Cdk1 Phosphorylation Research Articles

Ska3 Phosphorylated by Cdk1 Binds Ndc80 and Recruits Ska to Kinetochores to Promote Mitotic Progression

The spindle and kinetochore-associated (Ska) protein complex is required for accurate chromosome segregation during mitosis [1-6] and consists of two copies each of Ska1, Ska2, and Ska3 proteins [4, 7]. The Ska complex contains multiple microtubule-binding elements and promotes kinetochore-microtubule attachment [8-11]. The Ska1 C-terminal domain (CTD) recruits protein phosphatase 1 (PP1) to kinetochores to promote timely anaphase onset [12]. The Ska complex regulates, and is regulated by, Aurora B [13]. Aurora B phosphorylates both Ska1 and Ska3 to inhibit the kinetochore localization of the Ska complex [14]. Despite its multitude of functions at kinetochores, how the Ska complex itself is recruited to kinetochores is unclear. It is unknown whether any mitotic kinases positively regulate the localization of the Ska complex to kinetochores. Here, we show that Cdk1 phosphorylates Ska3 to promote its direct binding to the Ndc80 complex (Ndc80C), a core outer kinetochorecomponent. We also show that this phosphorylation occurs specifically during mitosis and is required for the kinetochore localization of the Ska complex. Ska3 mutants deficient in Cdk1 phosphorylation are defective in kinetochore localizationbut retain microtubule localization. These mutants support chromosome alignment but delay anaphase onset. We propose that Ska3 phosphorylated by Cdk1 in mitosis binds to Ndc80C and recruits the Ska complex to kinetochores where Ska1 can bind both PP1 and microtubules to promote anaphase onset.

β Cell Renewal versus Differentiation: Slow and Steady Wins the Race

Pancreatic endocrine progenitor cells proliferate and transiently express the bHLH transcription factor Ngn3. In recent issues of Developmental Cell, Azzarelli etal. (2017) and Krentz etal. (2017) demonstrate how CDK phosphorylation of Ngn3 governs the switch between their renewal and differentiation. Lengthening the cell cycle allows Ngn3 accumulation, outpacing phosphorylation-induced degradation.

JNKs function as CDK4-activating kinases by phosphorylating CDK4 and p21

Cyclin D-CDK4/6 are the first cyclin-dependent kinase (CDK) complexes to be activated by mitogenic/oncogenic pathways. They have a central role in the cell multiplication decision and in its deregulation in cancer cells. We identified T172 phosphorylation of CDK4 rather than cyclin D accumulation as the distinctly regulated step determining CDK4 activation. This finding challenges the view that the only identified metazoan CDK-activating kinase, cyclin H-CDK7-Mat1 (CAK), which is constitutively active, is responsible for the activating phosphorylation of all cell cycle CDKs. We previously showed that T172 phosphorylation of CDK4 is conditioned by an adjacent proline (P173), which is not present in CDK6 and CDK1/2. Although CDK7 activity was recently shown to be required for CDK4 activation, we proposed that proline-directed kinases might specifically initiate the activation of CDK4. Here, we report that JNKs, but not ERK1/2 or CAK, can be direct CDK4-activating kinases for cyclin D-CDK4 complexes that are inactivated by p21-mediated stabilization. JNKs and ERK1/2 also phosphorylated p21 at S130 and T57, which might facilitate CDK7-dependent activation of p21-bound CDK4, however, mutation of these sites did not impair the phosphorylation of CDK4 by JNKs. In two selected tumor cells, two different JNK inhibitors inhibited the phosphorylation and activation of cyclin D1-CDK4-p21 but not the activation of cyclin D3-CDK4 that is mainly associated to p27. Specific inhibition by chemical genetics in MEFs confirmed the involvement of JNK2 in cyclin D1-CDK4 activation. Therefore, JNKs could be activating kinases for cyclin D1-CDK4 bound to p21, by independently phosphorylating both CDK4 and p21.

Abstract IA21: Targeting WEE1 kinase to potentiate chemoradiation in the treatment of pancreatic cancer

Abstract Pancreas cancer is projected to cause approximately 42,000 deaths in 2016, making it the third greatest cause of cancer mortality (behind lung and colorectal, but greater even than breast). Systemic failure remains the dominant cause of death, although local failure is responsible for up to 1/3 of the pancreatic cancer related mortality. We have focused on improving the treatment of locally advanced pancreatic cancer, defined as disease which cannot be resected but has not overtly metastasized, where the issue of local control becomes even more important, and have hypothesized that the treatment of locally advanced pancreatic cancer will require control of both the gross primary tumor as well as the microscopic disease that exists in virtually every patient. Gemcitabine has been a mainstay of treatment for pancreatic cancer, and we have standardized combining it with radiation for locally advanced disease. Because our initial studies revealed that activation of the DNA damage response, particularly activation of CHK1 and WEE1, were crucial in the defense pancreatic cancers mount against chemotherapy and radiation, we have studied the effect of CHK1 and WEE1 inhibitors on gemcitabine and gemcitabine-radiation mediated cytotoxicity. These inhibitors abrogate the G2 checkpoint after DNA damage. The initial hypothesis for using CHK1 and WEE1 inhibitors was that cancer cells lacking a p53 checkpoint would be selectively sensitized, compared to normal cells, because normal cells would be protected by an intact G1 checkpoint. After initial studies with several agents, we have focused on the WEE1 inhibitor AZD1775, chiefly because it was available for a clinical trial. We have carried out both mechanistic and clinical studies using AZD1775. Mechanistically, we have evaluated both the contributions of homologous recombination repair (HRR) inhibition and G2 checkpoint abrogation by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistoneH3 flow cytometry, respectively in pancreatic cancer model systems. AZD1775 causes inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. Sensitization by AZD1775 is associated with persistent γH2AX and inhibition of RAD51 focus formation. Using BRCA2 isogenic cells, we have found that AZD1775 sensitizes to gemcitabine-radiation in BRCA2 wild type cells, while in BRCA2 null cells AZD1775 did not sensitize despite significant G2 checkpoint abrogation. In vivo, in patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibits tumor growth and impairs RAD51 focus formation in response to gemcitabine-radiation. We've concluded that, whereas sensitization is accompanied by inhibition of CDK1 phosphorylation and abrogation of the G2 checkpoint, this mechanism is not sufficient for sensitization. Our findings suggest that sensitization to chemoradiation by WEE1 inhibition also requires inhibition of HRR. The importance of HRR has also supported studies combining CHK1 or WEE1 inhibition with a PARP inhibitor, which would be anticipated to potentiate the effects of HRR inhibition. Based on these studies, we have launched a clinical trial combining gemcitabine and gemcitabine-radiation, at standard doses, with dose escalating AZD1775 for patients with locally advanced pancreatic cancer. Patients receive one cycle of gemcitabine with AZD1775, then 2 cycles of gemcitabine-radiation with AZD1775, then another cycle of AZD1775-gemcitabine, followed by assessment for response and toxicity. Optional cycles of AZD1775 and gemcitabine are then given to patients who have not progressed. AZD1775 has been escalated according to a Time-to-Event Continual Reassessment Model, in which the dose for each patient is based on the toxicity profile of all prior patients, and the goal is to establish a tolerable dose of the combination therapy and to estimate the response and survival. With 21 patients entered onto the study, the median survival has not been reached, and the 64% survival at the 18-month landmark point exceeds the 18-month median survival that was the agreed on endpoint for triggering a larger trial. The trial also includes a pharmacodynamic endpoint of a hair follicle biopsy to assess target engagement. In summary, these findings suggest that WEE1 inhibition may act through both G2 checkpoint abrogation but, more importantly, inhibition of HRR, and is a promising potentiator of gemcitabine-radiation in the treatment of locally advanced pancreatic cancer. Supported by P50 CA130810, R01 CA163895, P30 CA46592, and the Taubman Institute Citation Format: Theodore S. Lawrence, Meredith A. Morgan. Targeting WEE1 kinase to potentiate chemoradiation in the treatment of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA21.

Abstract P6-07-02: CDK4 phosphorylation status and corresponding gene expression profile predict sensitivity to Palbociclib

Abstract P6-07-02: CDK4 phosphorylation status and corresponding gene expression profile predict sensitivity to Palbociclib

PP2ACdc55 Phosphatase Imposes Ordered Cell-Cycle Phosphorylation by Opposing Threonine Phosphorylation

SummaryIn the quantitative model of cell-cycle control, progression from G1 through S phase and into mitosis is ordered by thresholds of increasing cyclin-dependent kinase (Cdk) activity. How such thresholds are read out by substrates that respond with the correct phosphorylation timing is not known. Here, using the budding yeast model, we show that the abundant PP2ACdc55 phosphatase counteracts Cdk phosphorylation during interphase and delays phosphorylation of late Cdk substrates. PP2ACdc55 specifically counteracts phosphorylation on threonine residues, and consequently, we find that threonine-directed phosphorylation occurs late in the cell cycle. Furthermore, the late phosphorylation of a model substrate, Ndd1, depends on threonine identity of its Cdk target sites. Our results support a model in which Cdk-counteracting phosphatases contribute to cell-cycle ordering by imposing Cdk thresholds. They also unveil a regulatory principle based on the phosphoacceptor amino acid, which is likely to apply to signaling pathways beyond cell-cycle control.

The c-Abl inhibitor in Parkinson disease

Parkinson's disease (PD) is an insidious onset neurodegenerative disease affecting approximately 1% of the population over the age of 65. So far available therapies for PD have only aimed at improving or alleviating symptoms, but not at slowing, preventing, and reversing the course of PD. Recently, some studies have indicated that the levels and activation of Abelson non-receptor tyrosine kinase (c-Abl, Abl1) were up-regulated in the brain tissue of patients with PD and demonstrated that c-Abl inhibitors could improve motor behavior, prevent the loss of dopamine neurons, inhibit phosphorylation of Cdk5, regulate α-synuclein phosphorylation and clearance, inhibit the tyrosine phosphorylation of parkin and decrease parkin substrate, for example, PARIS (zinc finger protein 746), AIMP2 (aminoacyl-tRNA synthetase-interacting multifunctional protein type2), FBP1 (fuse-binding protein 1), and synphilin-1. Therefore, we review the mechanism of the c-Abl inhibitor in PD and conclude that c-Abl inhibitors may be a potential treatment in PD and other neurodegenerative disease.

Re-examining the role of Cdc14 phosphatase in reversal of Cdk phosphorylation during mitotic exit.

Inactivation of cyclin-dependent kinase (Cdk) and reversal of Cdk phosphorylation are universally required for mitotic exit. In budding yeast (Saccharomyces cerevisiae), Cdc14 is essential for both and thought to be the major Cdk-counteracting phosphatase. However, Cdc14 is not required for mitotic exit in many eukaryotes, despite highly conserved biochemical properties. The question of how similar enzymes could have such disparate influences on mitotic exit prompted us to re-examine the contribution of budding yeast Cdc14. By using an auxin-inducible degron, we show that severe Cdc14 depletion has no effect on the kinetics of mitotic exit and bulk Cdk substrate dephosphorylation, but causes a cell separation defect and is ultimately lethal. Phosphoproteomic analysis revealed that Cdc14 is highly selective for distinct Cdk sites in vivo and does not catalyze widespread Cdk substrate dephosphorylation. We conclude that additional phosphatases likely contribute substantially to Cdk substrate dephosphorylation and coordination of mitotic exit in budding yeast, similar to in other eukaryotes, and the critical mitotic exit functions of Cdc14 require trace amounts of enzyme. We propose that Cdc14 plays very specific, and often different, roles in counteracting Cdk phosphorylation in all species.

Paris polyphylla Suppresses Proliferation and Vasculogenic Mimicry of Human Osteosarcoma Cells and Inhibits Tumor Growth In Vivo.

Paris polyphylla, a traditional antipyretic-detoxicate chinese medicinal herb, has been applied extensively in cancer treatments for nearly 2000 years. The purpose of the present study is to evaluate the potential anti-osteosarcoma effects of Paris polyphylla ethanol extract (PPEE) and to investigate its underlying mechanisms. The antiproliferation activity of PPEE was tested on 143B, MG-63, U-2 OS and hFOB1.19 cells using MTT assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by Hoechst 33342 staining and flow cytometry. The antimigratory, anti-invasive and antivasculogenic mimicry (VM) effects of PPEE were investigated by wound healing, Transwell and 3D culture assays. Mouse xenograft model was used to examine its anti-osteosarcoma efficacy in vivo. Hematologic profiles and hepatorenal functions were evaluated to assess the toxicity of PPEE.PPEE evidently suppressed cell proliferation of 143B, MG-63 and U-2 OS with IC50 values of 10-60[Formula: see text][Formula: see text]g/mL, but showed little cytotoxicity against normal osteoblastic cell. PPEE promoted apoptosis in 143B cell via caspase activation, increased Bax/Bcl-2 ratio and PARP cleavage. It also induced G2/M phase arrest associated with elevated phosphorylation of CDK1, Cdc25C, Chk2 and down-regulation of cyclin B1, CDK1, Cdc25C expression. Additionally, PPEE inhibited 143B cell migration, invasion and VM formation at noncytotoxic concentrations through decreasing the expression of FAK, Mig-7, MMP2 and MMP9. Finally, daily oral administration of PPEE for four weeks exhibits potent antitumor and anti-VM activity in 143B xenograft model with low toxicity. Taken together, these findings demonstrated PPEE possesses anti-osteosarcoma and anti-VM activity in vitro and in vivo, and therefore is a potential candidate for osteosarcoma treatment.

Cloning, localization and focus formation at DNA damage sites of canine Ku70

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair machinery, specifically non-homologous DNA-end joining (NHEJ), is crucial for developing next-generation radiotherapies and common chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, might play vital roles for regulation of NHEJ activity. The human Ku heterodimer (Ku70/Ku80) is a core NHEJ factor in the NHEJ pathway and is involved in sensing of DSBs. Companion animals, such as canines, have been proposed to be an excellent model for cancer research, including development of chemotherapeutics. However, the post-translational modifications, localization and complex formation of canine Ku70 have not been clarified. Here, we show that canine Ku70 localizes in the nuclei of interphase cells and that it is recruited quickly at laser-microirradiated DSB sites. Structurally, two DNA-PK phosphorylation sites (S6 and S51), an ubiquitination site (K114), two canonical sumoylation consensus motifs, a CDK phosphorylation motif, and a nuclear localization signal (NLS) in the human Ku70 are evolutionarily conserved in canine and mouse species, while the acetylation sites in human Ku70 are partially conserved. Intriguingly, the primary candidate nucleophile (K31) required for 5’dRP/AP lyase activity of human and mouse Ku70 is not conserved in canines, suggesting that canine Ku does not possess this activity. Our findings provide insights into the molecular mechanisms of Ku-dependent NHEJ in a canine model and form a platform for the development of next-generation common chemotherapeutics for human and animal cancers.

Cdk5 Regulation of the GRAB-Mediated Rab8-Rab11 Cascade in Axon Outgrowth.

While axon outgrowth requires membrane supply for surface expansion, the molecular mechanisms regulating the membrane transport in growing axons remain unclear. Here, we demonstrate that GRAB, a guanine nucleotide exchange factor for Rab8, is a novel regulator of axon outgrowth. GRAB promotes the axonal membrane transport by mediating the interaction between Rab11 and Rab8 in neurons. The activity of GRAB is regulated by phosphorylation with Cdk5. We describe an in vivo role for GRAB and its Cdk5 phosphorylation during neuronal migration and process formation in embryonic brains. Thus, the membrane supply for axonal outgrowth is regulated by Cdk5 through the Rab11-GRAB-Rab8 cascade.

Inhibition of WEE1 Induces Cell Death of Both Bortezomib- and Lenalidomide-Resistant Multiple Myeloma Cells: A Novel Therapeutic Approach Targeting Cell-Cycle Checkpoint Kinase

Multiple myeloma (MM) is a hematological malignancy that derives from the proliferation of unregulated plasma cells. Dramatic improvement in the clinical outcomes of both newly diagnosed and relapsed/refractory patients with MM has been achieved using many clinical approaches, including use of high-dose chemotherapy followed by hematopoietic stem cell transplantation, and new drugs, such as proteasome inhibitors, immunomodulatory drugs, and histone deacetylase inhibitors. However, most patients eventually relapse and develop drug resistance. Moreover, the prognosis of patients with bortezomib (BTZ) and/or lenalidomide (LEN)-resistant MM (key drugs in the treatment of MM) is very poor. Therefore, novel therapeutic approaches to overcome BTZ and LEN resistance are urgently needed in clinical settings. WEE1 is a cell-cycle checkpoint kinase and a key regulator of DNA damage surveillance pathways. In response to extrinsically induced DNA damage, WEE1 catalyzes inhibitory phosphorylation of both cyclin-dependent kinase1 and 2 (CDK1 and CDK2), leading to CDK1- and CDK2-induced cell cycle arrest at the G1, S, or G2-M phases. This cell-cycle arrest, in turn, allows for the damaged DNA to be repaired before the cell undergoes DNA replication, and prevents cells harboring unrepaired damaged DNA from mitotic lethality. Furthermore, recent research has shown that knockdown of WEE1 leads to DNA double-strand breaks specifically in S-phase cells undergoing DNA replication, and that WEE1 is most active in the S-phase, suggesting that WEE1 is involved in DNA synthesis. Overexpression of WEE1 has been observed in many types of cancers, including hepatic cancer, breast cancer, glioblastoma and gastric cancer, and high expression of WEE1 has been shown to correlate with poor prognosis. In addition, research has shown that inhibition of checkpoint kinase 1 (Chk1), a critical transducer of the DNA damage response, potentiates the cytotoxicity of chemotherapy on p53-deficient MM cells, which are regarded as chemotherapy-resistant, suggesting that inhibition of cell-cycle checkpoint kinase is involved in re-sensitization of refractory MM cells to anticancer drugs. These data suggest that WEE1 might be an attractive target for novel therapeutic agents against this incurable hematological malignancy. MK-1775 is a potent and highly-selective small-molecule inhibitor of WEE1. In the present study, we investigated the role of WEE1 in MM as a potential therapeutic target using MK-1775. MTSassays showed that single agent MK-1775 inhibited the proliferation of various MM cell lines, including the intrinsically LEN-resistant cell line, RPMI-8226, in a dose- (0 to 10 mM) and time- (0 to 72 h) dependent manner. Furthermore, the growth inhibition effect is irrespective of p53 status. To examine the mechanisms behind the growth inhibition effect induced by MK-1775, assays for apoptotic cell death were performed. These assays demonstrated that MK-1775 induces both early and late apoptosis in MM cells. To investigate the molecular mechanisms of MK-1775-induced cell death in MM cells, the expression of various cell death-associated proteins and downstream molecules of WEE1 were examined. Western blotting analysis showed that MK-1775 arrested cell growth and induced apoptotic cell death in MM cells in a dose-dependent manner by inhibiting both, the expression of the target molecules of Bcl-2 and MCL1, and the cleavage of PARP and Caspase 3. Similarly, there was a substantial inhibition of CDK1 phosphorylation downstream of WEE1. Moreover, an increased expression of histone H2AX was observed following administration of MK-1775, suggesting that MK-1775 results in cytotoxicity by direct DNA damage. Next, we examined the effects of MK-1775 on BTZ-resistant MM cells. Interestingly, MK-1775 inhibited the proliferation of both BTZ-sensitive wild-type MM cells and BTZ-resistant MM cells, suggesting that BTZ resistance can be overcome by targeting WEE1. Furthermore, in combination with BTZ, MK-1775 was able to re-sensitize BTZ-resistant MM cells to BTZ. These results indicate that inhibition of WEE1 might serve as an attractive therapeutic option for patients with both BTZ-resistant and LEN-resistant MM. In conclusion, our data suggest that WEE1 might be a promising molecular target for the treatment of MM. DisclosuresTokuhira:Bristol Myers Squibb Co., Ltd: Honoraria; Pfizer Co., Ltd: Honoraria; Eizai Co., Ltd: Honoraria.

AMG925, a Dual FLT3-CDK4/6 Inhibitor, Disrupts Survival Signaling and Triggers Apoptosis in AML Progenitor/Stem Cells

FLT3 mutations are detected in one third of patients diagnosed with acute myeloid leukemia (AML) and confer poor outcomes. Selective FLT3 inhibitors have been developed and are being tested at different stages of clinical development. Drug resistance triggered by inhibitor-induced secondary mutations is often observed, and is a major cause of treatment failure. AMG925 (FLX925) is a small molecule dual FLT3-CDK4/6 inhibitor, currently in clinical development. Because AMG925 concomitantly inhibits both FLT3 and CDK4/6, AMG925 treatment rarely causes development of secondary drug-resistant FLT3 mutations (Li C et. al Mol. Cancer Ther. 2015). In this study, we examined the anti-leukemic effect of AMG925 on AML, focusing on AML progenitor/stem cells. Utilizing the high-throughput technology of time-of-flight mass cytometry (CyTOF) and reverse phase protein array (RPPA), we investigated alterations of the FLT3-mediated signaling network trigged by AMG925 at the single cell level. While AMG925 caused cell growth inhibition and apoptosis in both, FLT3-mutated (MOLM13, MV4;11, Ba/F3-ITD, Ba/F3-D835Y and Ba/F3-ITD/D835Y) and -wild type (Ba/F3-wt, OCI-AML3 and U937) cell lines and primary AML blasts, it elicited maximal response in cells carrying ITD, D835Y and ITD/D835Y double mutations. Mechanistically, AMG925 attenuates CDK4 phosphorylation of retinoblastoma protein (Rb), inhibits FLT3 activation and abrogates FLT3-dependent and independent AKT/mTOR, MEK/ERK and STAT5 signaling. AMG925 at 10nM effectively inhibited colony formation of PDX-derived leukemic stem cells originating from 2 AML patients carrying FLT3-ITD mutation (54±8.7% inhibition, n=4). CyTOF analysis of cell surface and intracellular molecules measured by a panel of 36 antibodies revealed that the inhibitory effect of AMG925 on p-AKT, p-ERK and p-STAT5 is operational primarily in subpopulations expressing the stem cell markers CD123, CD117, CD34, CD133 and CD47. Using RPPA, we studied the expression and phosphorylation changes of 273 proteins from the same 2 AML-ITD xenograft samples in response to AMG925 ex-vivo. Heterogeneous expression signatures were observed in all samples isolated from bone marrows (n=1) and spleens (n=4). AMG925 treatment altered multiple signaling networks by inhibiting key molecules in the AKT/mTOR, cyclin D family, MEK/ERK and Src pathways, and triggered a stress-response by up-regulating STAT3 and TSC1 in all samples examined. The combinational consequences of targeting FLT3-CDK4/6 and the proteins that contribute to resistance are under investigation. Conclusions: combined FLT3-CDK4/6 inhibition by AMG925 results in growth inhibition and apoptosis of AML cells with or without FLT3 mutations and selectively inhibits signaling within PDX and phenotypically defined AML stem/progenitor cell populations. CyTOF analysis confirmed on-target effects of AMG925 on FLT3-regulated signaling in AML stem cell subsets. RPPA profiling of AML-ITD xenografts exposed ex-vivo to AMG925 revealed alterations in multiple pro-survival signaling pathways and identified the upregulation of alternative pathways potentially conferring resistance to AMG925. While our results indicate efficacy of combined FLT3-CDK4/6 inhibition it identifies unexpected resistance mechanisms that provide rationale for the additional mechanism-based combinatorial approaches to increase the therapeutic efficacy in AML DisclosuresShah:ARIAD: Research Funding; BMS: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Research Funding; Plexxikon: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

The mTOR Complex Controls HIV Latency

A population of CD4T lymphocytes harboring latent HIV genomes can persist in patients on antiretroviral therapy, posing a barrier to HIV eradication. To examine cellular complexes controlling HIV latency, we conducted a genome-wide screen with a pooled ultracomplex shRNA library and invitro system modeling HIV latency and identified the mTOR complex as a modulator of HIV latency. Knockdown of mTOR complex subunits or pharmacological inhibition of mTOR activity suppresses reversal of latency in various HIV-1 latency models and HIV-infected patient cells. mTOR inhibitors suppress HIV transcription both through the viral transactivator Tat and via Tat-independent mechanisms. This inhibition occurs at least in part via blocking the phosphorylation of CDK9, a p-TEFb complex member that serves as a cofactor for Tat-mediated transcription. The control of HIV latency by mTOR signaling identifies a pathway that may have significant therapeutic opportunities.

Targeting CCR3 to Reduce Amyloid-β Production, Tau Hyperphosphorylation, and Synaptic Loss in a Mouse Model of Alzheimer's Disease.

The majority of Alzheimer's disease (AD) patients have a late onset, and chronic neuroinflammation, characterized by glial activation and secretion of pro-inflammatory cytokines and chemokines, plays a role in the pathogenesis of AD. The chemokine CCL11 has been shown to be a causative factor of cognitive decline in the process of aging, but little is known whether it is involved in the pathogenesis of AD. In the present study, we showed that CCR3, the receptor for CCL11, was expressed by hippocampal neurons and treatment of primary hippocampal neuronal cultures (14days in vitro) with CCL11 resulted in activation of cyclin-dependent kinase 5 and glycogen synthase kinase-3β, associated with elevated tau phosphorylation at multiple sites. CCL11 treatment also induced the production of Aβ and dendritic spine loss in the hippocampal neuronal cultures. All these effects were blocked by the CCR3 specific antagonist, GW766994. An age-dependent increase in CCL11, predominantly expressed by the activated microglia, was observed in the cerebrospinal fluid of both APP/PS1 double transgenic mice and wild-type (WT) littermates, with a markedly higher level in APP/PS1 double transgenic mice than that in WT littermates. Deletion of CCR3 in APP/PS1 double transgenic mice significantly reduced the phosphorylation of CDK5 and GSK3β, tau hyperphosphorylation, Aβ deposition, microgliosis, astrogliosis, synaptic loss, and spatial learning and memory deficits. Thus, the age-related increase in CCL11 may be a risk factor of AD, and antagonizing CCR3 may bring therapeutic benefits to AD.

A Substrate Trapping Method for Identification of Direct Cdc14 Phosphatase Targets.

Mitotic exit requires the inactivation of cyclin-dependent kinase (Cdk) activity and reversal of Cdk-mediated phosphorylation events by protein phosphatases. In Saccharomyces cerevisiae the mitotic exit network (MEN) leads to activation and dispersal of the Cdc14 phosphatase throughout the cell following successful chromosome segregation. MEN-released Cdc14 is required for both full Cdk inactivation and dephosphorylation of Cdk substrates. While Cdc14 originally was thought to act broadly on mitotic Cdk substrates, recent biochemical studies revealed that Cdc14 possesses a strong preference for a subset of Cdk phosphorylation sites. This intrinsic specificity appears well conserved across fungi and animals. Identifying the direct physiological substrates of Cdc14 is an important step in fully understanding its biological functions, both in yeast and other species. Despite its strict specificity for phosphoserine Cdk sites, Cdc14 is structurally and mechanistically related to protein tyrosine phosphatases (PTPs). Like other PTPs, mutation of catalytic residues in the Cdc14 active site creates an inactive enzyme that retains high affinity substrate binding. Here we describe a protocol for using such "substrate trap" variants to biochemically isolate and detect direct substrates by co-immunopurification. The protocol is written for use in S. cerevisiae, but should be easily adaptable to other research organisms.

Dynamic Partitioning of Synaptic Vesicle Pools by the SNARE-Binding Protein Tomosyn.

Although information transfer at central synapses via sustained high-frequency neural activity requires coordinated synaptic vesicle (SV) recycling, the mechanism(s) by which synapses sense and dynamically modify SV pools to match network demands remains poorly defined. To advance understanding, we quantified SV pool sizes and their sensitivity to neural activity while altering Tomo1 expression, a putative regulator of the presynaptic Readily Releasable Pool. Remarkably, we find Tomo1 actions to extend beyond the Readily Releasable Pool to mediate the Total Recycling Pool and SV Resting Pool distribution, and this action is sensitive to neural activity through Cdk5 phosphorylation of Tomo1. Moreover, Tomo1 appears to exert these actions through interaction with Rab3A-GTP and synapsin proteins. Together, our results argue that Tomo1 is a central mediator of SV availability for neurotransmission.

Inhibitory phosphorylation of Cdk1 mediates prolonged prophase I arrest in female germ cells and is essential for female reproductive lifespan.

A unique feature of female germ cell development in mammals is their remarkably long arrest at the prophase of meiosis I, which lasts up to 50 years in humans. Both dormant and growing oocytes are arrested at prophase I and completely lack the ability to resume meiosis. Here, we show that the prolonged meiotic arrest of female germ cells is largely achieved via the inhibitory phosphorylation of Cdk1 (cyclin-dependent kinase 1). In two mouse models where we have introduced mutant Cdk1T14AY15F which cannot be inhibited by phosphorylation (Cdk1AF) in small meiotically incompetent oocytes, the prophase I arrest is interrupted, leading to a premature loss of female germ cells. We show that in growing oocytes, Cdk1AF leads to premature resumption of meiosis with condensed chromosomes and germinal vesicle breakdown followed by oocyte death, whereas in dormant oocytes, Cdk1AF leads to oocyte death directly, and both situations damage the ovarian reserve that maintains the female reproductive lifespan, which should be around 1 year in mice. Furthermore, interruption of the inhibitory phosphorylation of Cdk1 results in DNA damage, which is accompanied by induction of the Chk2 (checkpoint kinase 2)-p53/p63-dependent cell death pathway, which eventually causes global oocyte death. Together, our data demonstrate that the phosphorylation-mediated suppression of Cdk1 activity is one of the crucial factors that maintain the lengthy prophase arrest in mammalian female germ cells, which is essential for preserving the germ cell pool and reproductive lifespan in female mammals.

Suppression of Vimentin Phosphorylation by the Avian Reovirus p17 through Inhibition of CDK1 and Plk1 Impacting the G2/M Phase of the Cell Cycle

The p17 protein of avian reovirus (ARV) causes cell cycle retardation in a variety of cell lines; however, the underlying mechanism(s) by which p17 regulates the cell cycle remains largely unknown. We demonstrate for the first time that p17 interacts with CDK1 and vimentin as revealed by reciprocal co-immunoprecipitation and GST pull-down assays. Both in vitro and in vivo studies indicated that direct interaction of p17 and CDK1/vimentin was mapped within the amino terminus (aa 1–60) of p17 and central region (aa 27–118) of CDK1/vimentin. Furthermore, p17 was found to occupy the Plk1-binding site within the vimentin, thereby blocking Plk1 recruitment to CDK1-induced vimentin phosphorylation at Ser 56. Interaction of p17 to CDK1 or vimentin interferes with CDK1-catalyzed phosphorylation of vimentin at Ser 56 and subsequently vimentin phosphorylation at Ser 82 by Plk1. Furthermore, we have identified upstream signaling pathways and cellular factor(s) targeted by p17 and found that p17 regulates inhibitory phosphorylation of CDK1 and blocks vimentin phosphorylation at Ser 56 and Ser 82. The p17-mediated inactivation of CDK1 is dependent on several mechanisms, which include direct interaction with CDK1, p17-mediated suppression of Plk1 by activating the Tpr/p53 and ATM/Chk1/PP2A pathways, and p17-mediated cdc25C degradation via an ubiquitin- proteasome pathway. Additionally, depletion of p53 with a shRNA as well as inhibition of ATM and vimentin by inhibitors diminished virus yield while Tpr and CDK1 knockdown increased virus yield. Taken together, results demonstrate that p17 suppresses both CDK1 and Plk1functions, disrupts vimentin phosphorylation, causes G2/M cell cycle arrest and thus benefits virus replication.

A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit.

PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis.