Fig 2: CA inhibition of and binding to CDK8(a) CA inhibition of CDK8 module phosphorylation of CDK8 and STAT1 S727 substrate (mean ± s.e.m., n=3 biological replicates, one of two experiments shown, autorad in Supplementary Figure 1). (b) CA inhibition in vitro of CDK8 module activity but not CDK12:Cyclin K or CDK13:Cyclin K activity up to 10 µM. Equal amounts (silver stain) of GST-CTD were used as the substrate in in vitro kinase assays. The amount of each kinase used was empirically determined to give approximately the same GST-CTD signal under the assay conditions. “ns” is no substrate (kinase only) and “GST-CTD-P” is phosphorylated GST-CTD. One of four experiments shown. (c) Immunoblot showing that CA selectively and dose-dependently inhibits capture of native CDK8 (IC50 ~10 nM) and CDK19 (IC50 ~ 100 nM) from MOLM-14 lysates but did not inhibit capture of CDK9, CDK12, CDK13, ROCK1, ROCK2 or GSG2. One of two experiments shown, full scan in Supplementary Figure 1. (d) Immunoblots showing CA inhibition of CDK8-dependent IFN-?-stimulated STAT1 S727 phosphorylation in MOLM-14 cells and CA inhibition of TGF-ß-stimulated Smad2 T220 and Smad3 T179 phosphorylation in HaCaT cells (IC50 < 100 nM). One of two experiments shown, full scan in Supplementary Figure 1. (e) In vitro kinase activity profiling (mean for kinase reaction, n=2 biological replicates, experiment performed once). (f,g) CA dose-dependent inhibition of (f) CDK8/CCNC complex (IC50 = 5 nM) and (g) GSG2 (IC50 = 130 nM) as measured for the (e) (n=1, experiment performed once). (h) Dendrogram representation of results shown in Fig. 2c for 1 µM CA.

Fig 3: CycK/CDK13 phosphorylates SERINC5 at S360(A) SERINC5 was expressed with the indicated CDK13 or CDK12 proteins in the presence of WT or ?Nef HIV-1 in HEK293T cells. SERINC5, CDK13, and CDK12 proteins were detected by WB using an anti-FLAG antibody.(B) SERINC5 was expressed with WT or ?Nef HIV-1 in HEK293T cells and treated with THZ531 at 100 µM. SERINC5 expression was analyzed by WB using an anti-FLAG antibody.(C) SERINC5 and SERINC5S360A were expressed with WT or ?Nef HIV-1 in HEK293T cells and purified similar to as previously. After SDS-PAGE, proteins were analyzed after being stained with Coomassie brilliant blue or Phos-tag.(D) GST-ICL4 and GST-ICL4S360A recombinant proteins were expressed in E. coli and purified by glutathione resin. An in vitro kinase assay was conducted by incubating these proteins with affinity-purified CycK/CDK13. After SDS-PAGE, proteins were analyzed after being stained with Coomassie brilliant blue or Phos-tag.(E) Peptide 357FCFSPGGEDTEEQQPGK373, its S360-phosphorylated version, and its T366K-substituted version were synthesized. They were analyzed by high-resolution MS via direct infusion or analyzed by CZE-MS after treatment with affinity-purified CycK/CDK13.

Fig 4: Mediator kinases mediate the antiproliferative activity of CA(a) We evaluated point mutations to CDK8 residues lining the CA binding pocket: Ala155, His106, Asp103, and Trp105. Expression of CDK8 A155I, A155F, A155Q, H106K and D103E in MOLM-14 cells afforded only modest desensitization to CA. Differential sensitivity of MOLM-14 cells to CA upon expression of indicated mutant FLAG-CDK8 proteins (mean ± s.e.m., n=3 biological replicates, experiment performed once). (b) Immunoblots showing that FLAG-CDK8 or FLAG-CDK19 and FLAG-CDK8 W105M or FLAG-CDK19 W105M are expressed at similar levels in MOLM-14, MV4;11, and SKNO-1 cells (experiment performed once, full scan in Supplementary Figure 1). (c) Differential sensitivity of MV4;11 and SKNO-1 cells to CA upon expression of FLAG-CDK8, FLAG-CDK19, FLAG-CDK8 W105M and FLAG-CDK19 W105M, legend as in d (mean ± s.e.m., n=3 biological replicates, one of two experiments shown). (d) Control showing that expression of FLAG-CDK8 W105M or FLAG-CDK19 W105M in MOLM-14, MV4;11, and SKNO-1 cells does not confer resistance to antiproliferative agents paclitaxel and doxorubicin (mean ± s.e.m., n=3 biological replicates, one of two experiments shown). (e) Purified FLAG-CDK8 W105M and FLAG-CDK19 W105M remain catalytically active for phosphorylation of CTD in vitro but are resistant to inhibition by CA (mean ± s.e.m., n=3 biological replicates, experiment performed once). (f) Representative autorad and silver stain images supporting quantitation shown in (e). (g) Sequence alignment of human CDKs. Sequence alignment was performed on segments of CDK1-20 using Clustal Omega. The unique Trp105 residue in CDK8 and CDK19 is highlighted in red, and is absent from other CDKs (orange box). UniProt Knowledgebase entries: CDK1, P06493; CDK2, P24941; CDK3, Q00526; CDK4, P11802; CDK5, Q00535; CDK6, Q00534; CDK7, P50613; CDK8, P49336; CDK9, P50750; CDK10, Q15131; CDK11A, Q9UQ88; CDK11B, P21127; CDK12, Q9NYV4; CDK13, Q14004; CDK14, O94921; CDK15, Q96Q40; CDK16, Q00536; CDK17, Q00537; CDK18, Q07002; CDK19, Q9BWU1; CDK20, Q8IZL9.

Fig 5: Identification of CycK/CDK13 as a Nef-associated kinase complex via mass spectrometry(A) FLAG-tagged SERINC5 protein was expressed in HEK293T cells and purified by anti-FLAG M2 affinity chromatography. After SDS-PAGE, proteins from total cell lysate and three eluted fractions were analyzed after being stained with Coomassie brilliant blue. The monomeric SERINC5 band is labeled.(B) FLAG-tagged SERINC5 was expressed with a WT or ?Nef HIV-1 proviral vector in HEK293T cells and purified and analyzed similar to as in (A). The monomeric SERINC5 band is labeled and a protein band at ~170 kDa is indicated by an asterisk.(C) purified proteins from (B) were analyzed by liquid chromatography-mass spectrometry (LC-MS). Control experiments were conducted using beads that were not conjugated with any antibodies. Six proteins with molecular masses of 150–200 kDa that are not found in the control experiments are listed.(D) FLAG-tagged CycK was expressed with WT or ?Nef HIV-1 in HEK293T cells. Proteins were immunoprecipitated with an anti-FLAG antibody and analyzed by western blotting (WB). CycK was detected by an anti-FLAG antibody and Nef and GAPDH were detected by their specific antibodies. IP, immunoprecipitation; Input, cell lysate.(E) FLAG-tagged CycK was expressed with HA-tagged CDK12 or CDK13 in HEK293T cells. Proteins were immunoprecipitated and analyzed as in (D). CDK12 and CDK13 were detected by an anti-HA antibody.(F) FLAG-tagged CDK13 was expressed with HA-tagged SERINC5 in the presence of WT or ?Nef HIV-1 in HEK293T cells. Proteins were immunoprecipitated and analyzed as in (D) and (E).All experiments were repeated twice, and similar results were obtained.