Cdk Phosphorylation Sites Research Articles

Meiotic nuclear divisions in budding yeast require PP2ACdc55-mediated antagonism of Net1 phosphorylation by Cdk

During meiosis, one round of deoxyribonucleic acid replication is followed by two rounds of nuclear division. In Saccharomyces cerevisiae, activation of the Cdc14 early anaphase release (FEAR) network is required for exit from meiosis I but does not lead to the activation of origins of replication. The precise mechanism of how FEAR regulates meiosis is not understood. In this paper, we report that premature activation of FEAR during meiosis caused by loss of protein phosphatase PP2A(Cdc55) activity blocks bipolar spindle assembly and nuclear divisions. In cdc55 meiotic null (cdc55-mn) cells, the cyclin-dependent kinase (Cdk)-counteracting phosphatase Cdc14 was released prematurely from the nucleolus concomitant with hyperphosphorylation of its nucleolar anchor protein Net1. Crucially, a mutant form of Net1 that lacks six Cdk phosphorylation sites rescued the meiotic defect of cdc55-mn cells. Expression of a dominant mutant allele of CDC14 mimicked the cdc55-mn phenotype. We propose that phosphoregulation of Net1 by PP2A(Cdc55) is essential for preventing precocious exit from meiosis I.

The initial phase of chromosome condensation requires Cdk1-mediated phosphorylation of the CAP-D3 subunit of condensin II

The cell cycle transition from interphase into mitosis is best characterized by the appearance of condensed chromosomes that become microscopically visible as thread-like structures in nuclei. Biochemically, launching the mitotic program requires the activation of the mitotic cyclin-dependent kinase Cdk1 (cyclin-dependent kinase 1), but whether and how Cdk1 triggers chromosome assembly at mitotic entry are not well understood. Here we report that mitotic chromosome assembly in prophase depends on Cdk1-mediated phosphorylation of the condensin II complex. We identified Thr 1415 of the CAP-D3 subunit as a Cdk1 phosphorylation site, which proved crucial as it was required for the Polo kinase Plk1 (Polo-like kinase 1) to localize to chromosome axes through binding to CAP-D3 and thereby hyperphosphorylate the condensin II complex. Live-cell imaging analysis of cells carrying nonphosphorylatable CAP-D3 mutants in place of endogenous protein suggested that phosphorylation of Thr 1415 is required for timely chromosome condensation during prophase, and that the Plk1-mediated phosphorylation of condensin II facilitates its ability to assemble chromosomes properly. These observations provide an explanation for how Cdk1 induces chromosome assembly in cells entering mitosis, and underscore the significance of the cooperative action of Plk1 with Cdk1.

Cyclin E and Cdk2 Control GLD-1, the Mitosis/Meiosis Decision, and Germline Stem Cells in Caenorhabditis elegans

Coordination of the cell cycle with developmental events is crucial for generation of tissues during development and their maintenance in adults. Defects in that coordination can shift the balance of cell fates with devastating clinical effects. Yet our understanding of the molecular mechanisms integrating core cell cycle regulators with developmental regulators remains in its infancy. This work focuses on the interplay between cell cycle and developmental regulators in the Caenorhabditis elegans germline. Key developmental regulators control germline stem cells (GSCs) to self-renew or begin differentiation: FBF RNA–binding proteins promote self-renewal, while GLD RNA regulatory proteins promote meiotic entry. We first discovered that many but not all germ cells switch from the mitotic into the meiotic cell cycle after RNAi depletion of CYE-1 (C. elegans cyclin E) or CDK-2 (C. elegans Cdk2) in wild-type adults. Therefore, CYE-1/CDK-2 influences the mitosis/meiosis balance. We next found that GLD-1 is expressed ectopically in GSCs after CYE-1 or CDK-2 depletion and that GLD-1 removal can rescue cye-1/cdk-2 defects. Therefore, GLD-1 is crucial for the CYE-1/CDK-2 mitosis/meiosis control. Indeed, GLD-1 appears to be a direct substrate of CYE-1/CDK-2: GLD-1 is a phosphoprotein; CYE-1/CDK-2 regulates its phosphorylation in vivo; and human cyclin E/Cdk2 phosphorylates GLD-1 in vitro. Transgenic GLD-1(AAA) harbors alanine substitutions at three consensus CDK phosphorylation sites. GLD-1(AAA) is expressed ectopically in GSCs, and GLD-1(AAA) transgenic germlines have a smaller than normal mitotic zone. Together these findings forge a regulatory link between CYE-1/CDK-2 and GLD-1. Finally, we find that CYE-1/CDK-2 works with FBF-1 to maintain GSCs and prevent their meiotic entry, at least in part, by lowering GLD-1 abundance. Therefore, CYE-1/CDK-2 emerges as a critical regulator of stem cell maintenance. We suggest that cyclin E and Cdk-2 may be used broadly to control developmental regulators.

A cyclin-binding motif in human papillomavirus type 18 (HPV18) E1^E4 is necessary for association with CDK–cyclin complexes and G2/M cell cycle arrest of keratinocytes, but is not required for differentiation-dependent viral genome amplification or L1 capsid protein expression

The G2/M arrest function of human papillomavirus (HPV) E4 proteins is hypothesized to be necessary for viral genome amplification. Full-length HPV18 E1^E4 protein is essential for efficient viral genome amplification. Here we identify key determinants within a CDK-bipartite consensus recognition motif in HPV18 E1^E4 that are critical for association with active CDK–cyclin complexes and in vitro phosphorylation at the predicted CDK phosphorylation site (threonine 23). The optimal cyclin-binding sequence (43RRLL46) within this E4 motif is required for G2/M arrest of primary keratinocytes and correlates with cytoplasmic retention of cyclin B1, but not cyclin A. Disruption of this motif in the E4 ORF of HPV18 genomes, and the subsequent generation of stable cell lines in primary keratinocytes revealed that this motif was not essential for viral genome amplification or L1 capsid protein induction. We conclude that the HPV18 E4 G2/M arrest function does not play a role in early vegetative events.

Roscovitine Inhibits EBNA1 Serine 393 Phosphorylation, Nuclear Localization, Transcription, and Episome Maintenance

Latent Epstein-Barr virus (EBV) infection causes human lymphomas and carcinomas. EBV usually persists as an episome in malignant cells. EBV episome persistence, replication, and gene expression are dependent on EBNA1 binding to multiple cognate sites in oriP. To search for inhibitors of EBNA1- and oriP-dependent episome maintenance or transcription, a library of 40,550 small molecules was screened for compounds that inhibit EBNA1- and oriP-dependent transcription and do not inhibit EBNA1- and oriP-independent transcription. This screening identified roscovitine, a selective inhibitor of cyclin-dependent kinase 1 (CDK1), CDK2, CDK5, and CDK7. Based on motif predictions of EBNA1 serine 393 as a CDK phosphorylation site and (486)RALL(489) and (580)KDLVM(584) as potential cyclin binding domains, we hypothesized that cyclin binding to EBNA1 may enable CDK1, -2, -5, or -7 to phosphorylate serine 393. We found that Escherichia coli-expressed EBNA1 amino acids 387 to 641 were phosphorylated in vitro by CDK1-, -2-, -5-, and -7/cyclin complexes and serine 393 phosphorylation was roscovitine inhibited. Further, S393A mutation abrogated phosphorylation. S393A mutant EBNA1 was deficient in supporting EBNA1- and oriP-dependent transcription and episome persistence, and roscovitine had little further effect on the diminished S393A mutant EBNA1-mediated transcription or episome persistence. Immunoprecipitated FLAG-EBNA1 was phosphorylated in vitro, and roscovitine inhibited this phosphorylation. Moreover, roscovitine decreased nuclear EBNA1 and often increased cytoplasmic EBNA1, whereas S393A mutant EBNA1 was localized equally in the nucleus and cytoplasm and was unaffected by roscovitine treatment. These data indicate that roscovitine effects are serine 393 specific and that serine 393 is important in EBNA1- and oriPCp-dependent transcription and episome persistence.

Phosphorylation of Cdc5 regulates its accumulation

BackgroundCdc5 (polo kinase/Plk1) is a highly conserved key regulator of the S. cerevisiae cell cycle from S-phase until cytokinesis. However, much of the regulatory mechanisms that govern Cdc5 remain to be determined. Cdc5 is phosphorylated on up to 10 sites during mitosis. In this study, we investigated the function of phosphorylation site T23, the only full consensus Cdk1 (Cdc28) phosphorylation site present.FindingsCdc5T23A introduces a degron that reduces its cellular amount to undetectable levels, which are nevertheless sufficient for normal cell proliferation. The degron acts in cis and is reversed by N-terminal GFP-tagging. Cdk1 kinase activity is required to maintain Cdc5 levels during G2. This, Cdk1 inhibited, Cdc5 degradation is APC/CCdh1 independent and requires new protein synthesis. Cdc5T23E is hyperactive, and reduces the levels of Cdc5 (in trans) and drastically reduces Clb2 levels.ConclusionsPhosphorylation of Cdc5 by Cdk1 is required to maintain Cdc5 levels during G2. However, phosphorylation of T23 (probably by Cdk1) caps Cdc5 and other CLB2 cluster protein accumulation, preventing potential protein toxicity, which may arise from their overexpression or from APC/CCdh1 inactivation.

Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines

Smad3, a component of the TGFβ signaling pathway, contributes to G1 arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild type (WT) Smad3, or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1), or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers.

Efficient and rapid exact gene replacement without selection

We describe a highly efficient method for exact gene replacement in budding yeast. Induction of rapid and efficient recombination in an entire cell population results in at least 50% of the recombinants undergoing a switch of the endogenous copy to a specific mutated allele, with no remaining markers or remnant of foreign DNA, without selection. To accomplish this, a partial copy of the replacement allele, followed by an HO cut site, is installed adjacent to the wild-type locus, in a GAL-HO MATa-inc background. HO induction results in near-quantitative site cleavage and recombination/gene conversion, resulting in either regeneration of wild-type or switch of the endogenous allele to the mutant, with accompanying deletion of intervening marker sequences, yielding an exact replacement. Eliminating the need for selection (over days) of rare recombinants removes concerns about second-site suppressor mutations and also allows direct phenotypic analysis, even of lethal gene replacements, without the need of a method to make the lethality conditional or to employ regulated promoters of unknown strength compared to the endogenous promoter. To test this method, we tried two known lethal gene replacements, substituting the non-essential CDH1 gene with a dominantly lethal version mutated for its Cdk phosphorylation sites and substituting the essential CDC28 gene with two recessively lethal versions, one containing an early stop codon and another inactivating Cdc28 kinase activity. We also tested a gene replacement of unknown phenotypic consequences: replacing the non-essential CLB3 B-type cyclin with a version lacking its destruction box.

Cyclin-dependent kinase 4-mediated phosphorylation inhibits Smad3 activity in cyclin D-overexpressing breast cancer cells.

Smad3, a component of the transforming growth factor β signaling cascade, contributes to G(1) arrest in breast cancer cells. Cyclin D1/cyclin-dependent kinase 4 (CDK4) promotes G(1)-S-phase transition, and CDK phosphorylation of Smad3 has been associated with inhibition of Smad3 activity. We hypothesized that overexpression of cyclin D1 exerts tumorigenic effects in breast cancer cells through CDK4-mediated phosphorylation and inhibition of Smad3 and release of G(1) arrest. Real-time quantitative reverse transcription-PCR and immunoblotting were used to evaluate expression of study proteins in cyclin D1-overexpressing breast cancer cells. Smad3 transcriptional activity and cell cycle control were examined in cells transfected with wild-type (WT) Smad3 or Smad3 with single or multiple CDK phosphorylation site mutations (M) in the presence or absence of the CDK4 inhibitor or cotransfection with cdk4 small interfering RNA (siRNA). Transfection of the Smad3 5M construct resulted in decreased c-myc and higher p15(INK4B) expression. Compared with WT Smad3, overexpression of the Smad3 T8, T178, 4M, or 5M mutant constructs resulted in higher Smad3 transcriptional activity. Compared with cells transfected with WT Smad3, Smad3 transcriptional activity was higher in cells overexpressing Smad3 mutant constructs and treated with the CDK4 inhibitor or transfected with cdk4 siRNA. Cells transfected with Smad3 T8 or T178 and treated with the CDK4 inhibitor showed an increase in the G(1) cell population. Inhibition of CDK-mediated Smad3 phosphorylation released cyclin D1-regulated blockade of Smad3 transcriptional activity and recovered cell cycle arrest in breast cancer cells. Targeted inhibition of CDK4 activity may have a role in the treatment of cyclin D-overexpressing breast cancers.

Cdk Phosphorylation of a Nucleoporin Controls Localization of Active Genes through the Cell Cycle

Many inducible genes in yeast are targeted to the nuclear pore complex when active. We find that the peripheral localization of the INO1 and GAL1 genes is regulated through the cell cycle. Active INO1 and GAL1 localized at the nuclear periphery during G1, became nucleoplasmic during S-phase, and then returned to the nuclear periphery during G2/M. Loss of peripheral targeting followed the initiation of DNA replication and was lost in cells lacking a cyclin-dependent kinase (Cdk) inhibitor. Furthermore, the Cdk1 kinase and two Cdk phosphorylation sites in the nucleoporin Nup1 were required for peripheral targeting of INO1 and GAL1. Introduction of aspartic acid residues in place of either of these two sites in Nup1 bypassed the requirement for Cdk1 and resulted in targeting of INO1 and GAL1 to the nuclear periphery during S-phase. Thus, phosphorylation of a nuclear pore component by cyclin dependent kinase controls the localization of active genes to the nuclear periphery through the cell cycle.

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G(2)/M transition. We find that activation of Aurora A first occurs at centrosomes at late G(2) and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.

Cdk5-Mediated Phosphorylation of -Catenin Regulates Its Localization and GluR2-Mediated Synaptic Activity

Cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation plays an important role in proper synaptic function and transmission. Loss of Cdk5 activity results in abnormal development of the nervous system accompanied by massive disruptions in cortical migration and lamination, therefore impacting synaptic activity. The Cdk5 activator p35 associates with delta-catenin, the synaptic adherens junction protein that serves as part of the anchorage complex of AMPA receptor at the postsynaptic membrane. However, the implications of Cdk5-mediated phosphorylation of delta-catenin have not been fully elucidated. Here we show that Cdk5-mediated phosphorylation of delta-catenin regulates its subcellular localization accompanied by changes in dendritic morphogenesis and synaptic activity. We identified two Cdk5 phosphorylation sites in mouse delta-catenin, serines 300 and 357, and report that loss of Cdk5 phosphorylation of delta-catenin increased its localization to the membrane. Furthermore, mutations of the serines 300 and 357 to alanines to mimic nonphosphorylated delta-catenin resulted in increased dendritic protrusions accompanied by increased AMPA receptor subunit GluR2 localization at the membrane. Consistent with these observations, loss of Cdk5 phosphorylation of delta-catenin increased the AMPA/NMDA ratio. This study reveals how Cdk5 phosphorylation of the synaptic mediator protein delta-catenin can alter its localization at the synapse to impact neuronal synaptic activity.

Functional Comparison of H1 Histones in Xenopus Reveals Isoform-Specific Regulation by Cdk1 and RanGTP

H1 "linker" histones bind dynamically to nucleosomes and promote their compaction into chromatin fibers. Developmental H1 isoforms are evolutionarily conserved, but their function, regulation, and posttranslational modifications are poorly understood. In Xenopus egg extracts, the embryonic linker histone H1M does not affect nuclear assembly or replication but is required for proper chromosome architecture during mitosis. We report here that somatic H1 isoforms, which are more positively charged and feature multiple Cdk1 phosphorylation sites, cannot substitute for H1M at endogenous concentrations, instead causing chromatin compaction during interphase and dissociating from chromosomes at the onset of mitosis. Mitotic Cdk1 phosphorylation is not responsible for this dissociation and instead functions to enhance H1 binding in egg extracts and embryos. Nuclear import receptors RanBP7 and importin beta bind tightly to somatic H1 but not H1M, and addition of a constitutively active Ran mutant abolishes this interaction and enhances the ability of somatic H1 to rescue mitotic chromosome architecture. Our results reveal distinct regulatory mechanisms among linker histone isoforms and a specific role for H1M to compact chromosomes during egg meiotic arrest and early embryonic divisions.

B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast

Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.

Mitotic phosphorylation of Aki1 at Ser 208 by cyclin B1–Cdk1 complex

Akt kinase-interacting protein 1 (Aki1)/Freud-1/CC2D1A is localized in the cytosol, nucleus, and centrosome. Aki1 plays distinct roles depending on its localization. In the cytosol, it acts as a scaffold protein in the phosphoinositide 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase 1 (PDK1)/Akt pathway. In the nucleus, it is a transcriptional repressor of the serotonin-1A (5-HT1A) receptor. In the centrosome, it regulates spindle pole localization of the cohesin subunit Scc1, thereby mediating centriole cohesion during mitosis. Although the function of Aki1 has been well clarified, the regulatory machinery of Aki1 is poorly understood. We previously found that Aki1 in mitotic cells displayed reduced mobility on immunoblot analysis, but the reason for this was unclear. Here we show that the electrophoretic mobility shift of Aki1 is derived from mitotic phosphorylation. The cyclin B1–cyclin-dependent kinase 1 (Cdk1) complex was found to be one of the kinases responsible for Aki1 phosphorylation during mitosis. We identified the Ser 208 residue of Aki1 as a cyclin B1–Cdk1 phosphorylation site. Furthermore, cyclin B1–Cdk1 inhibitor treatment was shown to attenuate the level of Aki1 in complex with Scc1, suggesting that Aki1 phosphorylation by cyclin B1–Cdk1 contributes to Aki1–Scc1 complex formation. Our results indicate that cyclin B1–Cdk1 is a kinase of Aki1 during mitosis and that its phosphorylation of Aki1 may regulate mitotic function.

Mutual regulation of cyclin-dependent kinase and the mitotic exit network

The mitotic exit network (MEN) is a spindle pole body (SPB)-associated, GTPase-driven signaling cascade that controls mitotic exit. The inhibitory Bfa1-Bub2 GTPase-activating protein (GAP) only associates with the daughter SPB (dSPB), raising the question as to how the MEN is regulated on the mother SPB (mSPB). Here, we show mutual regulation of cyclin-dependent kinase 1 (Cdk1) and the MEN. In early anaphase Cdk1 becomes recruited to the mSPB depending on the activity of the MEN kinase Cdc15. Conversely, Cdk1 negatively regulates binding of Cdc15 to the mSPB. In addition, Cdk1 phosphorylates the Mob1 protein to inhibit the activity of Dbf2-Mob1 kinase that regulates Cdc14 phosphatase. Our data revise the understanding of the spatial regulation of the MEN. Although MEN activity in the daughter cells is controlled by Bfa1-Bub2, Cdk1 inhibits MEN activity at the mSPB. Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects.

Interaction of peptidyl-prolyl isomerase Pin1 with Tau at the Cdk5 phosphorylation sites

Interaction of peptidyl-prolyl isomerase Pin1 with Tau at the Cdk5 phosphorylation sites

CDK4 Phosphorylation Regulates Smad3 Activity in Cyclin D1 Overexpressing Breast Cancer Cells.

Abstract Introduction: Overexpression of cyclin D1 is associated with poor prognosis in breast cancer. Cyclin D1 regulates the noncanonical phosphorylation of Smad3, a member of the TGFΒ signaling cascade, through CDK4 and inhibits Smad3 activity in normal mouse fibroblasts and epithelial cells. We hypothesize that overexpression of cyclin D1 exerts tumorigenic effects in breast cancer cells through a CDK4 mediated inhibition of Smad3 cell cycle control.Methods: To examine the impact of cyclin D1 overexpression on Smad3 function, constructs containing empty vector (V), WT Smad3 (WT), and Smad3 with single or multiple CDK phosphorylation site mutations were cotransfected with a Smad3 reporter into cyclin D1 overexpressing MCF-7 and T47D cells. Smad3 containing mutant CDK sites is resistant to inhibitory CyclinD1/CDK4 phosphorylation. Study cells were also transfected with V, WT or mutant Smad3 and treated with a CDK4 inhibitor. Smad3 function was examined by luciferase reporter assays and cell cycle analysis. Levels of mRNA for cell cycle regulators cyclin dependent kinase inhibitor (cdki) p15 and c-myc were measured by qRT-PCR.Results: As expected, higher amounts of c-myc mRNA and lower amounts of cdki p15 mRNA were observed in parental, vector control and cyclin D1 overexpressing cells. While subsequent transfection with WT Smad3 resulted in a decrease in c-myc and an increase in p15 mRNA levels, the greatest decrease in c-myc and increase in p15 mRNA was found after transfection with CDK mutated Smad3, most prominently in the cyclin D1 overexpressing cells. Transfection of all individual Smad3 CDK phosphorylation site mutations resulted in an increase in Smad3 reporter activity as compared to transfection with WT Smad3. Transfection of Smad3 constructs with 4 and 5 CDK sites mutated resulted in highest overall Smad3 reporter activity, compared to WT Smad3, in the study cells. A dose dependent increase in Smad3 reporter activity was shown when MCF-7 and T47D cells were transfected with WT Smad3 and treated with increasing concentrations of a specific CDK4 inhibitor. Comparatively higher Smad3 reporter activity was found when the study cells were transfected with individual Smad3 mutant constructs and treated with the CDK4 inhibitor. Interestingly, transfection with the Smad3 construct with all CDK sites mutated and treatment with the CDK4 inhibitor resulted in inhibition of Smad3 reporter activity in the study cells. Lastly, cell cycle analysis revealed that MCF-7 and T47D cells treated with the CDK4 inhibitor showed an increase in the G1 cell cycle population.Conclusions: Mutation of CDK phosphorylation sites in the Smad3 construct or direct inhibition of CDK4 appear to facilitate release of cyclin D1/CDK4 mediated inhibition of Smad3 in breast cancer cells. To this end, restoration of Smad3 activity resulted in increasing p15 and decreasing c-myc mRNA levels in the study cells. However, mutation of all Smad3 CDK phosphorylation sites and treatment with the CDK4 inhibitor resulted in a decrease in Smad3 reporter activity, indicating that some CDK activity is necessary for Smad3 function. By helping to restore cell cycle control, inhibition of CDK4 activity may have a role in the treatment of primary and metastatic breast cancers overexpressing cyclin D. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2152.

Saccharomyces CDK1 Phosphorylates Rad53 Kinase in Metaphase, Influencing Cellular Morphogenesis

Rad53 is an essential protein kinase governing DNA damage and replication stress checkpoints in budding yeast. It also appears to be involved in cellular morphogenesis processes. Mass spectrometry analyses revealed that Rad53 is phosphorylated at multiple SQ/TQ and at SP/TP residues, which are typical consensus sites for phosphatidylinositol 3-kinase-related kinases and CDKs, respectively. Here we show that Clb-CDK1 phosphorylates Rad53 at Ser(774) in metaphase. This phosphorylation event does not influence the DNA damage and replication checkpoint roles of Rad53, and it is independent of the spindle assembly checkpoint network. Moreover, the Ser-to-Asp mutation, mimicking a constitutive phosphorylation state at site 774, causes sensitivity to calcofluor, supporting a functional linkage between Rad53 and cellular morphogenesis.

The maize HMGA protein is localized to the nucleolus and can be acetylated in vitro at its globular domain, and phosphorylation by CDK reduces its binding activity to AT-rich DNA

The high mobility group (HMG) proteins are nonhistone chromosomal proteins that participate in diverse nuclear activities including chromatin structure and gene regulation. We previously studied the biochemistry of the maize HMGA protein and its role in transcriptional regulation during maize endosperm development. Here, we extended our study and showed that a strong binding of ZmHMGA to AT-rich DNA requires at least three AT-hook motifs; two motifs showed a significant reduction whereas a single motif was not sufficient for binding. CDK phosphorylation sites situated between AT-hook3 and AT-hook4 were strongly phosphorylated by a SUC1-associated kinase; no in vitro phosphorylation is evident for the AtHMGA protein. Phosphorylation of ZmHMGA reduced its binding to AT-rich DNA in vitro. The maize HMGA protein fused to GFP was localized in the nucleus of transgenic Arabidopsis plants tending to concentrate within the nucleolus. Localization to the nucleolus was conferred by the C-terminal portion of the protein containing the AT-hooks. ZmHMGA was acetylated in vitro on its N-terminal globular domain by the human PCAF acetyltransferase. Our results suggest that ZmHMGA participates in nucleolar function and that its role may be regulated posttranslationally by phosphorylation and acetylation.