PMC

Arabidopsis CAK Complexes in Suspension Cells.
Arabidopsis protein extract from suspension cells was fractionated by Sephacryl S300 gel exclusion chromatography, and 15 μL of each fraction was immunoblotted with (A) anti-CAK1At, (B) anti-CAK2At, or (C) anti-CAK4At antibody. Each fraction was immunoprecipitated with the antibody and assayed for kinase activity using GST-CDK2 (K33R) or GST-CTD as substrate. Arrowheads indicate the elution positions of marker proteins with their molecular masses in kD.

CAK1At Substitutes for the Function of Csk1 in Fission Yeast.
(A) Complementation of mcs6-13 and csk1Δ mutations. The open reading frame of Arabidopsis CAKs cloned into the vector pREP3 were introduced into fission yeast strain JS12 carrying a mcs6-13 (L238R) mutation and a disrupted csk1Δ gene. Transformants were grown on thiamine-free minimal media at 27 or 36°C for 5 d. Morphology of yeast cells carrying the empty vector or pREP3-CAK1At was observed after culturing without thiamine at 36°C. Bar = 10 μm.
(B) Complementation of mcs6-SALR mutation. Each plasmid was introduced into mcs6-SALR cells carrying the S165A and L238R mutations in Mcs6. Transformants were grown on thiamine-free minimal media at 27 or 37°C for 5 d. Fission yeast Csk1 and Mcs6 cloned into the pREP1 vector were used as controls.

Phosphorylation of CAK2At and CAK4At by CAK1At.
(A) The CAK1At immunoprecipitates phosphorylate CAK2At and CAK4At. One hundred microliters of fraction No. 62 of gel exclusion chromatography () was immunoprecipitated with preimmune serum (PI) or with the anti-CAK1At antibody (αCAK1At), and the immunoprecipitates were subjected to kinase assays using each substrate.
(B) Recombinant CAK1At phosphorylates CAK2At and CAK4At. MBP-fused CAK2At, CAK3At, and CAK4At were incubated with 10 ng of MBP or MBP-CAK1At in the presence of [γ-³²P]ATP. MBP was used as a control.
(C) CAK1At phosphorylates the T-loop of CAK2At. Bold letters in amino acid sequences represent potential phosphorylation sites within the T-loop. In each mutant, these sites were substituted to Ala. One hundred microliters of fraction 62 of gel exclusion chromatography () was immunoprecipitated with preimmune serum (PI) or with the anti-CAK1At antibody (αCAK1At), and the immunoprecipitates were subjected to kinase assays using each mutant protein fused to MBP as substrate.
(D) CAK1At phosphorylates the T-loop of CAK4At. Details are the same as for (C).

Complementation Test of Budding Yeast cak1^ts Mutant with Deletion Derivatives of CAK1At.
(A) Schematic representation of wild-type (WT) and deletion mutants of CAK1At tentatively divided into three domains: A, B, and C. Domain B corresponds to the unusual insert of extra amino acids between positions 176 and 286. Lys50 (K50) at the kinase active site and Thr290 (T290) in the T-loop region are indicated. Solid bars represent truncated regions.
(B) Budding yeast strain GF2351 were transformed with each construct or the empty vector pYES2 and grown on MCGS. Yeast protein extracts were immunoblotted with anti-His antibody.
(C) Transformants were grown on MCD or on MCGS at 27, 34, or 37°C for 4 d.

At;CycH;1 Binds and Activates CAK2At and CAK4At.
(A) Yeast two-hybrid assay. Budding yeast strain Y190 was transformed with Arabidopsis CAKs and At;CycH;1 cloned into pACT2 (pACT-CAK) and pAS2-1 (pAS-CycH), respectively, or with an empty vector. Transformants were grown on a minimal medium for 4 d at 30°C, and β-galactosidase activity of colonies was detected using the filter-lift assay. pTA1-1 and pVA3-1 were used as controls.
(B) Quantitative β-galactosidase assay. Y190 strain was transformed with pACT-CAK only (red bars) or with both pACT-CAK and pAS-CycH (blue bars). The values of three independent assays were averaged.
(C) CDK- and CTD-kinase activities of Arabidopsis CAKs expressed in insect cells. FLAG-tagged CAKs (FLAG-CAK) and His-tagged At;CycH;1 (His-At;CycH;1) were expressed in insect Sf9 cells, and 100 ng of protein extract was immunoprecipitated using anti-FLAG antibody. In the case of FLAG-CAK1At, 10 ng of protein was used for immunoprecipitation. Kinase assay was performed using GST-CDK2 (K33R) or GST-CTD as substrate. Asterisks indicate bands of autophosphorylated FLAG-CAK1At.
(D) The cak1^ts suppressor activity of CAKs in budding yeast. CAK cDNAs cloned into pYX112 (pYX-CAK) were introduced into budding yeast strain GF2351 with (+) or without (−) At;CycH;1 in pAS2-1 (pAS-CycH). Transformants were grown on minimal medium at indicated temperatures for 4 d.

CAK1At activates the CTD-kinase activity of CAK4At.
(A) CAK4At activation in vitro. FLAG-CAK4At and/or His-At;CycH;1 were expressed in baculovirus-infected insect cells. Fifty nanograms of protein extract was immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-CAK4 antibody (top). The immunoprecipitates were incubated with MBP or MBP-CAK1At in the presence of [γ-³²P]ATP and autoradiographed (middle). After removal of MBP or MBP-CAK1At, the immunoprecipitates were subjected to kinase reaction using GST-CTD as substrate (bottom).
(B) CAK4At activation in Arabidopsis root protoplasts. The vector pMENCHU (lanes 1 to 3), pMENCHU-CAK4At (WT) (lanes 4 to 6) or pMENCHU-CAK4At (T168A) (lanes 7 to 9) was introduced into Arabidopsis root protoplasts. HA-CAK4At was coexpressed with 5 μg (lanes 2, 5, and 8) or 30 μg (lanes 3, 6, and 9) of pMESHI-CAK1At. Five micrograms of protein extract was immunoblotted with anti-CAK1At or anti-CAK4At antibody. Seventy micrograms of protein extract was immunoprecipitated with anti-HA antibody, followed by a kinase reaction using GST-CTD as substrate.
(C) Subcellular localization of GFP-fused CAKs in Arabidopsis protoplasts. Fluorescent microscopic images of CAK-GFP, and the corresponding images of DIC and Hoechst 33342 staining are shown. Merged images of Hoechst 33342 staining and GFP fluorescence are also shown. As a control, the nuclear localization signal (NLS) of Simian virus 40 fused to GFP was used. Bar = 10 μm.
(D) Subcellular localization of GFP-fused CAKs in onion epidermal cells. Fluorescent (left) and bright-field (right) images of cells are shown. Bar = 100 μm.