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The crystal structures of three protein kinases in various states of activity have recently been determined. Analysis of these structures is providing unprecedented insight into the precise atomic movements underlying protein kinase regulation.
We have isolated two murine cDNAs designated PCTAIRE-1 and -3 which encode putative serine/threonine-specific protein kinases. The predicted products of PCTAIRE-1 and -3 are 65% homologous and are organized into a core 295-residue kinase domain flanked by unique 161 and 117 amino acid N-terminal and 40 and 39 amino acid C-terminal domains respectively. The kinase domains are approximately 50-55% homologous to members of the cdc2/CDC28 kinase gene family, and each contains a cysteine-for-serine substitution within the conserved PSTAIRE motif. PCTAIRE-1 was ubiquitously expressed as a predominant 3.0-kb transcript and a minor 2.2-kb mRNA resulting from differential polyadenylation. In contrast, PCTAIRE-3 exhibited a more restricted pattern of expression with a single 3.0-kb mRNA detected in brain, kidney and intestine. The PCTAIRE-1 and -3 products produced by in vitro transcription-translation failed to bind to p13suc1 but were precipitated by antibodies directed to Schizosaccharomyces pombe p34cdc2 or to the human PSTAIRE motif. Thus, PCTAIRE-1 and -3 are members of a novel subfamily of cdc2/CDC28-related protein kinases.
The DAC2/FUS3 gene of Saccharomyces cerevisiae, which encodes a CDC28/cdc2-related protein kinase, is essential both for the arrest of cell division induced by mating pheromones and for cell fusion during conjugation. To elucidate the role of the DAC2 gene product in the pheromone response pathway, I determined the nucleotide sequence of the DAC2 gene and characterized two types of deletion mutants of the DAC2 gene. Here, I show that the DAC2 gene is identical to the FUS3 gene and that dac2/fus3 deletion mutants respond to mating pheromones by activating transcription. Therefore, the DAC2/FUS3 gene is not essential for transcriptional activation in the pheromone response pathway. The DAC2/FUS3 protein kinase has a positive role in cell fusion during sexual conjugation.
A cDNA clone, named R2, has been isolated by screening a rice cell cDNA library with a redundant oligonucleotide probe derived from the conserved ATP binding site of cdc2+/CDC28 protein kinases. The cDNA contained the entire coding sequence for a 424 amino acid polypeptide with a molecular mass of 47.6 kDa. The R2 mRNA, 2.1 kb in size, was expressed in both cultured rice cells and rice seedlings at similar levels. The predicted R2 protein has canonical motifs for ATP binding and catalysis, and is significantly homologous (up to 47%) to members of the cdc2+/CDC28 subfamily of serine/threonine protein kinase. The R2 protein is a novel member of the subfamily.
The E2F transcription factor has been found in association with the cyclin A protein, and this complex accumulates during the S phase of the cell cycle, suggesting that E2F may play a role in cell cycle control. In independent studies, cyclin A has been shown to be associated with two other proteins, the Rb-related p107 protein and the cdc2-related p33 cdk2 protein kinase. Through an analysis of the E2F-cyclin A complex, we now find that both the p107 protein and the cdc2-related p33cdk2 kinase are components of the previously described complex. Moreover, the complex possesses H1 kinase activity. These results thus define a cyclin A-cdk2 kinase complex that possesses sequence-specific DNA binding activity. This suggests that the cdk2 kinase may phosphorylate other DNA-bound substrates, and that one role of the E2F factor may be to localize this protein kinase to the DNA.
The transcription factor E2F controls the expression of several proliferation-related genes and is a target of the adenovirus E1A oncogene. In human cells, both cyclin A and the cdk2 protein kinase were found in complexes with E2F. Although the total amounts of cdk2 were constant in the cell cycle, binding to E2F was detected only when cells entered S phase, a time when the cdk2 kinase is activated. These data suggest that the interaction between cdk2 and E2F requires an active kinase that has cyclin A as a targeting component.
Numerous experiments have defined a critical role for the G1 cyclins and associated kinases in allowing a normal progression of cells from a quiescent state, through G1, and into S phase. We now demonstrate that G1 cyclin-dependent kinase activity is critical for the accumulation of E2F activity late in G1. Moreover, E2F-1 overexpression can overcome a G1 arrest caused by the inhibition of G1 cyclin-dependent kinase activity, consistent with E2F activation being an important consequence of the action of G1 cyclins. E2F-1 also overcomes a G1 block caused by gamma irradiation and leads to an apparent complete replication of the cellular genome and entry into mitosis. This E2F-1-mediated induction of S phase and mitosis is not accompanied by the rise in either cyclin D-associated kinase activity or cdk2 activity that is normally observed during the G1 phase of the cell cycle. We conclude that one key function for G1 cyclin-dependent kinase activity is the activation of E2F-1, that the accumulation of E2F activity may be sufficient to allow initiation and completion of S phase, but that additional events, including G1 cyclin kinase activity, are likely necessary for a normal proliferative event.
In budding yeast, Saccharomyces cerevisiae, the two principal cell cycle transitions, from G1 to S phase and from G2 to M phase, are controlled by the same protein from G2 to M phase, are controlled by the same protein kinase, CDC28, a homolog of the cdc2 protein kinase in fission yeast and other organisms. The G1 to S phase activity of the kinase is associated with accumulation of a novel family of G1 cyclins, distinct from cyclins that are required to activate the kinase for G2 to M phase functions. It remains to be determined whether G1 cyclins with similar functions exist in higher cells.
Cyclins are regulatory subunits which associate with kinases to form complexes that control many of the important steps in cell-cycle progression. The best characterized of the cyclin-containing complexes is the association of cyclin B with the p34cdc2 kinase. The p34cdc2/cyclin B complex is required for the G2 to M transition (see refs 1-4 for review), but the physiological role of other cyclin complexes is unclear. Human cyclin A binds independently to two kinases, associating with either p34cdc2 or a related protein, p33 (refs 5-7). In adenovirus-transformed cells, the viral E1A oncoprotein seems to associate with p33/cyclin A but not with p34cdc2/cyclin A (B. Faha, M.M., L-H.T. and E.H., manuscript submitted). To isolate the gene for p33, we have cloned several novel human cdc2-related genes. The protein product of one of these genes, cdk2 (cyclin-dependent kinase 2), shares 65% sequence identity with p34cdc2 (ref. 8) and 89% identity with the Xenopus Eg-1 gene product. Immunochemical characterization and partial proteolytic mapping show that the cdk2 gene product is the cyclin A-associated p33. Immunoprecipitations of the p33cdk2 protein suggest that it can act as a protein kinase in vitro. As p33cdk2 is bound to cyclin A and is targeted by the viral E1A protein, we suggest that the p33cdk2/cyclin A complex has a unique role in cell-cycle regulation of vertebrate cells.
Cyclin E is classified as a putative G1 cyclin on the basis of its cyclic pattern of mRNA expression, with maximal levels being detected near the G1/S boundary. We report here that cyclin E is found associated with the transcription factor E2F in a temporally regulated fashion. E2F is known to be a critical transcription factor for the expression of some S phase-specific proteins and is thought to be important for a series of others. Antisera specific for cyclin E were raised and used to demonstrate an association between cyclin E and E2F. This cyclin E/E2F complex was seen in a variety of human cell lines from various tissues, but its appearance was detected primarily during the G1 phase of the cell cycle. The cyclin E/E2F association decreased as cells entered S phase, just as the association of E2F with cyclin A became detectable. We characterized the cyclin E-E2F complex further to show that both the cyclin-dependent kinase-2 (cdk2) and p107 were present. Therefore, the p107/E2F complex is associated with two different cdk2 kinase complexes--one containing cyclin A and the other containing cyclin E--and the appearance of these complexes is temporally regulated during the cell cycle. The presence of cyclin E/E2F complexes in the G1 phase suggests a role for cyclin E in the control of genes required for the G1-to-S transition.
Human cyclin E, originally identified on the basis of its ability to function as a G1 cyclin in budding yeast, associated with a cell cycle-regulated protein kinase in human cells. The cyclin E-associated kinase activity peaked during G1, before the appearance of cyclin A, and was diminished during exit from the cell cycle after differentiation or serum withdrawal. The major cyclin E-associated kinase in human cells was Cdk2 (cyclin-dependent kinase 2). The abundance of the cyclin E protein and the cyclin E-Cdk2 complex was maximal in G1 cells. These results provide further evidence that in all eukaryotes assembly of a cyclin-Cdk complex is an important step in the biochemical pathway that controls cell proliferation during G1.
Progression through the cell cycle is monitored at two major points: during the G1/S and the G2/M transitions. In most cells, the G2/M transition is regulated by the timing of p34cdc2 dephosphorylation which results in the activation of the kinase activity of the cdc2-cyclin B complex. The timing of p34cdc2 dephosphorylation is determined by the balance between the activity of the kinase that phosphorylates p34cdc2 (wee1 in human cells) and the opposing phosphatase (cdc25C). Both enzymes are regulated and it has been shown that cdc25C is phosphorylated and activated by the cdc2-cyclin B complex. This creates a positive feed-back loop providing a switch used to control the onset of mitosis. Here, we show that another member of the human cdc25 family, cdc25A, undergoes phosphorylation during S phase, resulting in an increase of its phosphatase activity. The phosphorylation of cdc25A is dependent on the activity of the cdc2-cyclin E kinase. Microinjection of anti-cdc25A antibodies into G1 cells blocks entry into S phase. These results indicate that the cdc25A phosphatase is required to enter S phase in human cells and suggest that this enzyme is part of an auto-amplification loop analogous to that described at the G2/M transition. We discuss the nature of the in vivo substrate of the cdc25A phosphatase in S phase and the possible implications for the regulation of S phase entry.
The retinoblastoma gene product (the RB protein) is phosphorylated in a cell cycle-dependent manner and this modification is believed to be important for cells to progress through the cell cycle. We found that purified cdk2 (cyclin-dependent kinase/cell division kinase 2) can phosphorylate the RB protein in vitro at the sites phosphorylated in the cell. The timing of activation of cdk2 in the cell cycle was similar to that of the onset of phosphorylation of the RB protein. The kinase coprecipitated with the RB protein also exhibited a similar substrate specificity to cdk2 and a similar time course of activation during the cell cycle. We further showed that cdk2 formed a complex with the RB protein in vitro and that its formation was not competitively inhibited by the simian virus 40 large T antigen. These observations suggest that cdk2 or a cdk2-related protein is involved in the cell cycle-dependent phosphorylation of the RB protein.
Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone H1 kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.
Interferons (IFN) regulate transcription of certain genes playing a role in cell proliferation. Targets of IFN action may include tumor suppressor genes such as the retinoblastoma (RB) gene and cytokines such as transforming growth factor beta 1 (TGF beta 1) and IFN beta which are inhibitors of epithelial cell proliferation. Using reverse transcription followed by PCR amplification, an increase of those growth inhibitory gene mRNA levels (TGF beta 1, IFN beta and RB) were found after interferon treatment in condylomas harboring non-oncogenic human papilloma virus (HPV 6/11) types, in an oncogenic HPV 16-containing cell line, and in a HPV negative, epidermoid carcinoma cell line. In addition, immunodetection by Western blot demonstrated a higher proportion of underphosphorylated (active form) retinoblastoma gene protein (pRB) after IFN treatment due to the decrease in the phosphorylating cdc2 kinase levels. Changes in the phosphorylation pattern of pRB together with the increased expression of those inhibitory genes represent a growth inhibited state in those cells as demonstrated by diminished c-myc expression. Since the extent of c-myc inhibition was significantly lower in the case of oncogenic HPV infection, a role of viral oncoproteins in abrogation of the antiproliferative effect of IFN therapy could be considered. These results demonstrate a new mechanism via which IFNs exert their antiproliferative effect on HPV-infected cells by affecting the expression and phosphorylation of the RB tumor suppressor gene, through the inhibitory TGF beta 1/IFN beta cytokine pathway.
Cyclin-dependent kinase 2 (Cdk2), when bound to either cyclin A or cyclin E, recognizes the Ser/Thr-Pro-X-basic amino acid (motif A) as a phosphorylation site. In this study, we designed several peptides based on motif A and examined the substrate specificity of Cdk2-cyclin A and Cdk2-cyclin E using these peptides. Peptides containing a proline residue in the sequence Pro-X-Thr-Pro-X-basic amino acid (motif B) had higher affinity for both Cdk2 complexes than peptides containing motif A. Furthermore, differences in substrate affinity between the two Cdk2 complexes were caused by a proline residue adjacent to or three positions before the threonine residue. Similarly, the presence of different basic amino acids in motif B also had different effects on affinity for each complex. We demonstrate the possibility that the substrate specificity of Cdk2 bound to cyclin might be regulated by the species of cyclin.
Peripheral blood T lymphocytes require two sequential mitogenic signals to reenter the cell cycle from their natural, quiescent state. One signal is provided by stimulation of the T-cell antigen receptor, and this induces the synthesis of both cyclins and cyclin-dependent kinases (CDKs) that are necessary for progression through G1. Antigen receptor stimulation alone, however, is insufficient to promote activation of G1 cyclin-Cdk2 complexes. This is because quiescent lymphocytes contain an inhibitor of Cdk2 that binds directly to this kinase and prevents its activation by cyclins. The second mitogenic signal, which can be provided by the cytokine interleukin 2, leads to inactivation of this inhibitor, thereby allowing Cdk2 activation and progression into S phase. Enrichment of the Cdk2 inhibitor from G1 lymphocytes by cyclin-CDK affinity chromatography indicates that it may be p27Kip1. These observations show how sequentially acting mitogenic signals can combine to promote activation of cell cycle proteins and thereby cause cell proliferation to start. CDK inhibitors have been shown previously to be induced by signals that negatively regulate cell proliferation. Our new observations show that similar proteins are down-regulated by positively acting signals, such as interleukin 2. This finding suggests that both positive and negative growth signals converge on common targets which are regulators of G1 cyclin-CDK complexes. Inactivation of G1 cyclin-CDK inhibitors by mitogenic growth factors may be one biochemical pathway underlying cell cycle commitment at the restriction point in G1.
Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(delta 2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties.