Interactions of Cdk7 and Kin28 with Hint/PKCI-1 and Hnt1 Histidine Triad Proteins*

Cyclin-dependent kinase 7 (Cdk7) forms a trimeric complex with cyclin H and Mat1 to form the mammalian Cdk-activating kinase, CAK, as well as a part of the basal transcription factor TFIIH, where Cdk7 phosphorylates the C-terminal domain (CTD) of the large subunit of RNA polymerase II. Here, we report a novel interaction between Cdk7 and a histidine triad (HIT) family protein, Hint/PKCI-1. This interaction was initially observed in a yeast two-hybrid study and subsequently verified by co-immunoprecipitation and subcellular localization studies, where overexpression of Cdk7 leads to partial relocalization of Hint to the nucleus. The physical association is independent of cyclin H binding or Cdk7 kinase activity and is conserved between the relatedSacharomyces cerevisiae CTD kinase Kin28 and the HIT protein Hnt1. Furthermore, combination of a disruption ofHNT1 and a KIN28 temperature-sensitive allele in S. cerevisiae led to highly elongated cell morphology and reduced colony formation, indicating a genetic interaction between KIN28 and HNT1. The physical and genetic interactions of Hint and Hnt1 with Cdk7 and Kin28 suggest a role for this class of histidine triad proteins in the regulation of Cdk7 and Kin28 functions.

The mammalian cyclin-dependent kinase Cdk7 (also called MO15) is involved in two critical cellular functions (reviewed in Ref. 1). It activates Cdks 1 by phosphorylating a threonine residue in the T-loop and thus functions as the catalytic subunit of the Cdk-activating kinase (CAK). Cdk7 also regulates the functions of the large subunit of RNA polymerase II (RNA Pol II) through phosphorylation of its C-terminal domain (CTD) as a part of the basal transcription factor TFIIH. The two functions of Cdk7 may be mediated via specific protein complexes, because a trimeric complex containing Cdk7 together with cyclin H and Mat1 is efficient as a CAK, whereas CTD phosphorylation is mediated more efficiently by Cdk7 within TFIIH.
In Saccharomyces cerevisiae, the CAK and the TFIIH-associated CTD kinase activities reside in two distinct proteins (reviewed in Ref. 1). The Kin28 kinase forms a complex with a cyclin H homologue, Ccl1, and a Mat1 homologue, Tfb3/Rig2, and functions as a TFIIH kinase phosphorylating CTD. In contrast to the mammalian complex, Kin28⅐Ccl1⅐Tfb3/Rig2 does not possess CAK activity, but instead, a monomeric CAK Cak1/Civ1 phosphorylates and activates budding yeast Cdc28 and Kin28 Cdks.
Immunoprecipitation, Kinase Assay, and Western Blotting-Immunoprecipitations from yeast lysates with the indicated proteins and the subsequent kinase assays and Western blotting analysis were performed as described (20) using monoclonal ␣-HA (12CA5, Babco Inc., Berkeley, CA) or ␣-FLAG (M5, Kodak) antibodies.
Mammalian Cell Culture, Transfection, and Immunofluorescence-U2OS osteosarcoma cells were cultured using standard protocols and transfected with the calcium phosphate transfection method (22) using plasmids with CDK7 or HA-tagged HINT cDNAs in pCINeo (Promega). Transfected cells were fixed on coverslips with 3.5% (w/v) paraformaldehyde and permeabilized with 0.1% Triton X-100 for 5 min. Double immunofluorescence was performed using rabbit polyclonal ␣-Cdk7 or mouse monoclonal ␣-HA (12CA5) followed by rhodamine-conjugated goat ␣-rabbit and fluorescein-conjugated goat ␣-mouse secondary antibodies (Roche Molecular Biochemicals). The nuclei were visualized with Hoechst 33342 (Sigma; 0.5 g/ml), and the immunostainings were viewed and documented with a Zeiss Axiophot microscope.

RESULTS AND DISCUSSION
Cdk7 and Kin28 Interact with Hint and Hnt1-We were interested in characterizing novel proteins interacting with the Cdk7 kinase. For this purpose, we performed a yeast twohybrid screen using LexA-Cdk7 bait with the same strategy that had previously led to the identification of cyclin H (16). Screening of both HeLa and human keratinocyte cDNA libraries consistently led to the observation of characteristic small, intensely blue colonies (ignored in a previous screen (16)) that were found at a higher frequency than cyclin H. A streak from a representative clone is shown in Fig. 1A. Subsequent sequencing of the corresponding cDNAs indicated that these clones represented independent cDNAs of a gene termed either PKCI-1 (protein kinase C inhibitor-1 (23)) or HINT (histidine triad nucleotide-binding protein) (24). As PKCI-1/Hint apparently is not a protein kinase C inhibitor (25,26), it is referred to here as Hint. All identified HINT clones contained the entire open reading frame, suggesting that a full-length Hint is required for the interaction.
To date, Hint is the only other characterized human HIT protein in addition to the tumor suppressor FHIT (27). Likewise, the budding yeast genome has only two HIT proteins, where HNT1 (28) and HNT2/APH1 (29) represent homologues of HINT and FHIT, respectively. FHIT proteins have been shown to be involved in nucleotide metabolism (30 -32), and Hint has also been demonstrated to bind nucleotide and nucle-oside molecules (33) and to hydrolyze ADP in vitro (34). 2 As S. cerevisiae genes KIN28 and HNT1 represent apparent homologues of CDK7 and HINT, respectively, we subsequently tested whether the two-hybrid interaction was conserved from man to yeast. To this end, we used LexA-Kin28 and B42-Hnt1 proteins in the yeast two-hybrid system, noting that expression of these proteins also activated the lacZ reporter (Fig. 1A).
We next investigated whether the HIT proteins could be detected in Cdk7 or Kin28 immunocomplexes (Fig. 1B). We expressed HA epitope-tagged HA-Cdk7, HA-Kin28, HA-Hint, or HA-Hnt1 together with FLAG epitope-tagged FLAG-Hint or FLAG-Hnt1 in yeast cells in the combinations indicated in Fig.  1B. Complex formation was subsequently assessed by immunoprecipitation of HA-tagged proteins followed by Western blotting with ␣-FLAG antibodies. The results demonstrate that FLAG-Hint and FLAG-Hnt1 co-immunoprecipitate with HA-Cdk7 (Fig. 1B, lane 5) and HA-Kin28 (Fig. 1B, lane 6), respectively. Structural studies have demonstrated that Hint forms homodimers (24,35). This was also noted in our co-immunoprecipitation studies, where both FLAG-Hint and FLAG-Hnt1 efficiently co-precipitated with the corresponding HA-tagged proteins (Fig. 1B, lanes 3 and 4). The yeast and human HIT orthologues were also able to form a complex with each other (data not shown), indicating structural conservation between Hint and Hnt1 with 34% identity at the amino acid level. An association between Hint and endogenous mammalian Cdk7 could also be demonstrated, as Cdk7 specifically co-purified 2 N. Korsisaari and T. P. Mä kelä , unpublished results. with a GST-Hint protein from mammalian cell lysates (Fig. 1C).
Functional Characterization of the Cdk7-Hint Interaction-The evolutionary conservation of the interactions on one hand between human Cdk7 (a CAK and a CTD kinase) and Hint and on the other hand between yeast Kin28 (exclusively a CTD kinase) and Hnt1 suggests that the interaction is functionally significant. To address this possibility, we initially characterized the ability of Hint to associate with Cdk7 mutants using the two-hybrid assay ( Fig. 2A). Using the well characterized interaction between Cdk7 and cyclin H as a comparison, it was first noted that the Cdk7-Hint interaction appeared to be stronger based on lacZ expression ( Fig. 2A, top panels). As previously noted for cyclin H (16), Hint also efficiently associated with a kinase-deficient Cdk7 (Cdk7-K41M), although Hint did not associate with an N-terminally deleted Cdk7 (Cdk7⌬1-132). Hint association was also observed with a Cdk7 in which the T-loop activation site threonine had been mutated to glutamic acid (Cdk7-T170E) in an attempt to mimic phosphorylation of this site. As cyclin H was previously demonstrated not to associate with the T170E mutant (16), the results indicate that the Hint-Cdk7 interaction is independent of cyclin H binding. On the other hand, Hint-associated Cdk7 retained its kinase activity (Fig. 2B), suggesting that the association of Hint with Cdk7 does not exclude cyclin binding, which is required for Cdk7's kinase activity. This was further supported by the demonstration of Hint in cyclin H immunocomplexes in yeast cells also expressing Cdk7 (Fig. 2C). Thus, Hint apparently does not represent a Cdk inhibitor; rather it may be associated with modifying Cdk7 substrate specificity, as suggested by the preferential phosphorylation of a RNA Pol II CTD substrate by Hint-associated Cdk7 (Fig. 2B).
Redistribution of Hint in Cells Overexpressing Cdk7-Previous reports on the subcellular localization of Hint in mammalian cells have indicated either a cytoskeletal localization in LM217 human fibroblast cells (36) or a nuclear and cytoplasmic localization in rat basophilic leukemic cells (37), whereas Cdk7 is exclusively a nuclear protein (38). To investigate whether Hint and Cdk7 could be detected in the same subcellular compartments, U2OS human osteosarcoma cells were transfected with an HA-Hint-expressing plasmid alone or together with a Cdk7-expressing plasmid and subjected to immunofluorescence analysis (Fig. 3). In both cases, HA-Hint protein was detected throughout the cell including nuclei with a prominent perinuclear staining (Fig. 3A). However, in some cells also overexpressing Cdk7, HA-Hint staining was more prominent in the nuclei, where it co-localized with Cdk7 (Fig. 3D). This result demonstrates that overexpression of Cdk7 leads to redistribution of Hint in mammalian cells, perhaps because of the formation of direct complexes between these proteins. The results also demonstrate that in addition to the co-localization of Hint with Cdk7 in the nucleus, a significant fraction of Hint is cytoplasmic, suggesting that Hint is likely to be involved in additional functions not related to Cdk7. This suggestion is in line with previous studies identifying interactions between Hint and the ATDC (ataxia telangiectasia group D-complementing) gene product (18) and the mi transcription factor (37).
Genetic Interaction between KIN28 and HNT1-The extension of the association of Cdk7 and Hint to the S. cerevisiae Kin28 and Hnt1 proteins, together with the availability of temperature-sensitive alleles of KIN28 (15), prompted us to investigate whether KIN28 and HNT1 displayed genetic interactions. This analysis was initiated by generating yeast strain NKK1 in which a HIS3 selection cassette replaced amino acids 14 -146 (of 158) of HNT1 (Fig. 4A). The haploid disruptant strain proved to be viable and exhibited no apparent phenotype, indicating that HNT1 is not an essential gene. Subsequently, a double mutant haploid strain, NKK2, was generated
Analysis of the double mutant haploid indicated decreased colony formation compared with either parental strain at 30°C (Fig. 4B). Microscopic examination of the double mutant cells at 30°C revealed highly elongated cells forming clusters or filaments in which daughter cells had apparently remained attached to mother cells (Fig. 4C). This morphology was suppressed by ectopic expression of Hnt1 or Kin28 (Fig. 4C, lower  panels). Also, expression of the human Hint completely suppressed the phenotype (Fig. 4C, lower right panel) strongly suggesting complementation of HNT1 by HINT. The earlier observation that Hint and Hnt1 can form physical complexes is in agreement with this notion. Partial suppression was noted by expression of the Ccl1 cyclin, but expression of Tfb3/Rig2 did not rescue the morphology (data not shown).
These results provide genetic evidence to support the interaction between Kin28 and Hnt1. The morphology of the double mutant strain is somewhat reminiscent of diploid pseudohyphal growth (39), but the cells did not display invasive properties (data not shown). In addition, the morphology was more prominent when galactose was used as a carbon source, suggesting that the phenotype is related to metabolic stress and may involve improper control of the Ras/cyclic AMP pathway function (40). Further studies to investigate this possibility are under way.
The identification of physical and genetic interactions between the Cdk7 and Kin28 CTD kinases with the Hint and Hnt1 HIT proteins is interesting in the light of a recent report indicating that Hint associates with and negatively regulates the microphthalmia transcription factor mi (37). It raises the possibility that HIT proteins may bridge the interactions between basal transcription factors and transcription activators.