Abstract. The activity of cyclin-dependent kinases (cdl~) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34 ~c2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating _ki-nase), and it was shown to contain p40 M°15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40 M°15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40 M°tS, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coll. These tools were then used to demonstrate that p40 M°~5 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40 u°15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40 u°15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34 ~c2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).

The human Nek2 protein kinase is the closest known mammalian relative of the mitotic regulator NIMA of Aspergillus nidulans. The two kinases share 47% sequence identity over their catalytic domains and display a similar cell cycle-dependent expression peaking at the G2 to M phase transition. Hence, it is attractive to speculate that human Nek2 and fungal NIMA may carry out similar functions at the onset of mitosis. To study the biochemical properties and substrate specificity of human Nek2 and compare them to those reported previously for other NIMA-related protein kinases, we have expressed Nek2 in insect cells. We show that recombinant Nek2 is active as a serine/threonine-specific protein kinase and may undergo autophosphorylation. Both human Nek2 and fungal NIMA phosphorylate a similar, albeit not identical, set of proteins and synthetic peptides, and beta-casein was found to be a suitable substrate for assaying Nek2 in vitro. By exploiting these findings, we have studied the cell cycle regulation of Nek2 activity in HeLa cells. We show that Nek2 activity parallels its abundance, being low during M and G1 but high during S and G2 phase. Taken together, our results suggest that human Nek2 resembles fungal NIMA in its primary structure, cell cycle regulation of expression, and substrate specificity, but that Nek2 may function earlier in the cell cycle than NIMA.

This review compares the well-studied RNase H activities of human immunodeficiency virus, type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) reverse transcriptases. The RNase H domains of HIV-1 and MoMLV are structurally very similar, with functions assigned to conserved subregions like the RNase H primer grip and the connection subdomain, as well as to distinct features like the C-helix and loop in MoMLV RNase H. Like cellular RNases H, catalysis by the retroviral enzymes appears to involve a two-metal ion mechanism. Unlike cellular RNases H, the retroviral RNases H display three different modes of cleavage: internal, DNA 3′ end-directed, and RNA 5′ enddirected. All three modes of cleavage appear to have roles in reverse transcription. Nucleotide sequence is an important determinant of cleavage specificity with both enzymes exhibiting a preference for specific nucleotides at discrete positions flanking an internal cleavage site as well as during tRNA primer removal and plus-strand primer generation. RNA 5′ end-directed and DNA 3′ end-directed cleavages show similar sequence preferences at the positions closest to a cleavage site. A model for how RNase H selects cleavage sites is presented that incorporates both sequence preferences and the concept of a defined window for allowable cleavage from a recessed end. Finally, the RNase H activity of HIV-1 is considered as a target for anti-virals as well as a participant in drug resistance. KeywordsRNase H; reverse transcriptase; human immunodeficiency virus; type 1 (HIV -1); Moloney murine leukemia virus (MoMLV); reverse transcription; polypurine tract (PPT)

Metazoan cyclin C was originally isolated by virtue of its ability to rescue Saccharomyces cerevisiae cells deficient in G1 cyclin function. This suggested that cyclin C might play a role in cell cycle control, but progress toward understanding the function of this cyclin has been hampered by the lack of information on a potential kinase partner. Here we report the identification of a human protein kinase, K35 [cyclin-dependent kinase 8 (CDK8)], that is likely to be a physiological partner of cyclin C. A specific interaction between K35 and cyclin C could be demonstrated after translation of CDKs and cyclins in vitro. Furthermore, cyclin C could be detected in K35 immunoprecipitates prepared from HeLa cells, indicating that the two proteins form a complex also in vivo. The K35-cyclin C complex is structurally related to SRB10-SRB11, a CDK-cyclin pair recently shown to be part of the RNA polymerase II holoenzyme of S. cerevisiae.Hence, we propose that human K35(CDK8)-cyclin C might be functionally associated with the mammalian transcription apparatus, perhaps involved in relaying growth-regulatory signals.Complexes between cyclin-dependent kinases (CDKs) and cyclin regulatory subunits play a pivotal role in cell cycle regulation in all eukaryotes (1, 2). In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, cell cycle progression is controlled predominantly by a single CDK, termed p34CDC28 and p34cdc2, respectively (3-5). In metazoans, however, different cell cycle transitions require distinct CDKs (6-8). Cyclins are positive regulatory subunits of CDKs and constitute multiprotein families in yeasts and metazoans (9,10). Although the importance of many CDKs and cyclins in promoting the transitions between successive stages of the cell cycle is well established, it should not be assumed that all CDK-cyclin complexes function directly or exclusively in cell cycle control. This is illustrated best by studies on S. cerevisiae, where at least three distinct CDK-cyclin pairs, PHO85-PHO80, KIN28-CCL1, and SRB10-SRB11, have been implicated in the regulation of transcriptional events (refs. 11-13; for review, see ref. 14). PHO85 also functions in association with two additional cyclins, PCL1 and PCL2 (also termed HCS26 and ORF-D, respectively), possibly to integrate cell cycle progression with the availability of nutrients (15,16). In vertebrates, six of seven presently known CDKs have been implicated in cell cycle progression, but CDK5 may function primarily as a neurofilament kinase in postmitotic neurons (17)(18)(19)(20). Furthermore, the precise physiological role of the CDK7-cyclin H complex remains to be understood. Originally identified as a CDK-activating kinase (21-23), the CDK7-cyclin H complex was shown to form part of the general transcription factor TFIIH and to display kinase activity toward the C-terminal domain of RNA polymerase II (24-26), suggesting that it may also play a role in transcription and/or DNA repair.There is no doubt that additional vertebrate CDKs and cyclins aw...

At the time of its discovery in 1970, the presence of an RNA-dependent DNA polymerase activity in retrovirus particles provided strong and exciting support for the hypothesis that the single-stranded RNA genome of a retrovirus is replicated through a DNA intermediate [1,2]. Not only did this discovery of reverse transcriptase (as it was dubbed) challenge the existing dogma concerning the flow of genetic information in biology, it raised the critical question as to how the DNA ⁄ RNA hybrid created when the viral genome RNA is used as a template by reverse transcriptase is further processed. In retrospect, it is not surprising that an RNase H activity that degrades the RNA strand of a DNA ⁄ RNA hybrid is required to free the newly made DNA strand (called the minus strand because it is complementary to the plus genome RNA) for use as a template in the synthesis of the second or plus strand DNA. However, it was a surprise when the retroviral-specific RNase H activity turned out to be present in the same protein molecule as the polymerase activity [3]. This intimate association of the DNA polymerase and RNase H activities in reverse transcriptase has profound effects on the activities and capabilities of both enzymes.This minireview provides a summary of the salient features of retroviral RNases H with a focus on how the shared substrate-binding sites for the two activities of reverse transcriptase endow the retroviral RNases H with features not found in the cellular counterparts, and how these unusual properties are crucial for the multiple roles played by RNase H in reverse transcription. Although occasional reference is made to other retroviral enzymes, the primary focus is on the wellstudied RNase H activities associated with human Retroviral reverse transcriptases possess both a DNA polymerase and an RNase H activity. The linkage with the DNA polymerase activity endows the retroviral RNases H with unique properties not found in the cellular counterparts. In addition to the typical endonuclease activity on a DNA ⁄ RNA hybrid, cleavage by the retroviral enzymes is also directed by both DNA 3¢ recessed and RNA 5¢ recessed ends, and by certain nucleotide sequence preferences in the vicinity of the cleavage site. This spectrum of specificities enables retroviral RNases H to carry out a series of cleavage reactions during reverse transcription that degrade the viral RNA genome after minus-strand synthesis, precisely generate the primer for the initiation of plus strands, facilitate the initiation of plus-strand synthesis and remove both plus-and minus-strand primers after they have been extended.