Apoptosis of Central and Peripheral Neurons Can Be Prevented with Cyclin-Dependent Kinase/Mitogen-Activated Protein Kinase Inhibitors

Abstract: Previous studies have indicated that certain members of the cyclin-dependent kinase/mitogen-activated protein kinase superfamily are involved in apoptosis of neuronal cells. Here, we have examined programmed cell death induced by withdrawal of neurotrophic support from CNS (rat retinal) and PNS (chick sympathetic, sensory, and ciliary) neurons. All four neuron types were equally rescued by the purine analogues olomoucine and roscovitine. Olomoucine inhibits multiple cyclin-dependent and mitogen-activated protein kinases with similar potency. Roscovitine is a more selective cyclin-dependent kinase inhibitor; but, so is butyrolactone I, which did not prevent retinal ganglion cell death. The specific p38^MAPK inhibitor SB-203580 did not prevent apoptosis in retinal ganglion cells. Death of these cells in the absence of neurotrophic factors was accompanied by morphological changes indicative of apoptosis, including nuclear condensation and fragmentation. Treatment with olomoucine or roscovitine not only prevented these apoptotic changes in retinal ganglion cells but also blocked neurite outgrowth. The survival-promoting activity of olomoucine correlated with its in vitro IC₅₀ for c-Jun N-terminal kinase-1 and its potency to repress c- jun induction in live PC12 cells. Roscovitine was more potent in rescuing neurons than in inhibiting Jun kinase. Thus, the antiapoptotic action of roscovitine might be due to inhibition of additional kinases.     

Regulation of the Cyclin-Dependent Kinase Inhibitor p57Kip2 Expression by p63

The cyclin-dependent kinase (CDK) inhibitor p57^Kip2 is a negative regulator of cell proliferation, binding to a variety of cyclin-CDK complexes and inhibiting their kinase activities. The p57^Kip2 gene was recognized as a target gene for p73β, one member of the p53 family. In spite of this, the phenotypes of p73 and p57Kip2 knock out mice do not resemble each other while there is a phenotypic overlap betweeen the p57^Kip2 null mice, the p63 null mice and patients affected by p63 associated syndromes, suggesting that p57^Kip2 could be indeed a downstream target of p63. By ChIP we determined that in the HaCaT cell line the δNp63α protein is associated to three different regions of the p57Kip2 gene. δNp63 can activate both the endogenous p57^Kip2 gene and a reporter vector containing a -2191 promoter fragment of the p57^Kip2 gene. Natural p63 mutants, associated to the AEC syndrome, show a partial or complete lack of transactivation potential of the p57^Kip2 promoter, while three other natural p63 mutants, associated to the EEC, LMS and SHFM-4 syndromes, were less affected. These data suggests that p63 play an important role in the regulation of p57^Kip2 expression and that this regulation is subverted in AEC p63 mutants.     

A Novel Member of the Cyclin-Dependent Kinase Family in Paramecium tetraurelia

Passage through the cell cycle in eukaryotes requires the successive activation of different cyclin-dependent protein kinases. Here, we describe the identification and characterization of a novel class of cyclin-dependent protein kinase, termed Cdk2, in the ciliate Paramecium tetraurelia. It is 301 amino acids long, 7 amino acids shorter than Cdk1, the CDK that is associated with macronuclear DNA synthesis. All the catalytic domains typical of protein kinases can be located within the sequence and putative regulatory phosphorylation sites equivalent to Thr14, Tyr15, and Thr161 in human CDK1 are also conserved. The 'PSTAIRE' region characteristic of most CDKs is perfectly conserved. Cdk2 shares only 48% homology to Cdk1 at the amino acid level, suggesting that the evolutionary separation of Cdk1 and Cdk2 is ancient, and implying that they have different roles in cell cycle regulation. Like Cdk1, Cdk2 does not bind to yeast p13suc1, even though it has better conservation of p13suc1 binding sites than Cdk1 does. The Cdk2 protein level is relatively constant throughout the vegetative cell cycle. Cdk2 exhibits kinase activity towards bovine histone H1 in vitro with the maximal level late in the cell cycle, suggesting it may be involved in the regulation of cytokinesis. Our results further support the view that an analogue of the cyclin-dependent kinase cell cycle regulatory system like that of yeast and higher eukaryotic cells operates in Paramecium and that a family of cyclin-dependent kinases may control different aspects of the Paramecium cell cycle.     

Cell Cycle Regulation by the PRMT6 Arginine Methyltransferase through Repression of Cyclin-Dependent Kinase Inhibitors

PRMT6 belongs to the family of Protein Arginine Methyltransferase (PRMT) enzymes that catalyze the methylation of guanidino nitrogens of arginine residues. PRMT6 has been shown to modify the tail of histone H3, but the in vivo function of PRMT6 is largely unknown. Here, we show that PRMT6 regulates cell cycle progression. Knockdown of PRMT6 expression in the human osteosarcoma cell line U2OS results in an accumulation of cells at the G2 checkpoint. Loss of PRMT6 coincides with upregulation of p21 and p27, two members of the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors. Gene expression and promoter analysis show that p21 and p27 are direct targets of PRMT6, which involves methylation of arginine-2 of histone H3. Our findings imply arginine methylation of histones by PRMT6 in cell cycle regulation.     

膜结合鸟苷酸激酶蛋白质家族的结构与功能

MAGUKs蛋白家族成员位于细胞表面,通过其PDZ、SH₃、GK结构域调节离子通道受体的聚集,构建细胞骨架,参与信号转导,调控细胞周期进程,并与机体神经发育密切相关.     

Alp/Enigma Family Proteins Cooperate in Z-Disc Formation and Myofibril Assembly

The Drosophila Alp/Enigma family protein Zasp52 localizes to myotendinous junctions and Z-discs. It is required for terminal muscle differentiation and muscle attachment. Its vertebrate ortholog ZASP/Cypher also localizes to Z-discs, interacts with α-actinin through its PDZ domain, and is involved in Z-disc maintenance. Human mutations in ZASP cause myopathies and cardiomyopathies. Here we show that Drosophila Zasp52 is one of the earliest markers of Z-disc assembly, and we use a Zasp52-GFP fusion to document myofibril assembly by live imaging. We demonstrate that Zasp52 is required for adult Z-disc stability and pupal myofibril assembly. In addition, we show that two closely related proteins, Zasp66 and the newly identified Zasp67, are also required for adult Z-disc stability and are participating with Zasp52 in Z-disc assembly resulting in more severe, synergistic myofibril defects in double mutants. Zasp52 and Zasp66 directly bind to α-actinin, and they can also form a ternary complex. Our results indicate that Alp/Enigma family members cooperate in Z-disc assembly and myofibril formation; and we propose, based on sequence analysis, a novel class of PDZ domain likely involved in α-actinin binding.     

Bcl-2家族蛋白及其在细胞凋亡中的作用

Bcl-2家族蛋白是目前已知的细胞凋亡中最重要的调控因子,在细胞凋亡通路中起着重要的调节作用．对其作用机制的研究将有助于对肿瘤,自身免疫性和神经变性等疾病的治疗。该文介绍Bcl-2家族中主要的几种蛋白在凋亡中的作用,以及对线粒体膜通透性的调控作用。     

Cip/Kip cyclin-dependent kinase inhibitors: brakes of the cell cycle engine during development

Precise control of cell-cycle progression is believed to be critical for normal development, while oncogenesis may be a direct result of its disturbance. Cell-cycle progression is regulated predominantly by a series of serine/threonine kinases, the cyclin-dependent kinases (CDKs). The activities of the CDKs are controlled by a variety of mechanisms, and a group of molecules that inhibit CDK activity, CDK inhibitors (CKIs), has recently become the focus of interest, particularly in the fields of development and tumorigenesis. To date, seven CKIs have been identified in mammals and categorized into two families, the Cip/Kip and Ink4 families. The Cip/Kip family is well conserved phylogenetically, suggesting that it is biologically important. Despite the structural and biochemical similarities among the Cip/Kip members, the phenotypes of knockout mice of each Cip/Kip member are surprisingly different, which suggests that the Cip/Kip CKIs have a variety of physiological functions. In this review, the biological roles of Cip/Kip CKIs in development and tumor suppression are discussed.     

Association of a Genetic Variant in the Cyclin-Dependent Kinase Inhibitor 2B with Risk of Pancreatic Cancer

Background:Pancreatic cancer (PC) is among the most aggressive tumors with a poor prognosis, indicating the need for the identification of a novel prognostic biomarker for risk stratifications. Recent genome-wide association studies have demonstrated common genetic variants in a region on chromosome 9p21 associated with an increased risk of different malignancies.Methods:In the present study, we explore the possible relationship between genetic variant, rs10811661, and gene expression of CDKN2B in 75 pancreatic cancer patients, and 188 healthy individuals. DNAs were extracted and genotyping and gene expression were performed by TaqMan real-time PCR and RT-PCR, respectively. Logistic regression was used to assess the association between risk and genotypes, while the significant prognostic variables in the univariate analysis were included in multivariate analyses.Results:The patients with PDAC had a higher frequency of a TT genotype for rs10811661 than the control group. Also, PDAC patients with dominant genetic model, (TT + TC), was associated with increased risk of developing PDAC (OR= 14.71, 95% CI [1.96-110.35], p= 0.009). Moreover, patients with CC genotype had a higher expression of CDKN2B, in comparison with TT genotype.Conclusion:Our findings demonstrated that CDKN2A/B was associated with the risk of developing PDAC, supporting further investigations in the larger and multicenter setting to validate the potential value of this gene as an emerging marker for PDAC. Key Words: CDKN2A/B, Rs10811661, Pancreatic cancer, Prognostic biomarker     

Coevolutionary Landscape of Kinase Family Proteins: Sequence Probabilities and Functional Motifs

The protein kinase catalytic domain is one of the most abundant domains across all branches of life. Although kinases share a common core function of phosphoryl-transfer, they also have wide functional diversity and play varied roles in cell signaling networks, and for this reason are implicated in a number of human diseases. This functional diversity is primarily achieved through sequence variation, and uncovering the sequence-function relationships for the kinase family is a major challenge. In this study we use a statistical inference technique inspired by statistical physics, which builds a coevolutionary “Potts” Hamiltonian model of sequence variation in a protein family. We show how this model has sufficient power to predict the probability of specific subsequences in the highly diverged kinase family, which we verify by comparing the model’s predictions with experimental observations in the Uniprot database. We show that the pairwise (residue-residue) interaction terms of the statistical model are necessary and sufficient to capture higher-than-pairwise mutation patterns of natural kinase sequences. We observe that previously identified functional sets of residues have much stronger correlated interaction scores than are typical.     

The role of the LH subdomain in the function of the Cip/Kip cyclin-dependent kinase regulators

The Cip/Kip protein family, which includes p27, p21, and p57, modulates the activity of cyclin-dependent kinases (Cdks). A domain within these proteins, termed the kinase inhibitory domain (KID), is necessary and sufficient for Cdk inhibition. The KID consists of a cyclin-binding subdomain (termed D1) and a Cdk-binding subdomain (termed D2) joined by a 22-residue linker subdomain (termed LH). Before binding the Cdks, D1 and D2 are largely unstructured and the LH subdomain exhibits nascent helical characteristics. Curiously, although the sequence of the linker subdomain is not highly conserved within the family, its nascent helical structure is conserved. In this study, we explored the role of this structural conservation in interactions with cyclin-dependent kinase 2 (Cdk2) and cyclin A. We constructed chimeric p27-KID molecules in which the p27 LH subdomain was replaced with the corresponding segments of either p21 or p57. The chimeric molecules bind and inhibit Cdk2 in a manner similar to wild-type p27-KID. However, the extent of enthalpy/entropy compensation associated with these interactions was dramatically different, indicating different extents of LH subdomain folding upon binding. Our results indicate that the different LH subdomains, despite their sequence and thermodynamic differences, play similar roles in binding and inhibiting Cdk2/cyclin A.     

Isolation and Characterization of New Alleles of the Cyclin-Dependent Kinase Gene CDC28 with Cyclin-Specific Functional and Biochemical Defects

The G₁ cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 was significantly deficient in other aspects of genetic interaction with Cln2. The cdc28-csr1 mutation was determined to be Q188P, in the T loop distal to most of the probable Cdk-cyclin interaction regions. We performed random mutagenesis of CDC28 to identify additional alleles incapable of causing CLN2-dependent mating-factor resistance but capable of complementing cdc28 temperature-sensitive and null alleles. Two such mutants had highly defective Cln2-associated kinase, but, surprisingly, two other mutants had levels of Cln2-associated kinase near to wild-type levels. We performed a complementary screen for CDC28 mutants that could cause efficient Cln2-dependent mating-factor resistance but not complement a cdc28 null allele. Most such mutants were found to alter residues essential for kinase activity; the proteins had little or no associated kinase activity in bulk or in association with Cln2. Several of these mutants also functioned in another assay for CLN2-dependent function not involving the mating-factor pathway, complementing the temperature sensitivity of a cln1 cln3 cdc28-csr1 strain. These results could indicate that Cln2-Cdc28 kinase activity is not directly relevant to some CLN2-mediated functions. Mutants of this sort should be useful in differentiating the function of Cdc28 complexed with different cyclin regulatory subunits.