Different mechanisms of CDK5 and CDK2 activation as revealed by CDK5/p25 and CDK2/cyclin A dynamics

A detailed analysis is presented of the dynamics of human CDK5 in complexes with the protein activator p25 and the purine-like inhibitor roscovitine. These and other findings related to the activation of CDK5 are critically reviewed from a molecular perspective. In addition, the results obtained on the behavior of CDK5 are compared with data on CDK2 to assess the differences and similarities between the two kinases in terms of (i) roscovitine binding, (ii) regulatory subunit association, (iii) conformational changes in the T-loop following CDK/regulatory subunit complex formation, and (iv) specificity in CDK/regulatory subunit recognition. An energy decomposition analysis, used for these purposes, revealed why the binding of p25 alone is sufficient to stabilize the extended active T-loop conformation of CDK5, whereas the equivalent conformational change in CDK2 requires both the binding of cyclin A and phosphorylation of the Thr(160) residue. The interaction energy of the CDK5 T-loop with p25 is about 26 kcal.mol(-1) greater than that of the CDK2 T-loop with cyclin A. The binding pattern between CDK5 and p25 was compared with that of CDK2/cyclin A to find specific regions involved in CDK/regulatory subunit recognition. The analyses performed revealed that the alphaNT-helix of cyclin A interacts with the alpha6-alpha7 loop and the alpha7 helix of CDK2, but these regions do not interact in the CDK5/p25 complex. Further differences between the CDK5/p25 and CDK2/cyclin A systems studied are discussed with respect to their specific functionality.     

Homology Model of the CDK1/cyclin B Complex

Abstract

We describe a refined homology model of a CDK1/cyclin B complex that was previously used for the structure-based optimization of the Paullone class of inhibitors. The preliminary model was formed from the homologous regions of the deposited CDK2/cyclin A crystal structure. Further refinement of the CDK1/cyclin B complex was accomplished using molecular mechanics and hydropathic analysis with a protocol of constraints and local geometry searches. For the most part, our CKD1/cyclin B homology model is very similar to the high resolution CDK2/cyclin A crystal structure regarding secondary and tertiary features. However, minor discrepancies between the two kinase structures suggest the possibility that ligand design may be specifically tuned for either CDK1 or CDK2. Our examination of the CDK1/cyclin B model includes a comparison with the CDK2/cyclin A crystal structure in the PSTAIRE interface region, connecting portions to the ATP binding domain, as well as the ATP binding site itself.     

Homology model of the CDK1/cyclin B complex

We describe a refined homology model of a CDK1/cyclin B complex that was previously used for the structure-based optimization of the Paullone class of inhibitors. The preliminary model was formed from the homologous regions of the deposited CDK2/cyclin A crystal structure. Further refinement of the CDK1/cyclin B complex was accomplished using molecular mechanics and hydropathic analysis with a protocol of constraints and local geometry searches. For the most part, our CKD1/cyclin B homology model is very similar to the high resolution CDK2/cyclin A crystal structure regarding secondary and tertiary features. However, minor discrepancies between the two kinase structures suggest the possibility that ligand design may be specifically tuned for either CDK1 or CDK2. Our examination of the CDK1/cyclin B model includes a comparison with the CDK2/cyclin A crystal structure in the PSTAIRE interface region, connecting portions to the ATP binding domain, as well as the ATP binding site itself.     

Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover

Notch signaling releases the Notch receptor intracellular domain (ICD), which complexes with CBF1 and Mastermind (MAM) to activate responsive genes. We previously reported that MAM interacts with CBP/p300 and promotes hyperphosphorylation and degradation of the Notch ICD in vivo. Here we show that CycC:CDK8 and CycT1:CDK9/P-TEFb are recruited with Notch and associated coactivators (MAM, SKIP) to the HES1 promoter in signaling cells. MAM interacts directly with CDK8 and can cause it to localize to subnuclear foci. Purified recombinant CycC:CDK8 phosphorylates the Notch ICD within the TAD and PEST domains, and expression of CycC:CDK8 strongly enhances Notch ICD hyperphosphorylation and PEST-dependent degradation by the Fbw7/Sel10 ubiquitin ligase in vivo. Point mutations affecting conserved Ser residues within the ICD PEST motif prevent hyperphosphorylation by CycC:CDK8 and stabilize the ICD in vivo. These findings suggest a role for MAM and CycC:CDK8 in the turnover of the Notch enhancer complex at target genes.     

The CDK4/CDK6 inhibitor PD0332991 paradoxically stabilizes activated cyclin D3-CDK4/6 complexes

CDK4 and CDK6 bound to D-type cyclins are master integrators of G1 phase cell cycle regulations by initiating the inactivating phosphorylation of the central oncosuppressor pRb. Because of their frequent deregulation in cancer, cyclin D-CDK4/6 complexes are emerging as especially promising therapeutic targets. The specific CDK4/6 inhibitor PD0332991 is currently tested in a growing number of phase II/III clinical trials against a variety of pRb-proficient chemotherapy-resistant cancers. We have previously shown that PD0332991 inhibits not only CDK4/6 activity but also the activation by phosphorylation of the bulk of cyclin D-CDK4 complexes stabilized by p21 binding. Here we show that PD0332991 has either a positive or a negative impact on the activation of cyclin D-CDK4/6 complexes, depending on their binding to p21. Indeed, whereas PD0332991 inhibits the phosphorylation and activity of p21-bound CDK4/6, it specifically stabilized activated cyclin D3-CDK4/6 complexes devoid of p21 and p27. After elimination of PD0332991, these activated cyclin D3-CDK4/6 complexes persisted for at least 24 h, resulting in paradoxical cell cycle entry in the absence of a mitogenic stimulation. This unsuspected positive effect of PD0332991 on cyclin D3-CDK4/6 activation should be carefully assessed in the clinical evaluation of PD0332991, which until now only involves discontinuous administration protocols.     

The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles

Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) at 2.25 A resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates.     

A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His₆-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni²⁺-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004–14). Our protocol provides a novel systematic approach for the purification of these three (and possibly other) recombinant CDKs. Published: August 18, 2004.     

Activation mechanism of CDK2: role of cyclin binding versus phosphorylation

Activation of the cyclin-dependent kinases is a two-step process involving cyclin binding followed by phosphorylation at a conserved threonine residue within the kinase activation loop. In this study, we describe the separate roles of cyclin A binding versus phosphorylation in the overall activation mechanism of CDK2. Interaction of CDK2 with cyclin A results in a partially active complex that is moderately defective in the binding of the protein substrate, but not ATP, and severely defective in both phosphoryl group transfer and turnover. Alternatively, phosphorylation of the CDK2 monomer also results in a partially activated species, but one that is severely (> or = 480-fold) defective in substrate binding exclusively. Catalytic turnover in the phosphorylated CDK2 monomer is largely unimpaired (approximately 8-fold lower). Our data support a model for the activation of CDK2 in vivo, in which interaction of unphosphorylated CDK2 with cyclin A serves to configure the active site for ground-state binding of both ATP and the protein substrate, and further aligns ATP in the transition state for phosphoryl transfer. Optimizing the alignment of protein substrates in the phosphoryl transfer reaction is the principal role of phosphorylation at Thr(160).     

Increased p21 expression and complex formation with cyclin E/CDK2 in retinoid-induced pre-B lymphoma cell apoptosis

Cip/Kip family protein p21, a cyclin-dependent kinase (CDK) inhibitor, is directly transactivated by retinoic acid receptor alpha (RARalpha) upon retinoic acid (RA):RARalpha binding. Yet the role of p21 upregulation by RA in lymphoma cells remains unknown. Here, we show that, in human pre-B lymphoma Nalm6 cells, RA-induced proliferation inhibition results from massive cell death characterized by apoptosis. Upregulated p21 by RA accompanies caspase-3 activation and precedes the occurrence of apoptosis. p21 induction leads to increased p21 complex formation with cyclin E/CDK2, which occurs when cyclin E and CDK2 levels remain constant. CDK2 can alternatively promote apoptosis, but the mechanisms remain unknown. Data presented here suggest a novel RA-signaling, by which RA-induced p21 induction and complex formation with cyclin E/CDK2 diverts CDK2 function from normally driving proliferation to alternatively promoting apoptosis.     

Kinetic basis for activation of CDK2/cyclin A by phosphorylation

The activation of most protein kinases requires phosphorylation at a conserved site within a structurally defined segment termed the activation loop. A classic example is the regulation of the cell cycle control enzyme, CDK2/cyclin A, in which catalytic activation depends on phosphorylation at Thr(160) in CDK2. The structural consequences of phosphorylation have been revealed by x-ray crystallographic studies on CDK2/cyclin A and include changes in conformation, mainly of the activation loop. Here, we describe the kinetic basis for activation by phosphorylation in CDK2/cyclin A. Phosphorylation results in a 100,000-fold increase in catalytic efficiency and an approximate 1,000-fold increase in the overall turnover rate. The effects of phosphorylation on the individual steps in the catalytic reaction pathway were determined using solvent viscosometric techniques. It was found that the increase in catalytic power arises mainly from a 3,000-fold increase in the rate of the phosphoryl group transfer step with a more moderate increase in substrate binding affinity. In contrast, the rate of phosphoryl group transfer in the ATPase pathway was unaffected by phosphorylation, demonstrating that phosphorylation at Thr(160) does not serve to stabilize ATP in the ATPase reaction. Thus, we hypothesize that the role of phosphorylation in the kinase reaction may be to specifically stabilize the peptide phosphoacceptor group.