A ROS-dependent mechanism promotes CDK2 phosphorylation to drive progression through S phase

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Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.     

CDK phosphorylation of Drc1 regulates DNA replication in fission yeast

Cyclin-dependent kinases (CDKs) are absolutely required for DNA replication in eukaryotic cells. CDKs are thought to activate one or more replication factors, but the identities of these proteins are unknown. Here we describe fission yeast Drc1, a protein required for DNA replication that is phosphorylated by Cdc2. Drc1 depletion leads to catastrophic mitotic divisions with incompletely replicated DNA, indicating that Drc1 is required for DNA synthesis and S-M replication checkpoint control. Drc1 associates with Cdc2 and is phosphorylated at the onset of S phase when Cdc2 is activated. Mutant Drc1 that lacks CDK phosphorylation sites is nonfunctional and fails to interact with Cut5 replication factor. These data suggest that Cdc2 promotes DNA replication by phosphorylating Drc1 and regulating its association with Cut5.     

Multisite phosphorylation and the countdown to S phase.

Remarkably, SCF(Cdc4) ubiquitin ligase binds and ubiquitinates Sic1 decorated with six, but not five, phosphates. This numerical wizardry suggests how analog inputs can be rectified to digital outputs. Unraveling the counting mechanism promises to generate new insights into the architecture of protein nanoprocessors.     

Consequences of abnormal CDK activity in S phase

Cyclin Dependent Kinases (CDKs) are important regulators of DNA replication. In this work we have investigated the consequences of increasing or decreasing the CDK activity in S phase. To this end we identified S-phase regulators of the fission yeast CDK, Cdc2, and used appropriate mutants to modulate Cdc2 activity. In fission yeast Mik1 has been thought to be the main regulator of Cdc2 activity in S phase. However, we find that Wee1 has a major function in S phase and thus we used wee1 mutants to investigate the consequences of increased Cdc2 activity. These wee1 mutants display increased replication stress and, particularly in the absence of the S-phase checkpoint, accumulate DNA damage. Notably, more cells incorporate EdU in a wee1^? strain as compared to wildtype, suggesting altered regulation of DNA replication. In addition, a higher number of cells contain chromatin-bound Cdc45, an indicator of active replication forks. In addition, we found that Cdc25 is required to activate Cdc2 in S phase and used a cdc25 mutant to explore a situation where Cdc2 activity is reduced. Interestingly, a cdc25 mutant has a higher tolerance for replication stress than wild-type cells, suggesting that reduced CDK activity in S phase confers resistance to at least some forms of replication stress.     

Receptor association and tyrosine phosphorylation of S6 kinases

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB/Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein-protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme complex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR). The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Furthermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localization. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine/s in response to PDGF or serum.     

CDK1-mediated phosphorylation of Abi1 attenuates Bcr-Abl-induced F-actin assembly and tyrosine phosphorylation of WAVE complex during mitosis

Coordinated actin remodeling is crucial for cell entry into mitosis. The WAVE regulatory complex is a key regulator of actin assembly, yet how the WAVE signaling is regulated to coordinate actin assembly with mitotic entry is not clear. Here, we have uncovered a novel mechanism that regulates the WAVE complex at the onset of mitosis. We found that the Bcr-Abl-stimulated F-actin assembly is abrogated during mitosis. This mitotic inhibition of F-actin assembly is accompanied by an attenuation of Bcr-Abl-induced tyrosine phosphorylation of the WAVE complex. We identified serine 216 of Abi1 as a target of CDK1/cyclin B kinase that is phosphorylated in cells at the onset of mitosis. The Abi1 phosphorylated on serine 216 displayed greatly reduced tyrosine phosphorylation in the hematopoietic cells transformed by Bcr-Abl. Moreover, a phosphomimetic mutation of serine 216 to aspartic acid in Abi1 was sufficient to attenuate Bcr-Abl-induced tyrosine phosphorylation of the WAVE complex and F-actin assembly. Ectopic expression of Abi1 with serine 216 mutations interfered with cell cycle progression. Together, these data show that CDK1-mediated phosphorylation of serine 216 in Abi1 serves as a regulatory mechanism that may contribute to coordinated actin cytoskeleton remodeling during mitosis.     

Meiosis-dependent tyrosine phosphorylation of a yeast protein related to the mouse p51ferT

The FER locus of the mouse encodes two mRNA species: one is constitutively transcribed, giving rise to a 94 kDa tyrosine kinase (p94^ferT); the second is a meiosis-specific RNA that gives rise to a 51 kDa tyrosine kinase (p51^ferT). The p51^ferT RNA and protein accumulate in primary spermatocytes that are in prophase of the first meiotic division. By using polyclonal antibodies directed against synthetic peptides derived from the unique amino-terminus of the mouse p51^ferT, a 51 kDa phosphotyrosyl protein — p51^y — was identified in Saccharomyces cerevisiae. The p51^y protein is constitutively expressed in yeast, but in meiotic cells, concomitantly with commitment to meiotic recombination, its level of phosphorylation on tyrosine residues is increased. A different pattern of phosphorylation is observed on serine residues: at early meiotic times the level is decreased, while in later meiotic time the level increases, reaching the vegetative level. When p51^ferT is ectopically expressed in yeast, it is active, leading to preferential phosphorylation of an approx. 65 kDa protein. A similar pattern of phosphorylation by p51^ferT is seen in mammalian cells.     

The inhibitory function of CTLA-4 does not require its tyrosine phosphorylation

CTLA-4 is a negative regulator of T cell responses. Sequence analysis of this molecule reveals the presence of two cytoplasmic tyrosine residues at positions 165 and 182 that are potential Src homology (SH)-2 domain binding sites. The role of phosphorylation of these residues in CTLA-4-mediated signaling is unknown. Here, we show that sole TCR ligation induces zeta-associated protein (ZAP)-70-dependent tyrosine phosphorylation of CTLA-4 that is important for cell surface retention of this molecule. However, CTLA-4 tyrosine phosphorylation is not required for down-regulation of T cell activation following CD3-CTLA-4 coengagement. Specifically, inhibition of extracellular signal-regulated kinase (ERK) activation and of IL-2 production by CTLA-4-mediated signaling occurs in T cells expressing mutant CTLA-4 molecules lacking the cytoplasmic tyrosine residues, and in lck-deficient or ZAP-70-deficient T cells. Therefore, CTLA-4 function involves interplay between two different levels of regulation: phosphotyrosine-dependent cell surface retention and phosphotyrosine-independent association with signaling molecules.     

Differential regulation of the human tyrosine hydroxylase isoforms via hierarchical phosphorylation

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline, and adrenaline. In response to short term stimuli TH activity is primarily controlled by phosphorylation of serine 40. We have previously shown that phosphorylation of serine 19 in TH can indirectly activate TH via a hierarchical mechanism by increasing the rate of phosphorylation of serine 40. Here we show that phosphorylation of serine 31 in rat TH increases the rate of serine 40 phosphorylation 9-fold in vitro. Phosphorylation of serine 31 in intact bovine chromaffin cells potentiated the forskolin-induced increase in serine 40 phosphorylation and TH activity more than 2-fold. Humans are unique in that they contain four TH isoforms but to date no significant differences have been shown in the regulation of these isoforms. Phosphorylation of the human TH isoform 1 at serine 31 by extracellular signal-regulated protein kinase (ERK) also produced a 9-fold increase in the rate of phosphorylation of serine 40, whereas little effect was seen in the TH isoforms 3 and 4. ERK did not phosphorylate human TH isoform 2. The effect of serine 19 phosphorylation on serine 40 (44 in TH2) phosphorylation is stronger in TH2 than in TH1. Thus hierarchical phosphorylation provides a mechanism whereby the two major human TH isoforms (1 and 2) can be differentially regulated with only isoform 1 responding to the ERK pathway, whereas isoform 2 is more sensitive to calcium-mediated events.     

The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress

The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore-spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.     

Spontaneous clustering and tyrosine phosphorylation of NK cell inhibitory receptor induced by ligand binding

Inhibition of NK cell cytotoxicity by killer cell Ig-like receptors (KIR) depends on phosphorylation of cytoplasmic tyrosines in KIR, which recruit tyrosine phosphatase Src homology protein tyrosine phosphatase 1. It is not clear how KIR, whose function lies downstream of a tyrosine kinase, succeeds in blocking proximal NK cell activation signals upon binding HLA class I on target cells. Here we show that mixing NK cells with insect cells expressing HLA-C was sufficient to induce clustering of KIR, and phosphorylation of KIR and SHP-1. Transient phosphorylation of KIR was detected in the presence of pervanadate, an inhibitor of protein tyrosine phosphatases, at suboptimal concentration. Phosphorylation of KIR was specifically induced by ligand binding because it was detected only when HLA-C was loaded with a peptide that permits KIR binding. KIR phosphorylation was not dependent on ICAM-1-mediated adhesion and was not blocked by inhibition of actin polymerization, but required Zn(2+). Fluorescence resonance energy transfer between HLA-C molecules revealed close molecular interactions induced by KIR binding. These results demonstrate tight clustering of KIR and rapid KIR phosphorylation induced simply by binding to HLA-C. The unique property of KIR to become phosphorylated in the absence of adhesion and of actin cytoskeleton rearrangement explains how KIR can efficiently block early activation signals during NK-target cell contacts.     

Meiosis-dependent tyrosine phosphorylation of a yeast protein related to the mouse p51ferT

The FER locus of the mouse encodes two mRNA species: one is constitutively transcribed, giving rise to a 94 kDa tyrosine kinase (p94^ferT); the second is a meiosis-specific RNA that gives rise to a 51 kDa tyrosine kinase (p51^ferT). The p51^ferT RNA and protein accumulate in primary spermatocytes that are in prophase of the first meiotic division. By using polyclonal antibodies directed against synthetic peptides derived from the unique amino-terminus of the mouse p51^ferT, a 51 kDa phosphotyrosyl protein — p51^y — was identified in Saccharomyces cerevisiae. The p51^y protein is constitutively expressed in yeast, but in meiotic cells, concomitantly with commitment to meiotic recombination, its level of phosphorylation on tyrosine residues is increased. A different pattern of phosphorylation is observed on serine residues: at early meiotic times the level is decreased, while in later meiotic time the level increases, reaching the vegetative level. When p51^ferT is ectopically expressed in yeast, it is active, leading to preferential phosphorylation of an approx. 65 kDa protein. A similar pattern of phosphorylation by p51^ferT is seen in mammalian cells.     

Oncostatin M induces tyrosine phosphorylation in endothelial cells and activation of p62yes tyrosine kinase.

Oncostatin M is a polypeptide cytokine produced by activated and transformed T lymphocytes that has diverse biologic effects, including growth inhibition of tumor cells and induction of IL-6 expression in cultured human endothelial cells (HEC). HEC are highly responsive to oncostatin M and express high levels of oncostatin M receptors relative to other cell types. Oncostatin M has previously been found to bind a specific receptor of 150 to 160 kDa. We have found through the use of anti-phosphotyrosine immunoblotting that oncostatin M induces tyrosine phosphorylation in HEC. Anti-phosphotyrosine antibodies specifically immunoprecipitated labeled oncostatin M cross-linked to its receptor, demonstrating that the oncostatin M receptor is either directly phosphorylated on tyrosine after ligand binding or is tightly associated with a phosphotyrosyl protein in these cells. The tyrosine kinase inhibitor herbimycin A blocked the induction of IL-6 by oncostatin M in HEC. In addition, immune complex kinase assays showed that oncostatin M markedly increased the activity of the p62yes tyrosine kinase with a small increase in p59fyn but no increase in p56lyn tyrosine kinase activity in HEC. We conclude that oncostatin M utilizes a tyrosine phosphorylation signal transduction pathway in HEC involving the activation of the p62yes tyrosine kinase, and that this tyrosine phosphorylation pathway leads to the induction of IL-6 expression.     

Mps1 directs the assembly of Cdc20 inhibitory complexes during interphase and mitosis to control M phase timing and spindle checkpoint signaling

The spindle assembly checkpoint (SAC) in mammals uses cytosolic and kinetochore-based signaling pathways to inhibit anaphase. In this study, we use chemical genetics to show that the protein kinase Mps1 regulates both aspects of the SAC. Human MPS1-null cells were generated via gene targeting and reconstituted with either the wild-type kinase (Mps1^wt) or a mutant version (Mps1^as) sensitized to bulky purine analogues. Mps1 inhibition sharply accelerated anaphase onset, such that cells completed mitosis in 12 min, and prevented Cdc20’s association with either Mad2 or BubR1 during interphase, i.e., before the appearance of functional kinetochores. Furthermore, intramitotic Mps1 inhibition evicted Bub1 and all other known SAC transducers from the outer kinetochore, but contrary to a recent study, did not perturb aurora B–dependent phosphorylation. We conclude that Mps1 has two complementary roles in SAC regulation: (1) initial cytoplasmic activation of Cdc20 inhibitors and (2) recruitment of factors that promote sustained anaphase inhibition and chromosome biorientation to unattached kinetochores.     

Differential susceptibility of Plasmodium falciparum versus yeast and mammalian enolases to dissociation into active monomers

In the past, several unsuccessful attempts have been made to dissociate homodimeric enolases into their active monomeric forms. The main objective of these studies had been to understand whether intersubunit interactions are essential for the catalytic and structural stability of enolases. Further motivation to investigate the properties of monomeric enolase has arisen from several recent reports on the involvement of enolase in diverse nonglycolytic (moonlighting) functions, where it may occur in monomeric form. Here, we report successful dissociation of dimeric enolases from Plasmodium falciparum, yeast and rabbit muscle into active and isolatable monomers. Dimeric enolases could be dissociated into monomers by high concentrations ( approximately 250 mm) of imidazole and/or hydrogen ions. Two forms were separated using Superdex-75 gel filtration chromatography. A detailed comparison of the kinetic and structural properties of monomeric and dimeric forms of recombinant P. falciparum enolase showed differences in specific activity, salt-induced inhibition and inactivation, thermal stability, etc. Furthermore, we found that enolases from the three species differ in their dimer dissociation profiles. Specifically, on challenge with imidazole, Mg(II) protected the enolases of yeast and rabbit muscle but not of P. falciparum from dissociation. The observed differential stability of the P. falciparum enolase dimer interface with respect to mammalian enolases could be exploited to selectively dissociate the dimeric parasite enzyme into its catalytically inefficient, thermally unstable monomeric form. Thus enolase could be a novel therapeutic target for malaria.