorylation by PP2A, which triggers autophosphorylation and full kinase activity toward the CTD of RNA Pol II. This mechanism functions with or without RV-cyclin. RV-cyclin also binds the PP2A scaffold subunit, PR65, and excludes the regulatory B subunit. RV-cyclin may increase the PP2A-CDK8 activation process by colocalizing PP2A with its CDK8 substrate at active transcription sites. Known CDK8 substrates, histone H3, RNA Pol II CTD, E2F1, cyclin H, Med13, and CDK8 itself, are illustrated with asterisks.
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