Cdk Phosphorylation Sites Research Articles

Abstract P6-04-12: CDK inhibition mediates tumor suppressive Smad3 action and decreased metastatic phenotypes in triple negative breast cancer cells

Abstract Introduction: Breast cancer onset and disease progression has been linked to members of the TGFb superfamily and their downstream signaling components, the Smads. Changes in Smad signaling have been associated with the dichotomous role of TGFβ in malignancy, mediating both tumor suppressant and tumor promoting behaviors in breast cancer. Our previous work showed that cyclin/CDK mediated phosphorylation of Smad3 resulted in the inhibition of canonical tumor suppressant Smad3 action. We thus hypothesized that activation of CDKs leads to phosphorylation and inhibition of Smad3, releasing cell cycle arrest and promoting cell proliferation and metastasis. Using triple negative breast cancer cell lines, we examined the impact of specific CDK inhibitors (CDKis) on signaling patterns, metastatic phenotypes, and protein expression profiles. Methods: Triple negative cell lines Hs578T, MDA-MB-231 and MDA-MB-436 were treated with CDK2i or CDK4i and Smad3 activity was measured using a luciferase assay. Transwell migration and Matrigel invasion assays were used to show the effect of transfection with Smad3 CDK phosphorylation site mutants, resistant to inhibitory CDK phosphorylation, or treatment with CDK2i or CDK4i on the study cells. Immunoblotting was performed to measure protein expression levels of Smad3, pSmad3 T179, and MMP2. A novel live cell array was implemented to quantify changes in activity of 14 cancer-related transcription factors over a period of 3 days. TUNEL and Ki67 staining analysis and a xenograft mouse model were used to determine impact of CDKis, in combination with paclitaxel chemotherapy, on apoptosis and tumor growth respectively. Results: Treatment of study cells with CDK2i or CDK4i resulted in increased Smad3 activity and decreased migration and invasion in the study cells. Transfection with a 5M Smad3 mutant construct, containing mutations in all 5 CDK phosphorylation sites, resulted in the greatest decrease in cell migration, when compared with cells transfected with both vector control or WT Smad3. CDK2i or CDK4i therapy resulted in decreased Smad3 protein phosphorylation at the T179 site, while total Smad3 levels were unaffected. CDK2i/4i treatment also resulted in decreased MMP2 expression. The array studies revealed decreased activity of EMT transcription factors B-catenin, Snail, Twist, in MDA-MB-231 cells following CDK2i treatment. CDK2i and paclitaxel combination treatment resulted in increased apoptosis and decreased tumor volume and Ki67 staining compared to control treatment. Conclusions: We have shown for the first time that cyclin/CDK activity, in part, mediates the metastasis-associated role of Smad3 in triple negative breast cancer cells. Importantly, both CDK2i and CDK4i treatment resulted in decreased cell migration/invasion and also corresponded to decreased MMP2 expression. Lastly, the array studies revealed CDK2i mediated effects on key transcription factors associated with the promotion of EMT. Future studies will explore the significance of Smad3 interaction with these EMT factors and will pursue the clinical application of CDK2i for patients with triple negative/cyclin overexpressing breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-12.

Abstract P5-11-01: Impact of CDK mediated Smad3 phosphorylation and Pin1 in triple negative breast cancer cell migration

Abstract Introduction: Estrogen receptor (ER), progesterone receptor (PR), and Human Epidermal Growth Factor Receptor 2 (HER2) negative breast cancers, also known as triple negative breast cancers can overexpress cyclins, such as cyclin D and E, which have been linked to cancer progression and metastasis. Cyclin/CDK complexes can phosphorylate and regulate Smads, downstream signaling proteins in the transforming growth factor-beta (TGF-b) superfamily. CDKs phosphorylate Smad3, predominantly in the linker region, resulting in the decreased tumor suppressive role of this protein. The peptidyl-prolyl cis/trans isomerase Pin1 is also overexpressed in breast cancer. Pin1 can bind phosphorylated Smads contributing to TGF-β induced cell migration and invasion. Inhibition of CDK-mediated Smad3 phosphorylation has been shown to restore Smad3 activity and decrease tumor cell migration in vitro. Based on these findings, we hypothesized that inhibition of Pin1 or CDK-mediated Smad3 phosphorylation would result in decreased triple negative breast cancer cell migration. Methods: Hs578T, MDA-MB-436, and MDA-MB-231 triple negative breast cancer cells (TN) were transfected with either an si control (siCN) or an siPin1 plasmid to knockdown Pin1 protein expression. Transwell migration assays were used to determine the effect of Pin1 knockdown on migration. TN cells were treated with CDK4 or CDK2 inhibitors alone or in combination. The study cells were also stably transduced with lentiviral empty vector, wild-type (WT) Smad3, or a Smad3 mutant (5M) resistant to CDK-mediated phosphorylation. The effect of pharmacologically or genetically inhibiting CDK-mediated Smad3 phosphorylation on Pin1 binding to phosphorylated Smad3 (p-Smad3) or total Smad3 was determined using a co-immunoprecipitation assay (Co-IP). Results: Transfecting the study cells with siPin1 resulted in decreased cell migration when compared to the siCN transfected cells. Treatment with the CDK4 or CDK2 inhibitors alone or in combination did not affect Pin1 protein levels, but resulted in decreased Pin1 binding to p-Smad3 with the greatest decrease found after treatment with combination therapy. Similarly, transduction of the study cells with the WT or the 5M Smad3 constructs had no effect on Pin1 levels but resulted in decreased Pin1 binding to Smad3. The greatest decrease was observed in the 5M transduced cells. Concusions: Inhibition of CDK-mediated Smad3 phosphorylation and knock down of Pin1 resulted in decreased migration of TN breast cancer cells. Inhibition of CDK-mediated Smad3 phosphorylation by mutation of the CDK phosphorylation sites or by treatment with CDK4 and CDK2 inhibitors negatively impacted Pin1 binding to Smad3. Together, these findings indicate that CDK-mediated Smad3 phosphorylation enhances Pin1 binding to Smad3 and promotes tumor cell migration in triple negative breast cancer cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-11-01.

A Wee1 checkpoint inhibits anaphase onset

Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the budding yeast Wee1 kinase, Swe1, also restrains the metaphase-to-anaphase transition by preventing Cdk1 phosphorylation and activation of the mitotic form of the anaphase-promoting complex/cyclosome (APC(Cdc20)). Deletion of SWE1 or its opposing phosphatase MIH1 (the budding yeast cdc25(+)) altered the timing of anaphase onset, and activation of the Swe1-dependent morphogenesis checkpoint or overexpression of Swe1 blocked cells in metaphase with reduced APC activity in vivo and in vitro. The morphogenesis checkpoint also depended on Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). cdc55Δ checkpoint defects were rescued by mutating 12 Cdk1 phosphorylation sites on the APC, demonstrating that the APC is a target of this checkpoint. These data suggest a model in which stepwise activation of Cdk1 and inhibition of PP2A(Cdc55) triggers anaphase onset.

Comprehensive Proteomics Analysis Reveals New Substrates and Regulators of the Fission Yeast Clp1/Cdc14 Phosphatase

The conserved family of Cdc14 phosphatases targets cyclin-dependent kinase substrates in yeast, mediating late mitotic signaling events. To discover substrates and regulators of the Schizosaccharomyces pombe Cdc14 phosphatase Clp1, TAP-tagged Clp1, and a substrate trapping mutant (Clp1-C286S) were purified from asynchronous and mitotic (prometaphase and anaphase) cells and binding partners were identified by 2D-LC-MS/MS. Over 100 Clp1-interacting proteins were consistently identified, over 70 of these were enriched in Clp1-C286S-TAP (potential substrates) and we and others detected Cdk1 phosphorylation sites in over half (44/73) of these potential substrates. According to GO annotations, Clp1-interacting proteins are involved in many essential cellular processes including mitosis, cytokinesis, ribosome biogenesis, transcription, and trafficking among others. We confirmed association and dephosphorylation of multiple candidate substrates, including a key scaffolding component of the septation initiation network called Cdc11, an essential kinase of the conserved morphogenesis-related NDR kinase network named Shk1, and multiple Mlu1-binding factor transcriptional regulators. In addition, we identified Sal3, a nuclear β-importin, as the sole karyopherin required for Clp1 nucleoplasmic shuttling, a key mode of Cdc14 phosphatase regulation. Finally, a handful of proteins were more abundant in wild type Clp1-TAP versus Clp1-C286S-TAP, suggesting that they may directly regulate Clp1 signaling or serve as scaffolding platforms to localize Clp1 activity.

Abstract 1788: The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition.

Abstract Background: Triple Negative Breast Cancer (TNBC) comprises 15% of all breast cancers, and has a poor prognosis relative to other breast cancer subtypes. Inhibition of poly (ADP-ribose) polymerase (PARP) causes the degeneration of single-strand DNA breaks to more lethal double-strand breaks (DSBs), and also traps PARP-DNA complexes, which must be repaired or bypassed by homologous recombination (HR). BRCA1 plays a critical role in HR and its activity is in part regulated by cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation. PARP inhibition results in synthetic lethality in cells that have impaired HR, such as BRCA-deficient cells. Other CDK family members activate additional components of the HR pathway. Here, we show that HR-proficient TNBC cells can be sensitized to PARP inhibition through use of CDK inhibition to impair BRCA1 function and disrupt HR. Methods: We examined the effects of dinaciclib (CDK1, 2, 5 and 9 inhibitor), γ-irradiation and veliparib (PARP inhibitor) on a panel of BRCA1 wild type TNBC cell lines (MDA-MB-231, MDA-MB-468, and BT549). Levels of phospho-S1497 BRCA1 (pBRCA1; CDK phosphorylation site), γ-H2AX, and RAD51 were measured by western blot to assess effects on HR proteins. A direct assessment of DSB repair was measured using the U2OS-DR-GFP reporter system. Cell viability was measured using Cell-Titer Glo assays. Patient-derived TNBC xenograft models were established in immunocompromised NOD-SCID-IL2γ-/- mice, and implanted orthotopically into cohorts of mice for in vivo efficacy studies. Results: There was a significant reduction in total and pBRCA1 and RAD51 protein levels with increasing concentrations of dinaciclib in all TNBC cell lines. In response to 10 Gy γ-irradiation treatment, pretreatment with dinaciclib (20 nM) reduced the percentage of cells with greater than five BRCA1 foci from 54% to 5% (P < 0.001); and the percentage of cells with greater than 5 RAD51 foci from 26% to 4% (P = 0.001). In U2OS-DR-GFP assays, expression of GFP was detected in 3.4% of the vehicle group and in 1.9% of dinaciclib treated cells. Cell viability assays following five days of dinaciclib, veliparib or the combination revealed in vitro cytotoxic synergy in all three TNBC cell lines. In an orthotopic TNBC model derived from a primary patient sample, the relative tumor volumes over a 30-day treatment period for vehicle, dinaciclib, veliparib and combination- treated mice were 5.6-fold, 3.3-fold, 3.2-fold and 0.66-fold (P < 0.001) that of the initial tumor volume at the start of treatment. Tumor volume decreased only in the combination arm. Conclusion: CDK inhibition effectively inhibits HR, and renders BRCA-proficient TNBC cells sensitive to PARP inhibition. This combination represents an effective strategy for the treatment of TNBC. Citation Format: Shawn F. Johnson, Neil Johnson, David Chi, Benjamin Primack, Alan D. D'Andrea, Elgene Lim, Geoffrey I. Shapiro. The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1788. doi:10.1158/1538-7445.AM2013-1788

The Interaction of CtIP and Nbs1 Connects CDK and ATM to Regulate HR–Mediated Double-Strand Break Repair

CtIP plays an important role in homologous recombination (HR)–mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

Phosphorylation of Chs2p regulates interaction with COPII

Trafficking of the chitin synthase Chs2p from the endoplasmic reticulum (ER) to the bud-neck in late mitosis is tightly regulated by the cell cycle via phosphorylation of serine residues in the N-terminus of the protein. Here, we describe the effects of Chs2p phosphorylation on the interaction with coat protein complex II (COPII). Identification of a cdc5(ts) mutant, which fails to transport Chs2p-3xGFP to the bud-neck and instead accumulates the protein in intracellular puncta, led us to discover that Chs2p-3xGFP accumulates at ER exit sites in metaphase-arrested wild-type cells. Using an in vitro ER vesicle formation assay we showed that phosphorylation of Chs2p by the cyclin-dependent kinase CDK1 prevents packaging into COPII vesicles, whereas dephosphorylation of Chs2p by the phosphatase Cdc14p stimulates selection into the vesicles. We found that the cytoplasmic N-terminal domain of Chs2p, which contains the CDK1 phosphorylation sites, interacts with the COPII component Sec24p in a yeast two-hybrid assay and that phosphomimetic substitutions of serines at the CDK1 consensus sites reduces the interaction. Our data suggest that dephosphorylation functions as a molecular switch for regulated ER exit of Chs2p.

Direct binding between BubR1 and B56–PP2A phosphatase complexes regulate mitotic progression

BubR1 is a central component of the spindle assembly checkpoint that inhibits progression into anaphase in response to improper kinetochore-microtubule interactions. In addition, BubR1 also helps stabilize kinetochore-microtubule interactions by counteracting the Aurora B kinase but the mechanism behind this is not clear. Here we show that BubR1 directly binds to the B56 family of protein phosphatase 2A (PP2A) regulatory subunits through a conserved motif that is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and polo-like kinase 1 (Plk1). Two highly conserved hydrophobic residues surrounding the serine 670 Cdk1 phosphorylation site are required for B56 binding. Mutation of these residues prevents the establishment of a proper metaphase plate and delays cells in mitosis. Furthermore, we show that phosphorylation of serines 670 and 676 stimulates the binding of B56 to BubR1 and that BubR1 targets a pool of B56 to kinetochores. Our data suggest that BubR1 counteracts Aurora B kinase activity at improperly attached kinetochores by recruiting B56-PP2A phosphatase complexes.

Lipid-dependent and -independent regulation of nuclear envelope disassembly

The nuclear envelope(NE) isanorganelle ofuniquecomplexand dynamic structure, which serves not only as a physicalbarrier between the nucleus and the cytoplasm, but also as aregulator of many cellular events. During open mitosis, theNE is disassembled in prophase in order to allow the mitoticspindletoaccessthechromosomes[1].NEbreakdownentailsthe disassembly of all the NE components and is associatedwith a series of phosphorylations of proteins of the innernuclear membrane [2], nuclear pore complex [3]andlamina[4], the latest promoting depolymerisation of the lamin fila-ments. The A-type lamins are then solubilised in the cyto-plasm, whereas B-type lamins disperse in the endoplasmicreticulum membranes [5]. Multiple kinases, including cyclin-dependent kinase1(CDK1)anddiacylglycerol-activatedpro-tein kinase Cs (PKCs), have been suggested to play a role inlaminphosphorylationanddisassemblyinvariousmodelsandorganisms,mostlyin cell-free systems [6–17].InCaenorhab-ditiselegans,ahomologueoflipin,alipidphosphatasewhichconverts phosphatidic acid to DAG, is required for lamindisassembly during NE breakdown [18, 19]. To investigatemitotic lamin B1 disassembly, Mall et al. have developed ahigh-throughputlive-imagingassay[20].ThedataobtainedinHeLa cells using this assay confirm previous results suggest-ing the involvement of CDK1, PKCs and lipins in lamindisassembly.This assay is based on the automatic tracking and classi-fication intomitotic phases ofhundredsof individual mitoticcells expressing the chromatin marker H2B and lamin B1chimaeras, monitored by wide-field microscopy. Briefly,nuclei were detected in the H2B-mCherry channel,classified as previously described [21] and the chromatinwas segmented. A ring around the chromatin was defined tomeasure GFP-lamin B1 fluorescence intensity and deducethe assembled or disassembled (in the cytoplasm) state oflamin B1 throughout mitosis. Using a mathematical model,the authors determined the durations of lamin disassemblyin each condition. Mitotic progression from prophase toanaphase and chromosome congression (mean duration ofprometaphase and metaphase) were also calculated.Results obtained with transiently transfected wild-type ormutant lamin B1 reporter using this assay or higher resolu-tion images acquired by confocal microscopy showed thatmutation of the PKC consensus phosphorylation site did notalter lamin disassembly, whereas mutation of the CDK1phosphorylation site alone or combined to the mutation ofthe PKC motif significantly delayed lamin B1 disassemblycompared to controls. However, the differences in the meanduration of lamin disassembly between wild-type andCDK1- or CDK1/PKC-phosphorylation site mutants weresubtle, although significant (7.51±0.87 and 8.24±0.79 minp00.0146 or 8.86±0.80 min, p<0.0001, respectively). Theweakness of the mutants might be attributed to insufficientlevels of expression compared to endogenous proteins(Western blots omitted) and inability to form a network ofmutant proteins which would resist lamina disassembly.Another possibility is that these phosphorylation sites, onlyplay a minor role in lamin disassembly. Relatively highdoses (compared to IC50) of kinase inhibitors, i.e. classicalPKC inhibitor Go6976 and CDK1 inhibitor roscovitinealone or combined,caused significantperturbations of lamindisassembly and mitotic progression, although the twoevents were not correlated, and induced the apparition of‘cytoplasmic fragments’ of lamin B1. Those high intensity‘cytoplasmic fragments’, which were also observed by con-focal microscopy with the CDK1- or CDK1/PKC-sitemutants, could correspond to aggregates of lamin B1 in

Regulation of N-type Voltage-Gated Calcium Channels and Presynaptic Function by Cyclin-Dependent Kinase 5

N-type voltage-gated calcium channels localize topresynaptic nerve terminals and mediate key eventsincluding synaptogenesis and neurotransmission.While several kinases have been implicated in the modulation of calcium channels, their impact on presynaptic functions remains unclear. Here we report that the N-type calcium channel is a substrate for cyclin-dependent kinase 5 (Cdk5). The pore-forming α(1) subunit of the N-type calcium channel is phosphorylated in the C-terminal domain, and phosphorylation results in enhanced calcium influx due to increased channel open probability. Phosphorylation of the N-type calcium channel by Cdk5 facilitates neurotransmitter release and alters presynaptic plasticity by increasing the number of docked vesicles at the synaptic cleft. These effects are mediated by an altered interaction between N-type calcium channels and RIM1, which tethers presynaptic calcium channels to the active zone. Collectively, our results highlight a molecular mechanism by which N-type calcium channels are regulated by Cdk5 to affect presynaptic function.

Forced activation of Cdk1 via wee1 inhibition impairs homologous recombination

In response to DNA breaks, the 'DNA damage response' provokes a cell cycle arrest to facilitate DNA repair. Recent findings have indicated that cells can respond to DNA damage throughout the cell cycle, except during mitosis. Specifically, various mitotic kinases, including Cdk1, Aurora A and Plk1, were shown to inactivate key DNA damage checkpoint proteins when cells enter mitosis. Aberrant activation of mitotic kinases during interphase could therefore modulate cellular responses to DNA damage. In this study, our aim was to determine how aberrant activation of Cdk1 affects the cellular responses to DNA damage. We used Wee1 inhibition, using MK-1775, to force Cdk1 activation, which did not cause cytotoxicity in non-transformed cells. Instead, it accelerated mitotic entry and caused radio sensitization in p53-defective cancer cells, but not in p53-proficient cancer cells. Interestingly, we showed that Wee1 inhibition leads to elevation of Cdk1 activity in interphase cells. When we subsequently analyzed DNA damage responses in cells with forced Cdk1 activation, we observed a marked reduction of 53BP1 at sites of DNA damage along with an increase in γ-H2AX staining after irradiation, indicative of defective DNA repair. Indeed, when DNA repair was analyzed using in vivo endonuclease-induced homologous recombination (HR) assays, compromised DNA repair after Wee1 inhibition was confirmed. This defect in HR was accompanied by increased phosphorylation of BRCA2 at the Cdk1 phosphorylation site S3291. Taken together, our results indicate that Wee1 inhibition leads to forced Cdk1 activation in interphase cells, which interferes with normal DNA damage responses.

LMTK1/AATYK1 Is a Novel Regulator of Axonal Outgrowth That Acts via Rab11 in a Cdk5-Dependent Manner

Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1-deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WT or the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus, LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5-LMTK1-Rab11 is a novel signaling pathway involved in axonal outgrowth.

Role of phosphorylation of Cdc20 in p31 comet -stimulated disassembly of the mitotic checkpoint complex

The mitotic checkpoint system delays anaphase until all chromosomes are correctly attached to the mitotic spindle. When the checkpoint is turned on, it promotes the formation of the mitotic checkpoint complex (MCC), which inhibits the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2 bound to the APC/C activator Cdc20. When the checkpoint is satisfied, MCC is disassembled and APC/C becomes active. Previous studies have shown that the Mad2-binding protein p31(comet) promotes the dissociation of Cdc20 from BubR1 in MCC in a process that requires ATP. We now show that a part of MCC dissociation is blocked by inhibitors of cyclin-dependent kinases (Cdks) and that purified Cdk1-cyclin B stimulates this process. The mutation of all eight potential Cdk phosphorylation sites of Cdc20 partially prevented its release from BubR1. Furthermore, p31(comet) stimulated Cdk-catalyzed phosphorylation of Cdc20 in MCC. It is suggested that the binding of p31(comet) to Mad2 in MCC may trigger a conformational change in Cdc20 that facilitates its phosphorylation by Cdk, and that the latter process may promote its dissociation from BubR1.

Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism

Oxygen influences behaviour in many organisms, with low levels (hypoxia) having devastating consequences for neuron survival. How neurons respond physiologically to counter the effects of hypoxia is not fully understood. Here, we show that hypoxia regulates the trafficking of the glutamate receptor GLR-1 in C. elegans neurons. Either hypoxia or mutations in egl-9, a prolyl hydroxylase cellular oxygen sensor, result in the internalization of GLR-1, the reduction of glutamate-activated currents, and the depression of GLR-1-mediated behaviours. Surprisingly, hypoxia-inducible factor (HIF)-1, the canonical substrate of EGL-9, is not required for this effect. Instead, EGL-9 interacts with the Mint orthologue LIN-10, a mediator of GLR-1 membrane recycling, to promote LIN-10 subcellular localization in an oxygen-dependent manner. The observed effects of hypoxia and egl-9 mutations require the activity of the proline-directed CDK-5 kinase and the CDK-5 phosphorylation sites on LIN-10, suggesting that EGL-9 and CDK-5 compete in an oxygen-dependent manner to regulate LIN-10 activity and thus GLR-1 trafficking. Our findings demonstrate a novel mechanism by which neurons sense and respond to hypoxia.

P2-02-06: Inhibition of CDK4 Phosphorylation of Smad3 Decreases Cyclin D Overexpressing Breast Cancer Cell Proliferation and Migration.

Abstract Introduction: Several aspects of breast cancer onset and disease progression have been linked to members of the TGFβ superfamily and their associated downstream signaling components, the Smads. Alterations in Smad signaling have been directly implicated in the dichotomous role of TGFβ in malignancy, enacting both tumor suppressant and tumor promoting behaviors in breast carcinogenesis. Our previous work characterized Smad3 as a tumor suppressor and found that Smad3 action is inhibited upon phosphorylation by cyclin D/CDK4 in breast cancer cells. Others have shown that CDK phosphorylation of Smad3 can affect cell motility. Based on these findings, we hypothesized that activation of CDK4 leads to phosphorylation and inhibition of Smad3, thus releasing cell cycle arrest and promoting cell proliferation and metastasis. Methods: MCF7 parental and cyclin D overexpressing cells (MCF7 CD1) were treated with a CDK4 inhibitor alone or in combination with doxorubicin or paclitaxel and cell proliferation was determined using the MTS assay. MCF7 study cells were also transduced with lentiviral empty vector, wild-type (WT) Smad3, or Smad3 containing mutant CDK sites (T179V and 5M) resistant to inhibitory cyclin D/CDK phosphorylation. Stably transduced cells were plated in Matrigel 3-dimensional (3D) culture and treated with CDK4 inhibitor alone or with doxorubicin over a 12 day time course. Colony area was measured using MetaMorph software. Transwell migration and Matrigel invasion assays were used to determine the effect that transfection with Smad3 CDK phosphorylation site mutants or treatment with a CDK4 inhibitor had on highly metastatic cyclin D expressing MDA-MB-231 cells. Study wells were fixed/stained and the number of migrated or invaded cells was counted. Results: Treatment with the CDK4 inhibitor alone resulted in a growth inhibitory effect on proliferation of MCF7 parental and CD1 cells. Treatment with the CDK4 inhibitor in combination with chemotherapy resulted in the greatest inhibition of cell proliferation. In 3D culture, colonies formed by MCF7 study cells transduced with T179V or 5M Smad3 mutant constructs had smaller acinar size when compared with vector-transduced control cells. When compared to untreated cells, the MCF7 CD1 cells treated with CDK4 inhibitor or chemotherapy alone had smaller acinar size, with greatest decrease in size observed when the treatments were combined. Lastly, MDA-MB-231 cells transfected with the 5M Smad3 mutant construct, containing mutations in all 5 CDK phosphorylation sites, resulted in the greatest decrease in cell migration and invasion, when compared with cells transfected with both vector control or WT Smad3. Treatment of the MDA-MB-231 cells with the CDK4 inhibitor also resulted in a significant decrease in cell migration and invasion. Conclusions: Mutation of CDK phosphorylation sites in the Smad3 construct or direct inhibition of CDK4 resulted in a decrease in breast cancer cell proliferation, 3D colony formation and cell migration and invasion. Treatment with the CDK4 inhibitor in combination with chemotherapy further augmented these results. Inhibition of CDK4 and restoration of Smad3 activity may have a role in the treatment of breast cancers overexpressing cyclin D. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-06.

Glucose‐activated RUNX2 phosphorylation promotes endothelial cell proliferation and an angiogenic phenotype

The runt-related protein-2 (RUNX2) is a DNA-binding transcription factor that regulates bone formation, tumor cell metastasis, endothelial cell (EC) proliferation, and angiogenesis. RUNX2 DNA binding is glucose and cell cycle regulated. We propose that glucose may activate RUNX2 through changes in post-translational phosphorylation that are cell cycle-specific and will regulate EC function. Glucose increased cell cycle progression in EC through both G2/M and G1 phases with entry into S-phase occurring only in subconfluent cells. In the absence of nutrients and growth factors (starvation), subconfluent EC were delayed in G1 when RUNX2 expression was reduced. RUNX2 phosphorylation, activation of DNA binding, and pRb phosphorylation were stimulated by glucose and were necessary to promote cell cycle progression. Glucose increased RUNX2 localization at focal subnuclear sites, which co-incided with RUNX2 occupancy of the cyclin-dependent kinase (cdk) inhibitor p21(Cip1) promoter, a gene normally repressed by RUNX2. Mutation of the RUNX2 cdk phosphorylation site in the C-terminal domain (S451A.RUNX2) reduced RUNX2 phosphorylation and DNA binding. Expression of this cdk site mutant in EC inhibited glucose-stimulated differentiation (in vitro tube formation), monolayer wound healing, and proliferation. These results define a novel relationship between glucose-activated RUNX2 phosphorylation, cell cycle progression, and EC differentiation. These data suggest that inhibition of RUNX2 expression or DNA binding may be a useful strategy to inhibit EC proliferation in tumor angiogenesis.

Phosphorylation of NuSAP by Cdk1 regulates its interaction with microtubules in mitosis

Cell division in eukaryotes depends on a fine control of the dynamic changes of microtubules. Nucleolar and spindle-associated protein (NuSAP) is a microtubule-binding and -bundling protein essential for the integrity of the anaphase spindle and cell division. NuSAP contains two consensus cdk phosphorylation sites in its microtubule-binding domain. Here we show NuSAP is phosphorylated by cdk1 in early mitosis. This phosphorylation inhibits the binding of NuSAP to microtubules. During metaphase-to anaphase transition, NuSAP is dephosphorylated to promote spindle midzone formation and cell cycle progression. Expression of cdk1 phosphorylation-null mutant causes extensive bundling of microtubules in the prometaphase spindle. Our results suggest that phosphorylation and dephosphorylation of NuSAP during progression of mitosis regulate spindle organization through modulation of the dynamics of microtubules.

Cyclin B1 interacts with the BH3-only protein Bim and mediates its phosphorylation by Cdk1 during mitosis

Protracted mitotic arrest leads to cell death; however, the molecular signals that link these distinct processes remain poorly understood. Here we report that the pro-apoptotic BH3-only family member Bim undergoes phosphorylation in K562 cells following treatment with the microtubule targeting agents Taxol and Nocodazole. The phosphorylation of two Bim isoforms, BimEL and BimL, at the mitochondria correlates with mitotic arrest and precedes cell death induced by Taxol. It was also found that Bim undergoes transient phosphorylation during normal mitosis in K562 cells. In addition, siRNA silencing of Bim reduces sensitivity to Taxol-induced cell death. The transition of K562 cells from mitosis to G1 results in the loss of BimEL and BimL phosphorylation and correlates with the degradation of cyclin B1. The Cdk1 inhibitors, RO-3306 and Purvalanol A, block Bim phosphorylation in mitotically arrested cells. Importantly, it was found that cyclin B1 co-immunoprecipitates with endogenous Bim in mitotic extracts. Furthermore, active recombinant Cdk1/cyclin B1 phosphorylates BimEL and BimL in vitro and Serine 44 on BimL has been identified as a Cdk1 phosphorylation site. Collectively, these results suggest that Cdk1/cyclin B1-dependent hyper-phosphorylation of Bim during prolonged mitotic arrest is an important cell death signal.

Dependence of Chs2 ER export on dephosphorylation by cytoplasmic Cdc14 ensures that septum formation follows mitosis

Cytokinesis, which leads to the physical separation of two dividing cells, is normally restrained until after nuclear division. In Saccharomyces cerevisiae, chitin synthase 2 (Chs2), which lays down the primary septum at the mother-daughter neck, also ensures proper actomyosin ring constriction during cytokinesis. During the metaphase-to-anaphase transition, phosphorylation of Chs2 by the mitotic cyclin-dependent kinase (Cdk1) retains Chs2 at the endoplasmic reticulum (ER), thereby preventing its translocation to the neck. Upon Cdk1 inactivation at the end of mitosis, Chs2 is exported from the ER and targeted to the neck. The mechanism for triggering Chs2 ER export thus far is unknown. We show here that Chs2 ER export requires the direct reversal of the inhibitory Cdk1 phosphorylation sites by Cdc14 phosphatase, the ultimate effector of the mitotic exit network (MEN). We further show that only Cdc14 liberated by the MEN after completion of chromosome segregation, and not Cdc14 released in early anaphase by the Cdc fourteen early anaphase release pathway, triggers Chs2 ER exit. Presumably, the reduced Cdk1 activity in late mitosis further favors dephosphorylation of Chs2 by Cdc14. Thus, by requiring declining Cdk1 activity and Cdc14 nuclear release for Chs2 ER export, cells ensure that septum formation is contingent upon chromosome separation and exit from mitosis.

Regulation of sororin by Cdk1-mediated phosphorylation

Tumor cells are commonly aneuploid, a condition contributing to cancer progression and drug resistance. Understanding how chromatids are linked and separated at the appropriate time will help uncover the basis of aneuploidy and will shed light on the behavior of tumor cells. Cohesion of sister chromatids is maintained by the multi-protein complex cohesin, consisting of Smc1, Smc3, Scc1 and Scc3. Sororin associates with the cohesin complex and regulates the segregation of sister chromatids. Sororin is phosphorylated in mitosis; however, the role of this modification is unclear. Here we show that mutation of potential cyclin-dependent kinase 1 (Cdk1) phosphorylation sites leaves sororin stranded on chromosomes and bound to cohesin throughout mitosis. Sororin can be precipitated from cell lysates with DNA-cellulose, and only the hypophosphorylated form of sororin shows this association. These results suggest that phosphorylation of sororin causes its release from chromatin in mitosis. Also, the hypophosphorylated form of sororin increases cohesion between sister chromatids, suggesting that phosphorylation of sororin by Cdk1 influences sister chromatid cohesion. Finally, phosphorylation-deficient sororin can alleviate the mitotic block that occurs upon knockdown of endogenous sororin. This mitotic block is abolished by ZM447439, an Aurora kinase inhibitor, suggesting that prematurely separated sister chromatids activate the spindle assembly checkpoint through an Aurora kinase-dependent pathway.