Fig. 1. DmCAK contains cyclin H, MAT1 and XPD. (A) Immuno precipitations were carried out on 0–16 h embryonic extracts, and the isolated proteins were subjected to mass spectrometry. The identities of the four major proteins present in the immunoprecipitates are indicated on the right. Complete Drosophila cyclin H (AF024618) and MAT1 (AF071227) sequences can be obtained from GenBank. DmXPD has been described previously (Reynaud et al., 1999). (B) T-loop sequences and phosphorylation sites of Drosophila CDKs. In addition to the conserved threonine at position 170, Ser164 within the T-loop of CDK7 is also a target of phosphorylation.

Fig. 3. T-loop mutant forms of Drosophila CDK7 are temperature sensitive in vitro. CDK7 was immunoprecipitated from the various mutants; the immunoprecipitates were divided in half and either left on ice or incubated at 33°C for 30 min (hs) prior to kinase assays with either CDK2^D145N–cyclin A (A) or GST–CTD (B) as substrates. Note that the exposure times for experiments with CDK7^S164A/T170A were longer than for the wild-type and single mutants. For both substrates, incorporation was quantified by PhosphorImager and expressed as a percentage of incorporation by wild-type CDK7 not subjected to heat shock (defined as 100%). Measured values are indicated below each lane in (A) and (B). Top panel, anti-CDK7 immunoblot; bottom panel, ³²P incorporation into substrates.

Fig. 8. RNA polymerase II phosphorylation in CDK7 mutants. Third instar wandering larvae were homogenized directly in SDS sample buffer either prior to, or after a 30 min heat shock at 37°C (hs). Each sample was immunoblotted with antibodies to the CTD of the large subunit of RNA pol II: 8WG16, H5 (specific for phosphorylated Ser2 of the CTD heptad repeat) and H14 (specific for phosphorylated Ser5 of the CTD heptad repeat). Although the 8WG16 antibody preferentially recognizes unphosphorylated repeats (enriched in form IIA), it cross-reacts to some extent with form II0 that is not completely phosphorylated.

Fig. 7. T-loop phosphorylation of CDK7 regulates activity in a substrate-dependent manner. (A) CDK7 was immunoprecipitated from S180A, S164A and T170A mutants. Each sample was divided in half and tested for kinase activity towards either GST–CTD or CDK2–cyclin A (top panel). Incorporation was quantified and expressed as a percentage of incorporation by the wild-type (S180A) control immunoprecipitate. The T170A mutation results in a loss of activity that is specific to the CTD substrate. Both wild-type and T170A immunoprecipitates contain similar amounts of MAT1 protein, indicating that the substrate-specific effect is caused by the lack of phosphorylation rather than by loss of MAT1 from the complex (bottom panel). Some of the loss of activity by the S164A mutant with both substrates probably stems from the fact that S164A immuno precipitates contain significant amounts of completely dephosphoryl ated (monomeric) CDK7, which is paralleled by a reduced amount of MAT1 in S164A immunoprecipitates as compared with both wild-type and T170A. (B) The purified mammalian phosphorylated trimer exhibits increased activity (25- to 50-fold) towards the CTD substrate when compared with either phosphorylated dimer or unphosphorylated trimer, but the effect of phosphorylation on CAK activity is modest (∼2-fold).

Fig. 5. T-loop phosphorylation is required for the formation of a stable CDK7–cyclin H–MAT1 complex. (A) Embryonic extracts were fractionated on a Superdex 200 gel filtration column; each fraction was subjected to immunoprecipitation with anti-CDK7 antibodies and analyzed by immunoblot or assayed for phosphorylation of GST–CTD in vitro. Pure mammalian dimeric and trimeric CAK complexes were used as size standards in chromatography, and detected by immuno blotting with anti-CDK7 antibodies for the dimer and with anti-MAT1 antibodies for the trimer. (B) Size distribution of CDK7 protein in the various T-loop mutants. Embryonic extracts from the indicated mutants were fractionated as in (A); the fractions were immunoprecipitated with anti-CDK7 antibodies and subjected to SDS–PAGE followed by immunoblotting for CDK7 and MAT1 proteins. Due to the fragility of the complex in the total absence of CDK7 T-loop phosphorylation, no MAT1 could be detected in immunoprecipitates from the S164A/T170A mutant. (C) Decreased thermal stability of unphosphorylated CDK7-containing complexes. Embryonic extracts were fractionated as in (A) and analyzed directly (without immunoprecipitation) by SDS–PAGE and immunoblotting with anti-CDK7 antibodies. When S164A/T170A embryos are shifted from 18 to 29°C, the recovery of the complex is diminished further (bottom panel).

Fig. 4. T-loop phosphorylation protects mammalian CDK7 from thermal inactivation. (A) Anti-CDK7 immunoblots of recombinant CAK purified from Sf9 cells infected with baculoviruses encoding the mammalian CAK subunits. Double infections with CDK7 and cyclin H viruses produce CDK7 protein that is doubly phosphorylated on the T-loop (left: large arrowhead). The small arrowhead indicates a less abundant, probably singly phosphorylated CDK7 isoform. Triple infections with CDK7, cyclin H and MAT1 result in the isolation of CDK7 protein that is entirely unphosphorylated (right). (B) Time course of thermal inactivation of the different forms of CAK in vitro. We incubated the three complexes at 42°C for the indicated periods of time before we measured activity towards a CDK2–cyclin A substrate. (C) Relative thermal stability of the CAK dimer, unphosphorylated trimer and phosphorylated trimer quantified on the basis of triplicate experiments (represented by B). The phosphorylated trimer (circle) is resistant to heat inactivation compared with either the unphosphoryl ated trimer (diamond) or the phosphorylated dimer (square).

Fig. 6. Dephosphorylation of the T-loop destabilizes CDK7 complexes. (A) Anti-Cdk7 4A7 is specific for the monomeric form of CDK7, whereas the 20H5 antibody recognizes both complex and monomeric forms of CDK7. Antibody 20H5 can precipitate Cdk7 from an embryonic lysate whereas 4A7 cannot (Left). Following immuno precipitation with 20H5 and denaturation by boiling in SDS, 4A7 and 20H5 precipitate Cdk7 with equal efficiency (right). (B) The 4A7 antibody immunoprecipitates large amounts of protein only from the S164A/T170A mutant, and a small amount from the S164A mutant, whereas the 20H5 antibody precipitates similar amounts from all samples. Top panel, anti-CDK7 immunolot of total lysates prior to immunoprecipitation; second panel, anti-CDK7 immunoblot of immunoprecipitates; third panel, anti-CDK7 immunoblot of post-immunoprecipitation supernatants; bottom panel, anti-MAT1 immunoblot on the immunoprecipitated material. (C) Similar to (B) after 15 h incubation at 4°C. Detectable amounts of CDK7 can be immunoprecipitated from all samples with the 4A7 antibody. Both single mutants (S164A and T170A) dissociate to a much greater extent than does the wild-type (S180A) control. In each case, however, only the slow migrating (unphosphorylated) form can be precipitated, indicating that dephosphorylation of the T-loop is required to destabilize complexes. Dephosphorylation of the S164A protein correlates with the loss of MAT1 from the complex (bottom panel).

Fig. 2. Phospho-isoforms of Drosophila CDK7 detected by electro phoretic mobility shift. (A) CDK7 protein from ovaries of wild-type and mutant animals is shown. Dual T-loop phosphorylation (bottom arrowhead) results in a large increase in mobility compared with the unphosphorylated isoform (top arrowhead). Phosphorylation on Thr170 alone in the S164A mutant causes an intermediate shift (middle arrow). In this analysis, we could not distinguish the form singly phosphorylated on Ser164 from the completely unphosphorylated form. (B) The relative abundance of CDK7 protein and its state of T-loop phosphorylation vary little during embryonic development. (C) The state of CDK7 T-loop phosphorylation does not change appreciably during the embryonic cell cycles. Embryos were selected individually (embryonic cycle 9–13) after methanol fixation and staining for DNA, and identified by microscopic inspection as being in interphase (I), prophase (P) metaphase (M), anaphase (A) or telophase (T). (D) The state of CDK7 T-loop phosphorylation varies among different tissues: ovaries from wild-type (wt) and the double-mutant (S/T) animals, third instar larvae (L3), imaginal discs (Id), salivary glands (Sg) and ovaries (Ov). Note that the difference in isoform representation between the first and last lanes, which both contain extracts of wild-type ovaries, reflects differences in the age of the animals. We typically see more unphosphorylated CDK7 in more mature ovaries. Proteins were extracted by directly boiling the tissues in SDS sample buffer. (E) The unphosphorylated form of CDK7 exhibits low solubility. Embryos were homogenized in SDS sample buffer (Total) or HoB buffer. Following centrifugation, the supernatant (soluble) and pellet (insoluble) were boiled in SDS sample buffer. The unphosphorylated form of CDK7 is extracted efficiently only in the presence of SDS (lanes 1 and 3).