Cyclin H is a new binding partner for protein kinase CK2

PTP-FERM is a protein tyrosine phosphatase (PTP) of Caenorhabditis elegans containing a FERM domain and a PDZ domain. Here we report the characterization of PTP-FERM and the essential role of its FERM domain in the localization of PTP-FERM in the worm. There are at least three alternatively spliced PTP-FERM isoforms, all of which contain a band 4.1/FERM domain, a PDZ domain, and a catalytic domain. PTP-FERM possessed phosphatase activity. PTP-FERM was expressed predominantly in neurons in the nerve ring and the ventral nerve cord. PTP-FERM was found in the nerve processes and to be enriched in the peri-membrane region. Studies using various deletion mutants revealed that the FERM domain was essential and sufficient for the subcellular localization. These results suggest the essential role of the FERM domain in the function of PTP-FERM in the neurons of C. elegans.     

Cyclin A2/cyclin-dependent kinase 1-dependent phosphorylation of Top2a is required for S phase entry during retinal development in zebrafish

Cyclin-dependent kinase 1 (CDK1) plays an essential role in cell cycle regulation.However,as mouse Cdk1embryos die early,the role of CDK1 in regulating the cell cycle and embryo development remains unclear.Here,we showed that zebrafish cdk1~(-/-)embryos exhibit severe microphthalmia accompanied by multiple defects in S phase entry,M phase progression,and cell differentiation but not in interkinetic nuclear migration.We identified Top2a as a potential downstream target and cyclin A2 and cyclin B1 as partners of Cdk1 in cell cycle regulation via an in silico analysis.While depletion of either cyclin A2 or Top2a led to the decreased S phase entry in zebrafish retinal cells,the depletion of cyclin B1 led to M phase arrest.Moreover,phosphorylation of Top2a at serine 1213 (S1213) was nearly abolished in both cdk1 and ccna2mutants,but not in ccnb1 mutants.Furthermore,overexpression of TOP2A~(S1213D),the phosphomimetic form of human TOP2A,rescued S phase entry and alleviated the microphthalmia defects in both cdk1~(-/-)and ccna2~(-/-)embryos.Taken together,our data suggest that Cdk1 interacts with cyclin A2 to regulate S phase entry partially through Top2a phosphorylation and interacts with cyclin B1 to regulate M phase progression.     

Differential effect of kinase A and C blockers on lordosis facilitation by progesterone and its metabolites in ovariectomized estrogen-primed rats

Dose response curves for lordosis behavior was obtained for progesterone (P) and its two ring A-reduced metabolites: 5alpha-pregnanedione (alpha-DHP) and 5alpha,3alpha-pregnanolone (5alpha,3alpha-Pgl) by infusing these progestins in the right lateral ventricle (rlv) of ovariectomized (ovx) estradiol-treated rats (2 microg estradiol benzoate; EB), 40 h before intracerebro-ventricular (icv) injection. Effective doses 50 (ED50) revealed that ring A-reduced progestins were more potent than P itself to induce lordosis behavior. Two dose levels, one producing the maximal effect and the other one producing a submaximal response (ED50-ED60), were selected for testing the capacity of RpAMPS, a kinase A blocker, and H7, a kinase C blocker, to modify the response to the three progestins. rlv injection of RpAMPS significantly depressed the lordosis response to the two dose levels of P and alpha-DHP but failed to significantly inhibit that of 5alpha,3alpha-Pgl. The administration of H7 prevented the effect of both 5alpha-reduced progestins without affecting the response to P. The results suggest that P and its ring A-reduced metabolites stimulate lordosis behavior through different cellular mechanisms: P acting mainly through the cAMP-kinase system; alpha-DHP through both kinase A and kinase C signaling pathways and 5alpha,3alpha-Pgl through the kinase C system.     

New alternative phosphorylation sites on the cyclin dependent kinase 1/cyclin a complex in p53-deficient human cells treated with etoposide: possible association with etoposide-induced apoptosis

Cell cycle arrest is a major cellular response to DNA damage preceding the decision to repair or die. Many malignant cells have non-functional p53 rendering them more “aggressive” in nature. Arrest in p53-negative cells occurs at the G2M cell cycle checkpoint. Failure of DNA damaged cells to arrest at G2 results in entry into mitosis and potential death through aberrant mitosis and/or apoptosis. The pivotal kinase regulating the G2M checkpoint is Cdk1/cyclin B whose activity is controlled by phosphorylation. The p53-negative myeloid leukemia cell lines K562 and HL-60 were used to determine Cdk1 phosphorylation status during etoposide treatment. Cdk1 tyrosine 15 phosphorylation was associated with G2M arrest, but not with cell death. Cdk1 tyrosine 15 phosphorylation also led to suppression of nuclear cyclin B-associated Cdk1 kinase activity. However cell death, associated with broader tyrosine phosphorylation of Cdk1 was not attributed to tyrosine 15 alone. This broader phosphoryl isoform of Cdk1 was associated with cyclin A and not cyclin B. Alternative phosphorylations sites were predicted as tyrosines 4, 99 and 237 by computer analysis. No similar pattern was found on Cdk2. These findings suggest novel Cdk1 phosphorylation sites, which appear to be associated with p53-independent cell death following etoposide treatment.     

Phosphorylation of MCM3 protein by cyclin E/cyclin-dependent kinase 2 (Cdk2) regulates its function in cell cycle

MCM2-7 proteins form a stable heterohexamer with DNA helicase activity functioning in the DNA replication of eukaryotic cells. The MCM2-7 complex is loaded onto chromatin in a cell cycle-dependent manner. The phosphorylation of MCM2-7 proteins contributes to the formation of the MCM2-7 complex. However, the regulation of specific MCM phosphorylation still needs to be elucidated. In this study, we demonstrate that MCM3 is a substrate of cyclin E/Cdk2 and can be phosphorylated by cyclin E/Cdk2 at Thr-722. We find that the MCM3 T722A mutant binds chromatin much less efficiently when compared with wild type MCM3, suggesting that this phosphorylation site is involved in MCM3 loading onto chromatin. Interestingly, overexpression of MCM3, but not MCM3 T722A mutant, inhibits the S phase entry, whereas it does not affect the exit from mitosis. Knockdown of MCM3 does not affect S phase entry and progression, indicating that a small fraction of MCM3 is sufficient for normal S phase completion. These results suggest that excess accumulation of MCM3 protein onto chromatin may inhibit DNA replication. Other studies indicate that excess of MCM3 up-regulates the phosphorylation of CHK1 Ser-345 and CDK2 Thr-14. These data reveal that the phosphorylation of MCM3 contributes to its function in controlling the S phase checkpoint of cell cycle in addition to the regulation of formation of the MCM2-7 complex.     

CyclinE和cdk2在眼睑基底细胞癌组织中的表达及其意义

目的 探讨CyclinE和cdk2在眼睑基底细胞癌组织中的表达及其意义.方法 收集武汉市中心医院和武汉大学人民医院病理科2002-2009年手术切除及活检的眼睑基底细胞癌(basal cell carcinoma,BCC)标本其20例,另取癌周围组织5例作对照,采用免疫组织化学方法观察各组细胞内CyclinE和cdk2表达.利用HPIAS-2000图像分析系统测定CyclinE和cdk2在以上各组中表达的平均光密度和平均阳性面积率.结果 眼睑基底细胞癌组织中CyclinE和cdk2呈高表达;癌旁组织中CyclinE和cdk2呈低表达.图像分析结果显示:眼睑基底细胞癌组织与癌旁组织之间CyclinE和cdk2的平均光密度及阳性面积率的差异有显著性意义(P＜0.05).结论 CyclinE和cdk2的高表达,在眼睑基底细胞癌的发生、发展过程中起了重要作用,两者共同表达对于术后病人监测及治疗方案的制定具有指导意义.     

Proline-Directed Protein Kinase (p34cdc2/p58cyclin A) Phosphorylates Bovine Neurofilaments

Proline-directed protein kinase (PDPK), a complex of p34cdc2 and p58cyclin A, phosphorylates bovine neurofilaments (NFs) in vitro. Incubation of intact filaments with PDPK led to strong labeling of the heavy (NF-H) and middle (NF-M) molecular weight NF proteins and weaker labeling of the low molecular weight protein (NF-L). All three proteins were phosphorylated in solution, with the best substrate being NF-H. Proteins that had been dephosphorylated by enzymatic treatment were better substrates than native proteins--as many as 6 mol of phosphate were incorporated per mole of NF-H. Partial proteolytic cleavage experiments combined with two-dimensional peptide mapping indicated that NF-H and NF-M were phosphorylated predominantly in the tail domains, with some phosphate also appearing in the heads. Soluble NF-L is phosphorylated on the head domain peptide L-3, whereas NF-L within intact filaments is phosphorylated only on the tail domain peptide L-1. Phosphorylation does not lead to filament disassembly. A possible role for PDPK in NF phosphorylation in vivo is discussed.     

Human Cdc25A phosphatase has a non-redundant function in G2 phase by activating Cyclin A-dependent kinases

Cdc25 phosphatases activate Cdk/Cyclin complexes by dephosphorylation and thus promote cell cycle progression. We observed that the peak activity of Cdc25A precedes the one of Cdc25B in prophase and the maximum of Cyclin/Cdk kinase activity. Furthermore, Cdc25A activates both Cdk1-2/Cyclin A and Cdk1/Cyclin B complexes while Cdc25B seems to be involved only in activation of Cdk1/Cyclin B. Concomitantly, repression of Cdc25A led to a decrease in Cyclin A-associated kinase activity and attenuated Cdk1 activation. Our results indicate that Cdc25A acts before Cdc25B - at least in cancer cells, and has non-redundant functions in late G2/early M-phase as a major regulator of Cyclin A/kinase complexes.     

FOXM1c is activated by cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1, but repressed by GSK-3alpha

Two different inhibitory domains, N-terminus and central domain, keep FOXM1c almost inactive despite its strong transactivation domain. Here, we demonstrate that cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1 activate FOXM1c. Cyclin E/Cdk2 does not target its transactivation domain or its DNA-binding domain. Instead, its activating effect strictly depends on the presence of either the central domain or the N-terminus of FOXM1c and thus is completely lost if both inhibitory domains are deleted. Cyclin E/Cdk2 activates FOXM1c by releasing its transactivation domain from the repression by these two inhibitory domains. However, it does not directly increase the transactivation potential of the TAD. The DNA-binding is not affected by cyclin E/Cdk2, neither directly nor indirectly. These two activating effects of cyclin E/Cdk2 via central domain and N-terminus are additive. Cyclin A/Cdk2 and cyclin A/Cdk1 show similar characteristics. GSK-3alpha, another proliferation-associated kinase, represses FOXM1c.     

Integrated actions of progesterone receptor and cell cycle machinery regulate breast cancer cell proliferation

Multiple laboratories have investigated progesterone receptor (PR) involvement in breast cancer cell cycle progression. There is now a growing body of evidence demonstrating complex interactions between PR and cell cycle regulatory proteins. Here we review the current literature linking PR to cell cycle control and discuss gaps in the current knowledge. A more complete understanding of the relationships between PR and cell cycle regulatory molecules may reveal additional avenues for prevention and treatment of steroid receptor positive breast cancers.     

Coactivation of an endogenous progesterone receptor by TIF2 in COS-7 cells

Transfection experiments, a powerful tool to study the function of steroid hormone receptors and their coregulators, are often performed in COS-7 cells, because of high transfection efficiencies and expression levels. Here we report on the presence in COS-7 cells of an endogenous steroid hormone receptor, which is highly responsive to progesterone and the synthetic steroids R1881 and ORG2058, but not to 5 alpha-DHT. A 10-fold excess of the progesterone antagonist RU486 abolishes the stimulation by progesterone, while cotransfection with the coactivator TIF2 increases its activity 6- to 7-fold. A comparison of the ligand specificity with transfected androgen or progesterone receptors indicates that the endogenous receptor is a progesterone receptor. Its presence is confirmed by steroid-binding experiments, RT-PCR and Northern blot analysis. Consequently, progesterone receptor function may be studied conveniently in COS-7 cells without cotransfection of receptor, but the endogenous receptor may interfere in studies of ligand specificity and coactivation of cotransfected receptors.     

Molecular mechanisms involved in progesterone receptor regulation of uterine function

The ovarian steroid hormone progesterone is a major regulator of uterine function. The actions of this hormone is mediated through its cognate receptor, the progesterone receptor, Pgr. Ablation of the Pgr has shown that this receptor is critical for all female reproductive functions including the ability of the uterus to support and maintain the development of the implanting mouse embryo. High density DNA microarray analysis has identified direct and indirect targets of Pgr action. One of the targets of Pgr action is a member of the Hedgehog morphogen Indian Hedgehog, Ihh. Ihh and members of the Hh signaling cascade show a coordinate expression pattern in the mouse uterus during the preimplantation period of pregnancy. The expression of Ihh and its receptor Patched-1, Ptc1, as well as, down stream targets of Ihh-Ptch1 signaling, such as the orphan nuclear receptor COUP-TF II show that this morphogen pathway mediates communication between the uterine epithelial and stromal compartments. The members of the Ihh signaling axis may function to coordinate the proliferation, vascularization and differentiation of the uterine stroma during pregnancy. This analysis demonstrates that progesterone regulates uterine function in the mouse by coordinating the signals from the uterine epithelium to stroma in the preimplantation mouse uterus.     

Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.     

孕激素在自身免疫疾病中的免疫调节作用

孕激素是一种免疫调节分子,它主要通过与孕激素受体结合发挥免疫调节作用。大量研究证实,孕激素受体在多种淋巴细胞表达,孕激素在免疫调节中可诱导Th2细胞反应,抑制Th1细胞反应,抑制巨噬-单核细胞、自然杀伤细胞的杀伤活力。血清孕激素水平降低,孕激素受体表达调节障碍将影响自身免疫功能的调节,导致自身免疫性疾病的发生和发展。     

Distinct properties of cyclin-dependent kinase complexes containing cyclin A1 and cyclin A2

The distinct expression patterns of the two A-type cyclins during spermatogenesis and the absolute requirement for cyclin A1 in this biological process in vivo suggest that they may confer distinct biochemical properties to their CDK partners. We therefore compared human cyclin A1- and cyclin A2-containing CDK complexes in vitro by determining kinetic constants and by examining the complexes for their ability to phosphorylate pRb and p53. Differences in biochemical activity were observed in CDK2 but not CDK1 when complexed with cyclin A1 versus cyclin A2. Further, CDK1/cyclin A1 is a better kinase complex for phosphorylating potentially physiologically relevant substrates pRb and p53 than CDK2/cyclin A2. The activity of CDKs can therefore be regulated depending upon which A-type cyclin they bind and CDK1/cyclin A1 might be preferred in vivo.