Differences in the Conformational Energy Landscape of CDK1 and CDK2 Suggest a Mechanism for Achieving Selective CDK Inhibition.

Dysregulation of the cell cycle characterizes many cancer subtypes, providing a rationale for developing cyclin-dependent kinase (CDK) inhibitors. Potent CDK2 inhibitors might target certain cancers in which CCNE1 is amplified. However, current CDK2 inhibitors also inhibit CDK1, generating a toxicity liability. We have used biophysical measurements and X-ray crystallography to investigate the ATP-competitive inhibitor binding properties of cyclin-free and cyclin-bound CDK1 and CDK2. We show that these kinases can readily be distinguished by such inhibitors when cyclin-free, but not when cyclin-bound. The basis for this discrimination is unclear from either inspection or molecular dynamics simulation of ligand-bound CDKs, but is reflected in the contacts made between the kinase N- and C-lobes. We conclude that there is a subtle but profound difference between the conformational energy landscapes of cyclin-free CDK1 and CDK2. The unusual properties of CDK1 might be exploited to differentiate CDK1 from other CDKs in future cancer therapeutic design.

Combining the Optimized Yeast Cytosine Deaminase Protein Fragment Complementation Assay and an In Vitro Cdk1 Targeting Assay to Study the Regulation of the γ-Tubulin Complex.

Cdk1 is the essential cyclin-dependent kinase in the budding yeast Saccharomyces cerevisiae. Cdk1 orchestrates cell cycle control by phosphorylating target proteins with extraordinary temporal and spatial specificity by complexing with one of the nine cyclin regulatory subunits. The identification of the cyclin required for targeting Cdk1 to a substrate can help to place the regulation of that protein at a specific time point during the cell cycle and reveal information needed to elucidate the biological significance of the regulation. Here, we describe a combination of strategies to identify interaction partners of Cdk1, and associate these complexes to the appropriate cyclins using a cell-based protein-fragment complementation assay. Validation of the specific reliance of the OyCD interaction between Cdk1 and budding yeast γ-tubulin on the Clb3 cyclin, relative to the mitotic Clb2 cyclin, was performed by an in vitro kinase assay using the γ-tubulin complex as a substrate.

Expression and Purification of Recombinant Cyclins and CDKs for Activity Evaluation.

Cyclin-dependent kinases (Cdks) belong to a family of key regulators of cell division cycle and transcription. Their activity is mainly regulated by association with regulatory subunits named cyclins but their activities are also regulated by phosphorylation, acetylation, and the association with specific inhibitory proteins (CKIs). The activity of different Cdks is deregulated in many different type of tumors, and thus, Cdks are considered targets for antitumoral therapy. For large screenings of inhibitors the use of purified recombinant Cdks and cyclins is recommended. We report here the current methods to determine their in vitro activity for large screenings of inhibitors.

Human immunodeficiency virus type 1 Vpr binds to the N lobe of the Wee1 kinase domain and enhances kinase activity for CDC2.

Human immunodeficiency virus type 1 Vpr is a virion-associated accessory protein that has multiple activities within an infected cell. One of the most dramatic effects of Vpr is the induction of cell cycle arrest at the G(2)/M boundary, followed by apoptosis. This effect has implications for CD4(+) cell loss in AIDS. In normal cell cycle regulation, Wee1, a key regulator for G(2)-M progression, phosphorylates Tyr15 on Cdc2 and thereby blocks the progression of cells into M phase. We demonstrate that Vpr physically interacts with Wee1 at the N lobe of the kinase domain analogous to that present in other kinases. This interaction with Vpr enhances Wee1 kinase activity for Cdc2. Overexpression of Wee1 kinase-deficient mutants competes for Vpr-mediated cell cycle arrest, and deletion of the region of Wee1 that binds Vpr abrogates that competition. However, the Vpr mutants I74P and I81P, which fail to induce G(2) arrest, can bind to and increase the kinase activity of Wee1 to the same extent as wild-type Vpr. Therefore, we conclude that the binding of Vpr to Wee1 is not sufficient for Vpr to activate the G(2) checkpoint, and it may reflect an independent function of Vpr.

cdc2-cyclin B regulates eEF2 kinase activity in a cell cycle- and amino acid-dependent manner.

The calcium/calmodulin-dependent kinase that phosphorylates and inactivates eukaryotic elongation factor 2 (eEF2 kinase; eEF2K) is subject to multisite phosphorylation, which regulates its activity. Phosphorylation at Ser359 inhibits eEF2K activity even at high calcium concentrations. To identify the kinase that phosphorylates Ser359 in eEF2K, we developed an extensive purification protocol. Tryptic mass fingerprint analysis identified it as cdc2 (cyclin-dependent kinase 1). cdc2 co-purifies with Ser359 kinase activity and cdc2-cyclin B complexes phosphorylate eEF2K at Ser359. We demonstrate that cdc2 contributes to controlling eEF2 phosphorylation in cells. cdc2 is activated early in mitosis. Kinase activity against Ser359 in eEF2K also peaks at this stage of the cell cycle and eEF2 phosphorylation is low in mitotic cells. Inactivation of eEF2K by cdc2 may serve to keep eEF2 active during mitosis (where calcium levels rise) and thereby permit protein synthesis to proceed in mitotic cells. Amino-acid starvation decreases cdc2's activity against eEF2K, whereas loss of TSC2 (a negative regulator of mammalian target of rapamycin complex 1(mTORC1)) increases it. These data closely match the control of Ser359 phosphorylation and indicate that cdc2 may be regulated by mTORC1.

How tyrosine 15 phosphorylation inhibits the activity of cyclin-dependent kinase 2-cyclin A.

Inhibition of cyclin-dependent kinase 1 (CDK1) activity by Tyr-15 phosphorylation directly regulates entry into mitosis and is an important element in the control of the unperturbed cell cycle. Active site phosphorylation of other members of the CDK family that regulate cell cycle progression instates checkpoints that are fundamental to eukaryotic cell cycle regulation. Kinetic and crystallographic analyses of CDK2-cyclin A complexes reveal that this inhibitory mechanism operates through steric blockade of peptide substrate binding and through the creation of an environment that favors a non-productive conformation of the terminal group of ATP. By contrast, tyrosine phosphorylation of CDK2 alters neither its Km for ATP nor its significant intrinsic ATPase activity. Tyr-15-phosphorylated CDK2 retains trace protein phosphorylation activity that should be considered in quantitative and qualitative cell cycle models.

Measurement of Wee kinase activity.

The Wee kinases (Wee1, Wee2, and Myt1) are major regulators of mitotic entry. They function by phosphorylating Cdc2 and related Cdks on conserved tyrosine and threonine residues. This phosphorylation blocks the activity of the Cdc2 and prevents entry into mitosis. The abundance and activity of the Wee kinases are regulated during the cell cycle and development. In this chapter, we describe several procedures to measure the activity of the Wee kinases found either in crude extracts or in purified preparations. Specific protocols include the production and purification of recombinant Cdc2/Cyclin B substrate, the production of crude subcellular extract fractions, the purification of endogenous or recombinant Wee kinases, Wee kinase assays, and the Histone H1 kinase assay to measure Cdc2 activity. In addition, support protocols are provided that describe the use and production of Ni-IDA beads for the purification of Histidine-tagged proteins, and the use of the baculovirus expression system to produce recombinant proteins.

Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe.

We describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Schizosaccharomyces pombe. The tagging cassettes are designed for either carboxy- or amino-terminal tagging of proteins. The carboxyl terminal tags differ in that they contain either two or four repeats of IgG binding units. For tagging endogenous loci, the cassettes contain the kan MX6 module to allow for selection of G418-resistant cells. The amino-terminal tagging vectors allow for the regulated expression of proteins. Sz. pombe Cdc2p was chosen to test these new affinity tags. Several known binding proteins co-purified with both Cdc2p-CTAP and N-TAP-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz. pombe.

Acquisition of meiotic competence in mouse oocytes: absolute amounts of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent and competent oocytes.

M-Phase promoting factor (MPF) is a complex of p34(cdc2) and cyclin B. Results of previous studies in which relative mass amounts of these cell cycle regulators were determined suggested that the accumulation of p34(cdc2), rather than cyclin B, could be a limiting factor in the acquisition of meiotic competence in mouse oocytes. Nevertheless, in the absence of measurements of the absolute amount of these components of MPF, it is possible that the molar amount of p34(cdc2) is in excess to that of cyclin B, i.e., the accumulation of p34(cdc2) is not a limiting factor. We report measurements of the absolute mass of p34(cdc2) and cyclin B1, as well as the two proximal regulators of MPF, namely cdc25C and wee1, in meiotically incompetent and competent mouse oocytes. We find that the numbers of molecules of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent oocytes are 1.4 x 10(6), 11.3 x 10(6), 24.6 x 10(6), 15. 6 x 10(6), respectively, and in meiotically competent oocytes the numbers are 14.3 x 10(6), 95.5 x 10(6), 80.0 x 10(6), 40.1 x 10(6), respectively. Thus, the concentration of cyclin B1 is always in excess to that of p34(cdc2), and this is consistent with the hypothesis that the accumulation of p34(cdc2) plays a role in the acquisition of meiotic competence. Last, the concentration of cdc25C is greater than that of wee1 and the concentration of each is greater than that of p34(cdc2) in both meiotically incompetent and competent oocytes.

The Xenopus XMAP215 and its human homologue TOG proteins interact with cyclin B1 to target p34cdc2 to microtubules during mitosis.

Cytoskeleton reorganization, leading to mitotic spindle formation, is an M-phase-specific event and is controlled by maturation promoting factor (MPF: p34cdc2-cyclinB1 complex). It has previously been demonstrated that the p34cdc2-cyclin B complex associates with mitotic spindle microtubules and that microtubule-associated proteins (MAPs), in particular MAP4, might be responsible for this interaction. In this study, we report that another ubiquitous MAP, TOG in human and its homologue in Xenopus XMAP215, associates also with p34cdc2 kinase and directs it to the microtubule cytoskeleton. Costaining of Xenopus cells with anti-TOGp and anti-cyclin B1 antibodies demonstrated colocalization in interphase cells and also with microtubules throughout the cell cycle. Cyclin B1, TOG/XMAP215, and p34cdc2 proteins were recovered in microtubule pellets isolated from Xenopus egg extracts and were eluted with the same ionic strength. Cosedimentation of cyclin B1 with in vitro polymerized microtubules was detected only in the presence of purified TOG protein. Using a recombinant C-terminal TOG fragment containing a Pro-rich region, we showed that this domain is sufficient to mediate cosedimentation of cyclin B1 with microtubules. Finally, we demonstrated interaction between TOG/XMAP215 and cyclin B1 by co-immunoprecipitation assays. As XMAP215 was shown to be the only identified assembly promoting MAP which increases the rapid turnover of microtubules, the TOG/XMAP215-cyclin B1 interaction may be important for regulation of microtubule dynamics at mitosis.

Synthesis of C2 alkynylated purines, a new family of potent inhibitors of cyclin-dependent kinases.

The synthesis of a new family of inhibitors of the cell cycle regulating cyclin-dependent kinases (CDK's) is reported. These compounds, related to the purines olomoucine and roscovitine, are characterised by the presence of alkynylated side chains at C2. They inhibit CDK's with IC50's in the 200 nM range.

Phosphorylation by a cyclin-dependent kinase modulates DNA binding of the Arabidopsis heat-shock transcription factor HSF1 in vitro.

Phosphorylation is one of the mechanisms controlling the activity of heat-shock transcription factors in yeast and mammalian cells. Here we describe partial purification, identification, and characterization of a protein kinase that phosphorylates the Arabidopsis heat-shock factor AtHSF1 at multiple serine residues. The HSF1 kinase forms a stable complex with AtHSF1, which can be detected by kinase pull-down assays using a histidine-tagged AtHSF1 substrate. The HSF1 kinase interacts with the cell-cycle control protein Suc1p and is immunoprecipitated by an antibody specific for the Arabidopsis cyclin-dependent CDC2a kinase. Phosphorylation by CDC2a in vitro inhibits DNA binding of AtHSF1 to the cognate heat-shock elements, suggesting a possible regulatory interaction between heat-shock response and cell-cycle control in plants.

A novel cdc2-related protein kinase expressed in the nervous system.

We report the cloning and characterization of a cDNA encoding a cdc2-related protein kinase, named PFTAIRE, that is expressed primarily in the postnatal and adult nervous system. We have demonstrated by in situ hybridization and indirect immunofluorescence that several populations of terminally differentiated neurons and some neuroglia expressed PFTAIRE mRNA and protein. In neurons, PFTAIRE protein was localized in the nucleus and cytoplasm of cell bodies. The anatomical, cellular, and ontogenic patterns of PFTAIRE expression in the nervous system differed from those of p34cdc2 and cdk5, which are expressed in brain and several other mitotic tissues. Proteins of approximately 58-60 kDa coprecipitated specifically with PFTAIRE from cytosolic protein preparations of adult mouse brain and transfected cells. These proteins appeared to be the major endogenous substrates associated with this kinase activity. The temporal and spatial expression patterns of PFTAIRE in the postnatal and adult nervous system suggest that PFTAIRE kinase activity may be associated with the postmitotic and differentiated state of cells in the nervous system and that its function may be distinct from those of p34cdc2 and cdk5.

Interaction of the S phase regulator cdc18 with cyclin-dependent kinase in fission yeast.

The fission yeast gene cdc18(+) is required for entry into S phase and for coupling mitosis to the successful completion of S phase. Cdc18 is a highly unstable protein that is expressed only once per cell cycle at the G1/S boundary. Overexpression of Cdc18 causes a mitotic delay and reinitiation of DNA replication, suggesting that the inactivation of Cdc18 plays a role in preventing rereplication within a given cell cycle. In this paper, we present evidence that Cdc18 is associated with active cyclin-dependent kinase in vivo. We have expressed Cdc18 as a glutathione S-transferase fusion in fission yeast and demonstrated that the fusion protein is functional in vivo. We find that the Cdc18 fusion protein copurifies with a kinase activity capable of phosphorylating histone H1 and Cdc18. The activity was identified by a variety of methods as the cyclin-dependent kinase containing the product of the cdc2(+) gene. The amino terminus of Cdc18 is required for association with cyclin-dependent kinase, but the association does not require the consensus cyclin-dependent kinase phosphorylation sites in this region. Additionally, both G1/S and mitotic forms of cyclin-dependent kinase phosphorylate and interact with Cdc18. These interactions between Cdc18 and cyclin-dependent kinases suggest mechanisms by which cyclin-dependent kinases could activate the initiation of DNA replication and could prevent rereplication.

14-3-3 zeta protein binds to the carboxyl half of mouse wee1 kinase.

To identify proteins which bind to mouse wee1 kinase, the yeast "two-hybrid" system was used with a mouse cDNA library. Using the carboxyl half of weel kinase, the 14-3-3 zeta protein was isolated. Recombinant 14-3-3 zeta was demonstrated to bind to wee1 kinase in vitro. The wee1 kinase phosphorylated by cdc2 kinase also bound to 14-3-3 zeta protein. When both wee1 kinase and 14-3-3 zeta were transfected into COS-1 cells, they formed a complex in a cell. The sequence of wee1 kinase necessary for the binding was tested by a two hybrid system expressing different lengths of peptides derived from wee1 kinase. Both the entire kinase domain and a sequence in the carboxyl terminus was thought to be necessary for the binding. The function of 14-3-3 zeta protein remained to be elucidated in relation to the regulation of G2 to M phase transition through wee1 kinase.

The protein-tyrosine kinase Lck associates with and is phosphorylated by Cdc2.

The protein-tyrosine kinase Lck is essential for signaling through the T-cell antigen receptor. Treatment of T-cells with a variety of extracellular stimuli increases the phosphorylation of Lck on serine residues. This results in shifts in the apparent molecular weight of Lck to forms that exhibit reduced electrophoretic mobility on SDS-polyacrylamide gels. We found that as a result of arresting cells in mitosis, forms of Lck were generated that migrated with slower mobilities on SDS-polyacrylamide gels. This suggested that a serine/threonine kinase, active at mitosis, was phosphorylating Lck. Using antibodies to Lck and to the cyclin-dependent serine kinase, Cdc2, as well as the cyclin-dependent kinase affinity resin, Suc1-agarose, we detected a stable interaction between Lck and Cdc2. The interaction was mediated through the Src homology 3 domain of Lck and was selective, as only the active form of Cdc2 was found to associate with Lck. Moreover, Cdc2 was able to phosphorylate Lck in vitro and shift its electrophoretic mobility to a more slowly migrating form. An association between active Cdc2 and the Src-related kinases Lyn and Fyn was also demonstrated, although Cdc2 was not found associated with the tyrosine kinases, Csk and Syk. These results demonstrate that at mitosis, Cdc2 associates with and phosphorylates Lck.

A predictive scale for evaluating cyclin-dependent kinase substrates. A comparison of p34cdc2 and p33cdk2.

Protein phosphorylation by members of the Cdk (cyclin-dependent kinase) family of protein kinases is necessary for progression through the cell cycle. However, the primary sequence determinants of Cdk substrate specificity have yet to be examined quantitatively. We have used a panel of glutathione S-transferase peptide fusions to investigate the fine-structure specificity of p33(cdk2) and p34(cdc2). Our data indicate that the generally held consensus sequences for p34(cdc2) represent a significant oversimplification of its true specificity and that this specificity is conserved between species. p33(cdk2) and p34(cdc2) have similar but distinct substrate specificities that are affected modestly by the associated cyclin subunit. We derive specific values of phosphorylation efficiencies by these enzymes that can be used to estimate the phosphorylation potential of proposed Cdk substrates.

A cdc2-like kinase distinct from cdk5 is associated with neurofilaments.

An immunoprecipitation assay was used to identify protein kinases which are physically associated with neurofilaments (NF) in mouse brain extracts. Using this approach, we show that a cdc2-related kinase is associated with NF. The cdc2-related kinase was found to be distinct from cdk5 and the authentic cdc2 by a number of criteria. Firstly, it has a molecular mass on SDS-PAGE gels of 34 kDa, similar to that of cdc2, but differing from cdk5 (31 kDa). Secondly, it is not recognized by an antibody specific for cdk5. Thirdly, it is recognized by an antibody raised against the C-terminal region of authentic cdc2, but not by an antibody specific for the PSTAIRE motif. Using immunoblotting, we further show that the cdc2-related kinase copurifies with NF isolated from rat tissues. In vitro kinase assays further demonstrated that immunoprecipitated cdc2-related kinase phosphorylates recombinant NF-H protein. Phosphorylation of NF-H by the cdc2-like activity was not affected by 3 microM olomoucine but was inhibited by 10 microM of this kinase inhibitor. Phosphoamino acid analysis of in vitro phosphorylated NF-H indicates that the immunoprecipitated cdc2-related kinase phosphorylates serine residues.