Cyclin-Dependent Kinase 1-Mediated Bcl-xL/Bcl-2 Phosphorylation Acts as a Functional Link Coupling Mitotic Arrest and Apoptosis

Despite detailed knowledge of the components of the spindle assembly checkpoint, a molecular explanation of how cells die after prolonged spindle checkpoint activation, and thus how microtubule inhibitors and other antimitotic drugs ultimately elicit their lethal effects, has yet to emerge. Mitotically arrested cells typically display extensive phosphorylation of two key antiapoptotic proteins, Bcl-x_L and Bcl-2, and evidence suggests that phosphorylation disables their antiapoptotic activity. However, the responsible kinase has remained elusive. In this report, evidence is presented that cyclin-dependent kinase 1 (CDK1)/cyclin B catalyzes mitotic-arrest-induced Bcl-x_L/Bcl-2 phosphorylation. Furthermore, we show that CDK1 transiently and incompletely phosphorylates these proteins during normal mitosis. When mitosis is prolonged in the absence of microtubule inhibition, Bcl-x_L and Bcl-2 become highly phosphorylated. Transient overexpression of nondegradable cyclin B1 caused apoptotic death, which was blocked by a phosphodefective Bcl-x_L mutant but not by a phosphomimetic Bcl-x_L mutant, confirming Bcl-x_L as a key target of proapoptotic CDK1 signaling. These findings suggest a model whereby a switch in the duration of CDK1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-x_L/Bcl-2 phosphorylation, resulting in inactivation of their antiapoptotic function. Thus, phosphorylation of antiapoptotic Bcl-2 proteins acts as a sensor for CDK1 signal duration and as a functional link coupling mitotic arrest to apoptosis.The cell division cycle is controlled by checkpoints, which ensure the fidelity of chromosome replication and segregation, as well as orderly progression through the cell cycle. If these critical events cannot be completed as scheduled, damaged cells, which might otherwise pose a threat to the organism as precancerous cells, are eliminated (16). The mitotic checkpoint, for example, produces a “prevent anaphase” signal until all the chromosomes are properly attached to kinetochores (22). Microtubule inhibitors (MTIs) and other antimitotic agents prolong the activation of this checkpoint, causing mitotic arrest, which culminates in cell death generally via intrinsic apoptosis, providing a rationale for the use of these agents as antitumor agents (20, 31). Intrinsic or mitochondrial apoptosis is regulated by the Bcl-2 family of proteins, which exhibit either pro- or antiapoptotic properties (17, 37). The BH3-only proapoptotic members act as essential initiators of intrinsic apoptosis, whereas the multidomain proapoptotic members, Bax and Bak, act as essential mediators of mitochondrial membrane permeability. Antiapoptotic Bcl-2 family members, including Bcl-x_L, Bcl-2, and Mcl-1, oppose apoptosis by binding to the proapoptotic members and neutralizing their activity.The molecular mechanisms leading to cell death in response to spindle checkpoint activation have yet to be established. Indeed, how the spindle checkpoint couples to pathways regulating cell survival and death still represents an unresolved issue in cell biology (26, 35). Nonetheless, it seems reasonable to hypothesize that signals generated in response to prolonged mitotic arrest are eventually transduced to the apoptotic machinery. In this regard, it is striking that MTIs consistently induce the phosphorylation of two key antiapoptotic proteins, Bcl-2 and Bcl-x_L, whereas other apoptotic stimuli fail to do so (9, 13, 25). The results of studies with phosphodefective mutants of Bcl-2 and Bcl-x_L indicate that phosphorylation antagonizes their antiapoptotic function (2, 33, 36), but the precise mechanism(s) has yet to be fully clarified.The identity of the kinase responsible for the extensive phosphorylation of Bcl-x_L and Bcl-2 that occurs in response to sustained spindle checkpoint activation is unresolved. Identification of this kinase is considered to be of critical importance, since it will provide insight into the molecular links between mitotic arrest and cell death, as well as the molecular mechanism of action of antimitotic drugs. Several candidates have been proposed, including Raf-1 (3), Jun N-terminal protein kinase (JNK) (2, 11, 36), protein kinase A (PKA) (32), cyclin-dependent kinase 1 (CDK1) (24), and mammalian target of rapamycin (mTOR) (4). In general, however, conclusions have been correlative or have been based on the use of kinase inhibitors tested under conditions that precluded mitotic arrest and thus indirectly blocked the effects of MTIs. Thus, strong experimental evidence supporting identification is lacking.Here we present evidence that the CDK1/cyclin B kinase complex is responsible for mitotic arrest-induced Bcl-x_L/Bcl-2 phosphorylation. Furthermore, we show that CDK1 transiently and incompletely phosphorylates these proteins during normal mitosis. The findings suggest a model whereby a switch in the duration of CDK1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-x_L/Bcl-2 phosphorylation, resulting in inactivation of the antiapoptotic function of Bcl-x_L/Bcl-2. Thus, CDK1-mediated phosphorylation of antiapoptotic Bcl-2 proteins acts as a key link coupling mitotic arrest to apoptosis.     

Online immunoaffinity liquid chromatography/tandem mass spectrometry determination of a type II collagen peptide biomarker in rat urine: Investigation of the impact of collision-induced dissociation fluctuation on peptide quantitation

Proteolytic fragments of type II collagen, a major component of joint tissue, have recently been identified as biomarkers for osteoarthritis, a progressive disease associated with cartilage degeneration. A liquid chromatography/tandem mass spectrometry (MS/MS) assay that utilizes online immunoaffinity chromatography and column switching was developed in our laboratory for the neoepitope of type II collagen (NET2C). During method development, peptide collision-induced dissociation (CID) was found to be a significant source of assay variation, which exceeded 10% CV, despite the fact that a stable-isotope-labeled (SIL) internal standard was used to minimize imprecision. This phenomenon was studied in detail using peptides and associated SIL internal standards of varying lengths and amino acid compositions. Variability in peptide CID necessitated the monitoring of multiple MS/MS transitions to obtain acceptable assay precision. The assay was subsequently validated to measure NET2C concentrations in rat urine over the range of 0.1 to 10 ng/mL. The interday accuracy and precision ranged from 3.9 to 13.1 (%CV) and 10.7 to 5.3 (%RE), respectively, across the range of validated concentrations. A specific application of the assay is presented in which the role of estrogen deficiency in the development and progression of osteoarthritis was investigated. In this study, the effect of estrogen on lowering NET2C concentrations in urine in ovariectomized rats was demonstrated.     

Effects of phosphorylation of P-glycoprotein on multidrug resistance

Cells expressing elevated levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug resistance phenotype. Studies involving protein kinase activators and inhibitors have implied that covalent modification of P-glycoprotein by phosphorylation may modulate its biological activity as a multidrug transporter. Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells independent of, or in addition to their effects on the state of phosphorylation of P-glycoprotein. The protein kinase(s) responsible for P-glycoprotein phosphorylation has(ve) not been unambiguously identified, although several possible candidates have been suggested. Recent biochemical analyses demonstrate that the major sites of phosphorylation are clustered within the linker region that connects the two homologous halves of P-glycoprotein. Mutational analyses have been initiated to confirm this finding. Preliminary data obtained from phosphorylation- and dephosphorylation-defective mutants suggest that phosphorylation of P-glycoprotein is not essential to confer multidrug resistance.     

Identification and partial characterization of α2-macroglobulin from the tuatara (Sphenodon punctatus)

α₂-Macroglobulin (α₂-M), a large molecular mass proteinase-binding protein, was identified in plasma from tuatara (Sphenodon), a rare reptile endemic to New Zealand. In this genus, α₂-M constitutes 11–13% of total plasma protein (∼2.2–3.9 mg/ml). Analysis of blood samples collected at approximately monthly intervals from individual tuatara indicated that the plasma level of α₂-M remains fairly constant. The subunits of tuatara α₂-M have an apparent molecular mass of ∼160 kDa as determined by SDS-polyacrylamide gel electrophoresis and the intact protein is an oligomer that contains inter-chain disulfide bonds. N-terminal sequence analyses of tuatara α₂-M revealed a distinct similarity to α-macroglobulins of other vertebrates and that at least two types of α₂-M subunits are present in plasma of tuatara.     

A sliding docking interaction is essential for sequential and processive phosphorylation of an SR protein by SRPK1

The 2.9 A crystal structure of the core SRPK1:ASF/SF2 complex reveals that the N-terminal half of the basic RS domain of ASF/SF2, which is destined to be phosphorylated, is bound to an acidic docking groove of SRPK1 distal to the active site. Phosphorylation of ASF/SF2 at a single site in the C-terminal end of the RS domain generates a primed phosphoserine that binds to a basic site in the kinase. Biochemical experiments support a directional sliding of the RS peptide through the docking groove to the active site during phosphorylation, which ends with the unfolding of a beta strand of the RRM domain and binding of the unfolded region to the docking groove. We further suggest that the priming of the first serine facilitates directional substrate translocation and efficient phosphorylation.     

Mitochondrial morphodynamics alteration induced by influenza virus infection as a new antiviral strategy

Influenza virus infections are major public health threats due to their high rates of morbidity and mortality. Upon influenza virus entry, host cells experience modifications of endomembranes, including those used for virus trafficking and replication. Here we report that influenza virus infection modifies mitochondrial morphodynamics by promoting mitochondria elongation and altering endoplasmic reticulum-mitochondria tethering in host cells. Expression of the viral RNA recapitulates these modifications inside cells. Virus induced mitochondria hyper-elongation was promoted by fission associated protein DRP1 relocalization to the cytosol, enhancing a pro-fusion status. We show that altering mitochondrial hyper-fusion with Mito-C, a novel pro-fission compound, not only restores mitochondrial morphodynamics and endoplasmic reticulum-mitochondria contact sites but also dramatically reduces influenza replication. Finally, we demonstrate that the observed Mito-C antiviral property is directly connected with the innate immunity signaling RIG-I complex at mitochondria. Our data highlight the importance of a functional interchange between mitochondrial morphodynamics and innate immunity machineries in the context of influenza viral infection.     

Restriction enzyme analysis of DNA methylation in "condensed" chromatin of Ha-ras-transformed NIH 3T3 cells.

Increased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha-ras oncogene. The structural basis for this oncogene-mediated alteration in nuclear organization is unknown. Since DNA methylation is likely to be involved in regulating the nucleosomal level of DNA packaging, we studied the role of DNA methylation in higher-order chromatin organization induced by Ha-ras. CpG-methylated DNA content was estimated in "condensed" chromatin of Ha-ras-transformed NIH 3T3 cell lines which differ in ras expression and ras-induced metastatic ability but present approximately the same values of "condensed" chromatin areas. The question posed was that if DNA methylation were involved with the chromatin higher-order organization induced by Ha-ras in these cell lines, the methylated DNA density in the "condensed" chromatin would also be the same. The DNA evaluation was performed by video image analysis in Feulgen-stained cells previously subjected to treatment with Msp I and Hpa II restriction enzymes, which distinguish between methylated and non-methylated DNA. The amount of methylated CpG sequences not digested by Hpa II in "condensed" chromatin regions was found to vary in the studied ras-transformed cell lines. DNA CpG methylation status is thus suggested not to be involved with the higher order chromatin condensation induced by ras transformation in the mentioned NIH 3T3 cell lines.     

Concurrent reductions in blood pressure and metabolic rate during fasting in the unrestrained SHR

Arctic ground squirrels (Spermophilus parryii) overwinter in hibernaculum conditions that are substantially below freezing. During torpor, captive arctic ground squirrels displayed ambient temperature (T(a))-dependent patterns of core body temperature (T(b)), metabolic rate (TMR), and metabolic fuel use, as determined by respiratory quotient (RQ). At T(a) 0 to -16 degrees C, T(b) remained relatively constant, and TMR rose proportionally with the expanding gradient between T(b) and T(a), increasing >15-fold from a minimum of 0.0115 +/- 0.0012 ml O(2). g(-1). h(-1). At T(a) 0-20 degrees C, T(b) increased with T(a); however, TMR did not change significantly from T(b) 0 to 12 degrees C, indicating temperature-independent inhibition of metabolic rate. The overall change in TMR from T(b) 4 to 20 degrees equates to a Q(10) of 2.4, but within this range of T(b), Q(10) changed from 1.0 to 14.1. During steady-state torpor at T(a) 4 and 8 degrees C, RQ averaged 0.70 +/- 0.013, indicating exclusive lipid catabolism. At T(a) -16 and 20 degrees C, RQ increased significantly to >0.85, consistent with recruitment of nonlipid fuels. RQ was negatively correlated with maximum torpor bout length. For T(a) values <0 degrees C, this relationship supports the hypothesis that availability of nonlipid metabolic fuels limits torpor duration in hibernating mammals; for T(a) values >0 degrees C, hypotheses linked to body temperature are supported. Because anterior body temperatures differ from core, overall, the duration torpor can be extended in hibernating mammals may be dependent on brain temperature.