Regulatory phosphorylation of cyclin-dependent kinase 2: insights from molecular dynamics simulations

The structures of fully active cyclin-dependent kinase-2 (CDK2) complexed with ATP and peptide substrate, CDK2 after the catalytic reaction, and CDK2 inhibited by phosphorylation at Thr14/Tyr15 were studied using molecular dynamics (MD) simulations. The structural details of the CDK2 catalytic site and CDK2 substrate binding box were described. Comparison of MD simulations of inhibited complexes of CDK2 was used to help understand the role of inhibitory phosphorylation at Thr14/Tyr15. Phosphorylation at Thr14/Tyr15 causes ATP misalignment for the phosphate-group transfer, changes in the Mg²⁺ coordination sphere, and changes in the H-bond network formed by CDK2 catalytic residues (Asp127, Lys129, Asn132). The inhibitory phosphorylation causes the G-loop to shift from the ATP binding site, which leads to opening of the CDK2 substrate binding box, thus probably weakening substrate binding. All these effects explain the decrease in kinase activity observed after inhibitory phosphorylation at Thr14/Tyr15 in the G-loop. Interaction of the peptide substrate, and the phosphorylated peptide product, with CDK2 was also studied and compared. These results broaden hypotheses drawn from our previous MD studies as to why a basic residue (Arg/Lys) is preferred at the P₊₂ substrate position. Figure View of the substrate binding site of the fully active cyclin-dependent kinase-2 (CDK2) (pT160-CDK2/cyclin A/ATP). The pThr160 activation site is located in the T-loop (yellow secondary structure). The G-loop, which partly forms the ATP binding site, is shown in blue. The Thr14 and Tyr15 inhibitory phosphorylation sites located in the G-loop are shown in licorice representation     

The mechanism of inhibition of the cyclin-dependent kinase-2 as revealed by the molecular dynamics study on the complex CDK2 with the peptide substrate HHASPRK

Molecular dynamics (MD) simulations were used to explain structural details of cyclin-dependent kinase-2 (CDK2) inhibition by phosphorylation at T14 and/or Y15 located in the glycine-rich loop (G-loop). Ten-nanosecond-long simulations of fully active CDK2 in a complex with a short peptide (HHASPRK) substrate and of CDK2 inhibited by phosphorylation of T14 and/or Y15 were produced. The inhibitory phosphorylations at T14 and/or Y15 show namely an ATP misalignment and a G-loop shift (~5 A) causing the opening of the substrate binding box. The biological functions of the G-loop and GxGxxG motif evolutionary conservation in protein kinases are discussed. The position of the ATP gamma-phosphate relative to the phosphorylation site (S/T) of the peptide substrate in the active CDK2 is described and compared with inhibited forms of CDK2. The MD results clearly provide an explanation previously not known as to why a basic residue (R/K) is preferred at the P(2) position in phosphorylated S/T peptide substrates.     

Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15.

We have examined the role of phosphorylation in the regulation of human cyclin-dependent kinase-2 (CDK2), a protein closely related to the cell cycle regulatory kinase CDC2. We find that CDK2 from HeLa cells contains three major tryptic phosphopeptides. Analysis of site-directed mutant proteins, expressed by transient transfection of COS cells, demonstrates that the two major phosphorylation sites are Tyr15 (Y15) and Thr160 (T160). Additional phosphorylation probably occurs on Thr14 (T14). Replacement of T160 with alanine abolishes the kinase activity of CDK2, indicating that phosphorylation at this site (as in CDC2) is required for kinase activity. Mutation of Y15 and T14 stimulates kinase activity, demonstrating that phosphorylation at these sites (as in CDC2) is inhibitory. Similarly, CDK2 is activated in vitro by dephosphorylation of Y15 and T14 by the phosphatase CDC25. Analysis of HeLa cells synchronized at various cell cycle stages indicates that CDK2 phosphorylation on T160 increases during S phase and G2, when CDK2 is most active. Phosphorylation on the inhibitory sites T14 and Y15 is also maximal during S phase and G2. Thus, the activity of a subpopulation of CDK2 molecules is inhibited at a time in the cell cycle when overall CDK2 activity is increased.     

Molecular dynamics simulation of the substitution of serine for the conserved glycine in the G-loop in the cdc28-srm yeast mutant using the crystal lattice of human CDK2 kinase

Two-nanosecond molecular dynamics simulations of the crystal lattice of an active complex of pT160-CDK2 kinase/cyclin A/ATP-Mg²⁺/substrate were performed. The simulations showed that the structures of the wild-type CDK2 complex and the mutant CDK2 complex involving the substitution G16S-CDK2 corresponding to the yeast substitution G20S-CDC28 differ noticeably and the differences between the structural conformations are most pronounced in the regions that play a key role in the kinase functioning. The results of the computer calculations were used to consider the structural elements that may affect the kinase activity, the regulatory phosphorylation, and the binding of protein kinase with cyclins and substrates.     

Coupling of T161 and T14 phosphorylations protects cyclin B-CDK1 from premature activation

Mitosis is triggered by the abrupt dephosphorylation of inhibitory Y15 and T14 residues of cyclin B1-bound cyclin-dependent kinase (CDK)1 that is also phosphorylated at T161 in its activation loop. The sequence of events leading to the accumulation of fully phosphorylated cyclin B1-CDK1 complexes remains unclear. Two-dimensional gel electrophoresis allowed us to determine whether T14, Y15, and T161 phosphorylations occur on same CDK1 molecules and to characterize the physiological occurrence of their seven phosphorylation combinations. Intriguingly, in cyclin B1-CDK1, the activating T161 phosphorylation never occurred without the T14 phosphorylation. This strict association could not be uncoupled by a substantial reduction of T14 phosphorylation in response to Myt1 knockdown, suggesting some causal relationship. However, T14 phosphorylation was not directly required for T161 phosphorylation, because Myt1 knockdown did uncouple these phosphorylations when leptomycin B prevented cyclin B1-CDK1 complexes from accumulating in cytoplasm. The coupling mechanism therefore depended on unperturbed cyclin B1-CDK1 traffic. The unexpected observation that the activating phosphorylation of cyclin B1-CDK1 was tightly coupled to its T14 phosphorylation, but not Y15 phosphorylation, suggests a mechanism that prevents premature activation by constitutively active CDK-activating kinase. This explained the opposite effects of reduced expression of Myt1 and Wee1, with only the latter inducing catastrophic mitoses.     

Targeting Conformational Activation of CDK2 Kinase

Cyclin‐dependent kinases constitute attractive pharmacological targets for cancer therapeutics, yet inhibitors in clinical trials target the ATP‐binding pocket of the CDK and therefore suffer from limited selectivity and emergence of resistance. The more recent development of allosteric inhibitors targeting conformational plasticity of protein kinases offers promising perspectives for therapeutics. In particular tampering with T‐loop dynamics of CDK2 kinase would provide a selective means of inhibiting this kinase, by preventing its conformational activation. To this aim we engineered a fluorescent biosensor that specifically reports on conformational changes of CDK2 activation loop and is insensitive to ATP or ATP‐competitive inhibitors, which constitutes a highly sensitive probe for identification of selective T‐loop modulators. This biosensor was successfully applied to screen a library of small chemical compounds leading to discovery of a family of quinacridine analogs, which potently inhibit cancer cell proliferation, and promote accumulation of cells in S phase and G2. These compounds bind CDK2/ Cyclin A, inhibit its kinase activity, compete with substrate binding, but not with ATP, and dock onto the T‐loop of CDK2. The best compound also binds CDK4 and CDK4/Cyclin D1, but not CDK1. The strategy we describe opens new doors for the discovery of a new class of allosteric CDK inhibitors for cancer therapeutics.     

Molecular dynamics modeling of the substitution of serine for the conservative glycine in the G loop in the yeast cdc28-srm mutant using the crystalline lattice of human kinase CDK2

Two-nanosecond molecular dynamics modeling of the crystalline lattice of an active complex of kinase pT160-CDK2/cyclin A/ATP-Mg2+ substrate has been performed. The results of modeling indicated that the structures of the nonmutant CDK2 complex and mutant CDK2 complex, which involves the G 16S-CD K2 substitution corresponding to that of yeast, markedly differ, the differences in structural conformations being particularly well pronounced in those regions that play a key role in the functioning of kinase. Based on the results of computations, structural elements are considered that may affect the kinase activity and regulatory phosphorylation, and the binding of protein kinase to cyclins and substrates.     

Role of phosphorylated Thr160 for the activation of the CDK2/Cyclin A complex

The enzymatic activity of the CDK2/Cyclin A complex increases upon the specific phosphorylation of Thr160@CDK2. In the present study, we have performed a comparative molecular dynamics (MD) study of models of the complex CDK2/Cyclin A/Substrate, which differ for the presence or absence of the phosphate group bound to Thr160. The models are based on two X-ray structures available for CDK2/CyclinA and pCDK2/CyclinA/Substrate complexes. In this way, we analyze the influence of the phosphorylated Thr160 (pThr160) on both the flexibility of CDK2 activation loop (AL) and substrate binding in CDK2. Our calculations point to a decreased flexibility of the AL in the phosphorylated model, in fairly good agreement with experimental data, and to a key role of pThr160 for substrate recognition and stability. Multiple alignments of the CDKs sequences point to the very high conservation of the AL sequence among the CDKs, thus extending our results to all CDKs.     

Phosphorylations of cyclin-dependent kinase 2 revisited using two-dimensional gel electrophoresis

To control the G1/S transition and the progression through the S phase, the activation of the cyclin-dependent kinase (CDK) 2 involves the binding of cyclin E then cyclin A, the activating Thr-160 phosphorylation within the T-loop by CDK-activating kinase (CAK), inhibitory phosphorylations within the ATP binding region at Tyr-15 and Thr-14, dephosphorylation of these sites by cdc25A, and release from Cip/Kip family (p27kip1 and p21cip1) CDK inhibitors. To re-assess the precise relationship between the different phosphorylations of CDK2, and the influence of cyclins and CDK inhibitors upon them, we introduce here the use of the high resolution power of two-dimensional gel electrophoresis, combined to Tyr-15- or Thr-160-phosphospecific antibodies. The relative proportions of the potentially active forms of CDK2 (phosphorylated at Thr-160 but not Tyr-15) and inactive forms (non-phosphorylated, phosphorylated only at Tyr-15, or at both Tyr-15 and Thr-160), and their respective association with cyclin E, cyclin A, p21, and p27, were demonstrated during the mitogenic stimulation of normal human fibroblasts. Novel observations modify the current model of the sequential CDK2 activation process: (i) Tyr-15 phosphorylation induced by serum was not restricted to cyclin-bound CDK2; (ii) Thr-160 phosphorylation engaged the entirety of Tyr-15-phosphorylated CDK2 associated not only with a cyclin but also with p27 and p21, suggesting that Cip/Kip proteins do not prevent CDK2 activity by impairing its phosphorylation by CAK; (iii) the potentially active CDK2 phosphorylated at Thr-160 but not Tyr-15 represented a tiny fraction of total CDK2 and a minor fraction of cyclin A-bound CDK2, underscoring the rate-limiting role of Tyr-15 dephosphorylation by cdc25A.     

Phosphorylation of cyclin Y by CDK14 induces its ubiquitination and degradation

Cyclin Y, a membrane associated cyclin, is capable of binding and activating CDK14. Here we report that human cyclin Y (CCNY) is a phosphoprotein in vivo and that phosphorylation of CCNY by CDK14 triggers its ubiquitination and degradation. Inactivation of either CDK14 or Cul1 results in accumulation of CCNY. An in vivo and in vitro mapping of CCNY phosphorylation sites by mass spectrometry revealed that the flanking regions of the conserved cyclin box are heavily phosphorylated. Phosphorylation of CCNY at Serines 71 and 73 creates a putative phospho-degron that controls its association with an SCF complex. Mutation of serine to alanine at these two sites stabilized CCNY and enhanced the activity of CCNY/CDK14 on phosphorylation of LRP6. Our results provide insight into autoregulation of the cyclin Y/CDK14 pair in CDK14 activation and cyclin Y turnover which is a process that is involved in membrane proximal signaling.     

The activation and inhibition of cyclin-dependent kinase-5 by phosphorylation

Despite the very similar 3-dimensional structures as reflected by the more than 60% identity in amino acid sequences, CDK2 and CDK5 have very different functions and characteristics. Phosphorylation on a conserved Thr14 can inhibit activities of both the kinases, but phosphorylating another conserved Tyr15, however, can lead to totally opposite inhibition and stimulation consequences in CDK2 and CDK5. Our molecular dynamics (MD) simulations suggest a similar inhibition mechanism of phosphorylation on the Thr14 as in the CDK2 system. In both the systems, the kinase activities are inhibited by the phosphorylation because it causes ATP phosphate moiety misalignment and changes in the Mg2+ ion coordination sphere, which have been proven to be critical for the phosphate group of the ATP transferring to the hydroxyl group on the serine in the substrate peptide. The calculations indicate that ATP adopts a more favorable conformation and location in the phosphorylated Tyr15 complex to facilitate the interactions with the substrate and the Mg2+ is wrapped more strongly by the phosphate group than in the unphosphorylated system, which might be favored by the transfer reaction.     

Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2

Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.     

Endogenous and synthetic inhibitors of the Src-family protein tyrosine kinases

Src-family kinases (SFKs) are protooncogenic enzymes controlling mammalian cell growth and proliferation. The activity of SFKs is primarily regulated by two tyrosine phosphorylation sites: autophosphorylation of a conserved tyrosine (Y(A)) in the kinase domain results in activation while phosphorylation of the regulatory tyrosine (Y(T)) near the C-terminus leads to inactivation. The phosphorylated Y(T) (pY(T)) engages in intramolecular interactions that stabilise the inactive conformation of SFKs. These inhibitory intramolecular interactions include the binding of pY(T) to the SH2 domain and the binding of the SH2-kinase linker to the SH3 domain. Thus, SFKs are active upon (i) disruption of the inhibitory intramolecular interactions, (ii) autophosphorylation of Y(A) and/or (iii) dephosphorylation of pY(T). Since aberrant activation of SFKs contributes to cancer, SFKs in normal cells are kept inactive by multiple endogenous inhibitors classified as catalytic and non-catalytic inhibitors. The catalytic inhibitors include C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) that phosphorylate Y(T) of SFKs, as well as the protein tyrosine phosphatases that dephosphorylate pY(A) of the activated SFKs. The non-catalytic inhibitors inactivate SFKs by direct binding. CHK is unique among these inhibitors because it employs both catalytic and non-catalytic mechanisms to inhibit SFKs. Other known non-catalytic inhibitors include WASP, caveolin and RACK1, which function to down-regulate SFKs in specific subcellular locations. This review discusses how the various endogenous SFK inhibitors cooperate to regulate SFKs in normal cells. As chemical compounds that can selectively inhibit SFKs in vivo are potential anti-cancer therapeutics, this review also discusses how investigation into the inhibitory mechanisms of the endogenous inhibitors will benefit the design and screening of these compounds.     

Targeting the cyclin-binding groove site to inhibit the catalytic activity of CDK2/cyclin A complex using p27KIP1-derived peptidomimetic inhibitors

Functionally activated cyclin-dependent kinase 2 (CDK2)/cyclin A complex has been validated as an interesting therapeutic target to develop the efficient antineoplastic drug based on the cell cycle arrest. Cyclin A binds to CDK2 and activates the kinases as well as recruits the substrate and inhibitors using a hydrophobic cyclin-binding groove (CBG). Blocking the cyclin substrate recruitment on CBG is an alternative approach to override the specificity hurdle of the currently available ATP site targeting CDK2 inhibitors. Greater understanding of the interaction of CDK2/cyclin A complex with p27 (negative regulator) reveals that the Leu-Phe-Gly (LFG) motif region of p27 binds with the CBG site of cyclin A to arrest the malignant cell proliferation that induces apoptosis. In the present study, Replacement with Partial Ligand Alternatives through Computational Enrichment (REPLACE) drug design strategies have been applied to acquire LFG peptide-derived peptidomimetics library. The peptidomimetics function is equivalent with respect to substrate p27 protein fashion but does not act as an ATP antagonist. The combined approach of molecular docking, molecular dynamics (MD), and molecular electrostatic potential and ADME/T prediction were carried out to evaluate the peptidomimetics. Resultant interaction and electrostatic potential maps suggested that smaller substituent is desirable at the position of phenyl ring to interact with Trp217, Arg250, and Gln254 residues in the active site. The best docked poses were refined by the MD simulations which resulted in conformational changes. After equilibration, the structure of the peptidomimetic and receptor complex was stable. The results revealed that the various substrate protein-derived peptidomimetics could serve as perfect leads against CDK2 protein.

Electronic supplementary material

The online version of this article (doi:10.1007/s12154-014-0124-y) contains supplementary material, which is available to authorized users.     

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.     

Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7

The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK⿿s interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK⿿s binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK⿿s interaction with Importin 7.     

Activation of Cdk2/Cyclin E complexes is dependent on the origin of replication licensing factor Cdc6 in mammalian cells

Cyclin E-associated CDK2 activity is required for the initiation of DNA synthesis in human cells. CDK2 activity is tightly regulated; CDK2 must be in the nucleus, bound to a cyclin, phosphorylated on T160, and dephosphorylated on T14/Y15 for complete kinase activation. Nuclear localization exposes CDK2 to activating enzymes (CAK, Cdc25A) in stimulated cells. Previous studies from our lab indicate CDK2 nuclear localization and cyclin E co-expression are insufficient to cause CDK2 activation or T160 phosphorylation in stimulated IIC9 cells; these activities still require serum stimulation and ERK kinase activity. Recent studies have implicated a role for origin of replication (ORC) licensing proteins in the activation of G1/S Cdks. In this study, we show that CDK2 associates with chromatin and Cdc6 in an ERK-dependent manner following stimulation of IIC9 CHEF cells. We show that nuclear-localized CDK2 (CDK2-NLS) ectopically expressed with cyclin E requires mitogenic stimulation and ERK activation for chromatin association, in addition to previously shown kinase activation and T160 phosphorylation in IIC9 cells. Additionally, we show that expression of Cdc6 in stimulated IIC9 cells treated with ERK inhibitor rescues CDK2-NLS chromatin association, kinase activation, and T160 phosphorylation. From the above data, we deduce ERK-dependent CDK2 activation is due in part to ERK-dependent Cdc6 expression. To examine the role of Cdc6 directly in stimulated primary human fibroblasts, we used RNA interference to attenuate the expression of Cdc6. We show that Cdc6 expression is required for CDK2 chromatin association and kinase activation in stimulated primary human fibroblasts. Additionally, we show that Cdc6 expression is required for the initiation of DNA synthesis and S phase entry in stimulated primary human fibroblasts. Ultimately, this data implicates Cdc6 expression as an important mitogen-induced mechanism in the activation of CDK2/cyclin E, the initiation of DNA synthesis, and the regulation of G1-S phase progression.     

Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis

Cyclin B1 is a key regulatory protein controlling cell cycle progression in vertebrates. Cyclin B1 binds CDK1, a cy-clin-dependent kinase catalytic subunit, forming a complex that orchestrates mitosis through phosphorylation of key proteins. Cyclin B1 regulates both the activation of CDK1 and its subcellular localization, which may be critical for substrate selection. Here, we demonstrate that cyclin B1 is concentrated on the outer plate of the kinetochore during prometaphase. This localization requires the cyclin box region of the protein. Cyclin B1 is displaced from individual kinetochores to the spindle poles by microtubule attachment to the kinetochores, and this displacement is dependent on the dynein/dynactin complex. Depletion of cyclin B1 by vector-based siRNA causes inefficient attachment between kinetochores and microtubules, and chromosome alignment defects, and delays the onset of anaphase. We conclude that cyclin B1 accumulates at kinetochores during prometaphase, where it contributes to the correct attachment of mi- crotubules to kinetochores and efficient alignment of the chromosomes, most likely through localized phosphorylation of specific substrates by cyclin B1-CDK1. Cyclin B1 is then transported from each kinetochore as microtubule attachment is completed, and this relocalization may redirect the activity of cyclin B1-CDK1 and contribute to inactivation of the spindle assembly checkpoint.     

Stimulation of the Raf/MEK/ERK cascade is necessary and sufficient for activation and Thr-160 phosphorylation of a nuclear-targeted CDK2

The activity of cyclin-dependent kinase 2 is required for G(1)-S-phase progression of the eukaryotic cell cycle. In this study, we examine the activation of CDK2-cyclin E by constructing a CDK2 that is constitutively targeted to the nucleus. Activation of CDK2 requires the removal of two inhibitory phosphates (Thr-14 and Tyr-15) and the addition of one activating phosphate (Thr-160) by a nuclear localized CDK-activating kinase, which is thought to be constitutively active. Surprisingly, nuclear localized CDK2-NLS and CDK2-NLS(A14,F15), which lacks the inhibitory phosphorylation sites, require serum to become active, despite complexing with expressed cyclin E. We show that inhibition of mitogen-mediated ERK activation by treatment with U0126, a selective MEK inhibitor, or expression of dominant-negative ERK markedly reduces the phosphorylation of Thr-160 and enzymatic activity of both CDK2-NLS constructs. Consistent with a role for ERK in Thr-160 phosphorylation, expression of constitutively active Raf-1 induces Thr-160 phosphorylation of CDK2-NLS in serum-arrested cells, an effect that is blocked by treatment with U0126. Taken together, these data show a new role for ERK in G1 cell cycle progression: In addition to its role in stimulating cyclin D1 expression and nuclear translocation of CDK2, ERK regulates Thr-160 phosphorylation of CDK2-cyclin E.