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.     

Mechanism of CDK5 activation revealed by steered molecular dynamics simulations and energy calculations

In the current work, CDK5/p25 complexes were pulled apart by applying external forces with steered molecular dynamics (SMD) simulations. The crucial interactions between the kinase and the activation protein were investigated and the SMD simulations showed that several activation-relevant motifs of CDK5 leave p25 in sequence during the pulling and lead to an apo-CDK2 like CDK5 structure after separation. Based on systematic examination of hydrogen bond breaking and classical MD/molecular mechanics-generalized Born/surface area) (MM-GBSA) calculations, a CDK5 activation mechanism by p25 is suggested. This is the first step towards the systemic development of CDK inhibitors and the mechanism proposed could lead to a better understanding of the protein–protein recognition characteristics between the kinase and its activator.     

HDACs and the senescent phenotype of WI-38 cells

Background  

Normal cells possess a limited proliferative life span after which they enter a state of irreversible growth arrest. This process, known as replicative senescence, is accompanied by changes in gene expression that give rise to a variety of senescence-associated phenotypes. It has been suggested that these gene expression changes result in part from alterations in the histone acetylation machinery. Here we examine the influence of HDAC inhibitors on the expression of senescent markers in pre- and post-senescent WI-38 cells.     

ING1a expression increases during replicative senescence and induces a senescent phenotype

The ING family of tumor suppressor proteins affects cell growth, apoptosis and response to DNA damage by modulating chromatin structure through association with different HAT and HDAC complexes. The major splicing isoforms of the ING1 locus are ING1a and INGlb. While INGlb plays a role in inducing apoptosis, the function of ING1a is currently unknown. Here we show that alternative splicing of the ING1 message alters the INGla:INGlb ratio by approximately 30-fold in senescent compared to low passage primary fibroblasts. INGla antagonizes INGlb function in apoptosis, induces the formation of structures resembling senescence-associated heterochromatic foci containing heterochromatin protein 1 gamma, the accumulation of senescence-associated beta-galactosidase activity and promotes senescent cell morphology and cell cycle arrest. Phenotypic effects may result from differential effects on gene expression since ING1a increases levels of both retinoblastoma and the p16 cyclin-dependent kinase inhibitor and ING1a and ING1b have opposite effects on the expression of proliferating nuclear cell antigen (PCNA), which is required for cell growth. Gene expression appears to be altered by targeting of HDAC complexes to gene promoters since INGla associates with several-fold higher levels of HDAC1 in senescent, compared to replication-competent cells and ING1 is found on the PCNA promoter by chromatin immunoprecipitation analysis. These data demonstrate a novel role for the ING1 proteins in differentially regulating senescence-associated chromatin remodeling vs. apoptosis and support the idea that altered ratios of the ING1 splicing isoforms may contribute to establishing the senescent phenotype through HDAC and HAT complex-mediated effects on chromatin structure.     

Aberrant activation of CDK5 is involved in the pathogenesis of OPIDN

Exposure to triorthocresyl phosphate (TOCP) may result in a late neurological complication, i.e. organophosphate-induced delayed neuropathy (OPIDN). The aim of this study was to examine changes in levels of cyclin-dependent kinase 5 (CDK5) and of its activator, p35/p25, in the spinal cord of hens treated by TOCP. After exposure to a single dose of TOCP, groups of adult hens were examined in 3, 5, 7, 9, 14, and 18 days after exposure. CDK5, p35/p25 expression and distribution in the lumbar spinal cord were evaluated by immunohistochemistry and Western blotting. The hens showed signs of OPIDN around day 9 after exposure. The number of p (phosphorylated) -CDK5 and p35 positive cells increased significantly. Co-localization and mislocalization of p-CDK5 and p35/p25 was identified and became evident in neurons around the 9th day. Meanwhile, CDK5, p-CDK5, p35, p25 protein levels and p25/p35 ratio were increased, and peaked around the 9th day, then decreased. Some hens' unilateral common peroneal was treated by roscovitine 3 days after TOCP exposure. Axonal transport of these nerves was faster than of their opposite side and of those simply treated by TOCP. These findings indicate aberrant activation of CDK5 may be involved in the pathogenesis of OPIDN.     

Genetic modulation of the senescent phenotype in Homo sapiens

While it is important to search for unifying mechanisms of aging among a variety of model systems, evolutionary arguments suggest that the pathophysiological details of senescence may be, to some extent, species specific. Moreover, in species that are characterized by extensive genetic heterogeneity, such as our own, one is likely to find kindreds with both "private" and "public" markers of aging. Crude estimates of the number of loci with the potential to modulate aspects of the senescent phenotype of man suggest that thousands of genes could be involved. No single locus appears to modulate all features. Some affect predominantly a single aspect ("unimodal progeroid syndromes"); familial Alzheimer's disease is discussed as a prototype. Linkage studies indicate genetic heterogeneity for autosomal dominant forms of the disease. Some loci affect multiple aspects of the phenotype ("segmental progeroid disorders"); the prototype is Werner's syndrome, an autosomal recessive. Cells from homozygotes behave like mutator strains and undergo accelerated senescence in vitro. Elucidation of the biochemical genetic basis of such abiotrophic disorders may shed light on specific aging processes in man.     

Cloning, characterization and expression of CDK5RAP1_v3 and CDK5RAP1_v4, two novel splice variants of human CDK5RAP1

Two novel splice variants of CDK5RAP1, named CDK5RAP1_v3 and CDK5RAP1_v4, were isolated through the large-scale sequencing analysis of a human fetal brain cDNA library. The CDK5RAP1_v3 and CDK5RAP1_v4 cDNAs are 1923bp and 1792bp in length, respectively. Sequence analysis revealed that CDK5RAP1_v4 lacked 1 exon, which was present in CDK5RAP1_v3, with the result that these cDNAs encoded different putative proteins. The deduced proteins were 574 amino acids (designated as CDK5RAP1_v3) and 426 amino acids (CDK5RAP1_v4) in length, and shared the 420 N-terminal amino acids. RT-PCR analysis showed that human CDK5RAP1_v3 was widely expressed in human tissues. The expression level of CDK5RAP1_v3 was relatively high in placenta and lung, whereas low levels of expression were detected in heart, brain, liver, skeletal muscle, pancreas, spleen, thymus, small intestine and peripheral blood leukocytes. In contrast, human CDK5RAP1_v4 was mainly expressed in brain, placenta and testis.     

Spatial recruitment and activation of the Fes kinase by ezrin promotes HGF-induced cell scattering

The remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) results in the reorganization of actin cytoskeleton and cellular junctions. We previously showed that the membrane-cytoskeleton linker ezrin plays a major role in HGF-induced morphogenic effects. Here we identified a novel partner of phosphorylated ezrin, the Fes kinase, that acts downstream of ezrin in HGF-mediated cell scattering. We found that Fes interacts directly, through its SH2 domain, with ezrin phosphorylated at tyrosine 477. We show that in epithelial cells, activated Fes localizes either to focal adhesions or cell-cell contacts depending on cell confluency. The recruitment and the activation of Fes to the cell-cell contacts in confluent cells depend on its interaction with ezrin. When this interaction is impaired, Fes remains in focal adhesions and as a consequence the cells show defective spreading and scattering in response to HGF stimulation. Altogether, these results provide a novel mechanism whereby ezrin/Fes interaction at cell-cell contacts plays an essential role in HGF-induced cell scattering and implicates Fes in the cross-talk between cell-cell and cell-matrix adhesion.     

CDK5 interacts with Slo and affects its surface expression and kinetics through direct phosphorylation

Large-conductance calcium-activated potassium (BK) channels are ubiquitous and play an important role in a number of diseases. In hair cells of the ear, they play a critical role in electrical tuning, a mechanism of frequency discrimination. These channels show variable kinetics and expression along the tonotopic axis. Although the molecular underpinnings to its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Here we identify CDK5, a member of the cyclin-dependent kinase family, as an interacting partner of Slo. We show CDK5 to be present in hair cells and expressed in high concentrations in the cuticular plate and in the circumferential zone. In human embryonic kidney cells, we show that CDK5 inhibits surface expression of Slo by direct phosphorylation of Slo. Similarly, we note that CDK5 affects Slo voltage activation and deactivation kinetics, by a direct phosphorylation of T847. Taken together with its increasing expression along the tonotopic axis, these data suggest that CDK5 likely plays a critical role in electrical tuning and surface expression of Slo in hair cells.     

TRPC5 activation kinetics are modulated by the scaffolding protein ezrin/radixin/moesin-binding phosphoprotein-50 (EBP50)

TRPC1-7 proteins are members of a family of mammalian non-specific cation channels that mediate receptor-operated, phospholipase Cbeta/Cgamma dependent Ca(2+) influx in various cell types. TRPC4 and TRPC5 form a subfamily within TRPCs. Uniquely in the TRPC family, these channels possess a C-terminal "VTTRL" motif that binds to PDZ-domains of the scaffolding protein, EBP50 (NHERF1; Tang et al., J Biol Chem 275:37559-37564). The functional effects of EBP50 on TRPC4/5 activity have not been investigated. We have cloned rat TRPC5 (rTRPC5), functionally expressed it in HEK293 cell, and studied channel regulation with patch-clamp techniques. Both rTRPC5 and its VTTRL deletion mutant (r5dV) were localized to the plasma membrane. rTRPC5 did not display any significant basal activity in unstimulated HEK293 cells. In cells co-expressing rTRPC5 and H1 histamine receptor, rTRPC5 current evoked by GTPgammaS or histamine developed in two phases: a slowly developing, small inward current was followed by a rapidly developing, transient, large inward current. Each phase had a characteristic non-linear current-voltage (I-V) relationship. Deletion of the VTTRL motif had no detectable effect on the biophysical properties of the channel. Co-expression of EBP50 with rTRPC5 caused a significant delay in the time-to-peak of the histamine-evoked, transient large inward current. EBP50 did not modify the activation kinetics of the VTTRL-deletion mutant. We conclude that the VTTRL motif is not necessary for activation of TRPC5, but may mediate the modulatory effect of EBP50 on TRPC5 activation kinetics.     

Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin

Ezrin, a membrane-actin cytoskeleton linker, which participates in epithelial cell morphogenesis, is held inactive in the cytoplasm through an intramolecular interaction. Phosphatidylinositol 4,5-bisphosphate (PIP2) binding and the phosphorylation of threonine 567 (T567) are involved in the activation process that unmasks both membrane and actin binding sites. Here, we demonstrate that ezrin binding to PIP2, through its NH2-terminal domain, is required for T567 phosphorylation and thus for the conformational activation of ezrin in vivo. Furthermore, we found that the T567D mutation mimicking T567 phosphorylation bypasses the need for PIP2 binding for unmasking both membrane and actin binding sites. However, PIP2 binding and T567 phosphorylation are both necessary for the correct apical localization of ezrin and for its role in epithelial cell morphogenesis. These results establish that PIP2 binding and T567 phosphorylation act sequentially to allow ezrin to exert its cellular functions.     

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.     

Increased expression of TGF-beta 2 in osteoblasts results in an osteoporosis-like phenotype

The development of the skeleton requires the coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts. The activities of these two cell types are likely to be regulated by TGF-beta, which is abundant in bone matrix. We have used transgenic mice to evaluate the role of TGF-beta 2 in bone development and turnover. Osteoblast- specific overexpression of TGF-beta 2 from the osteocalcin promoter resulted in progressive bone loss associated with increases in osteoblastic matrix deposition and osteoclastic bone resorption. This phenotype closely resembles the bone abnormalities seen in human hyperparathyroidism and osteoporosis. Furthermore, a high level of TGF- beta 2 overexpression resulted in defective bone mineralization and severe hypoplasia of the clavicles, a hallmark of the developmental disease cleidocranial dysplasia. Our results suggest that TGF-beta 2 functions as a local positive regulator of bone remodeling and that alterations in TGF-beta 2 synthesis by bone cells, or in their responsiveness to TGF-beta 2, may contribute to the pathogenesis of metabolic bone disease.     

Ca2+-dependent binding and activation of dormant ezrin by dimeric S100P

S100 proteins are EF hand type Ca2+ binding proteins thought to function in stimulus-response coupling by binding to and thereby regulating cellular targets in a Ca2+-dependent manner. To isolate such target(s) of the S100P protein we devised an affinity chromatography approach that selects for S100 protein ligands requiring the biologically active S100 dimer for interaction. Hereby we identify ezrin, a membrane/F-actin cross-linking protein, as a dimer-specific S100P ligand. S100P-ezrin complex formation is Ca2+ dependent and most likely occurs within cells because both proteins colocalize at the plasma membrane after growth factor or Ca2+ ionophore stimulation. The S100P binding site is located in the N-terminal domain of ezrin and is accessible for interaction in dormant ezrin, in which binding sites for F-actin and transmembrane proteins are masked through an association between the N- and C-terminal domains. Interestingly, S100P binding unmasks the F-actin binding site, thereby at least partially activating the ezrin molecule. This identifies S100P as a novel activator of ezrin and indicates that activation of ezrin's cross-linking function can occur directly in response to Ca2+ transients.     

siRNAs target sites selection of ezrin and the influence of RNA interference on ezrin expression and biological characters of osteosarcoma cells

Tissue kallikrein 1 cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in inhibiting neointimal hyperplasia in rat carotid arteries after balloon injury. However, its effects on the proliferation, cell cycle and its mechanisms, for example, cyclin-dependent kinase inhibitors, p27(Kip1) and p2l(Cip1) in vascular biology are poorly understood. The objective of this study was to explore the effects of human tissue kallikrein 1 (hTK1) mediated by recombinant adenovirus (Ad-hTK1) on proliferation and cell cycle of vascular smooth muscle cells (VSMCs) derived from spontaneously hypertensive rats induced by platelet-derived growth factor-BB (PDGF-BB) in vitro. The results showed that, within a given multiplicity of infection (MOI) and time, the hTK1 gene delivery inhibited PDGF-BB-stimulating VSMCs growth in a concentration-dependent (20-100 MOI) and time-dependent (2-5 days) manner by cell counting, with a peak inhibition rate at 36.3% at 72 h (P < 0.01). In addition, hTK1 gene delivery significantly suppressed PDGF-BB-induced proliferation of VSMCs by methyl thiazolyl tetrazoliuin assay, and decreased the percentage of cells in the S phase and in DNA synthesis by flow cytometry, with a peak inhibition rate at 30.2 and 36.4%, respectively (P < 0.01). Western blot assay showed that the protein levels of p27(Kip1) and p2l(Cip1) in cells infected with Ad-hTK1 were much more abundant than those in cells only induced by PDGF-BB, with up-modulating rates at 51.8 and 58.7%, respectively (P < 0.001). We also observed that the effects of hTK1 gene delivery in inhibiting VSMCs proliferation, arresting cell cycling in G(0)/G(1) phase and up-regulating the expression of p27(Kip1) and p2l(Cip1) could be blocked by icatibant (Hoe 140), a specific bradykinin B(2) receptor antagonist. Taken together, these results demonstrated that hTK1 overexpressed by recombinant adenovirus potently inhibits VSMCs proliferation that is required for neointimal hyperplasia and restenosis, and may activate p27(Kip1) and p2l(Cip1) signaling pathways via bradykinin B(2) receptor.