Polo-like kinase 1-mediated phosphorylation stabilizes Pin1 by inhibiting its ubiquitination in human cells

The Polo-like kinase 1 (Plk1) is a key regulator of mitosis. It is reported that the human peptidyl-prolyl cis/trans-isomerase Pin1 binds to Plk1 from mitotic cell extracts in vitro. Here we demonstrate that Ser-65 in Pin1 is the major site for Plk1-specific phosphorylation, and the polo-box domain of Plk1 is required for this phosphorylation. Interestingly, the phosphorylation of Pin1 by Plk1 does not affect its isomerase activity but rather is linked to its protein stability. Pin1 is ubiquitinated in HeLa S3 cells, and substitution of Glu for Ser-65 reduces the ubiquitination of Pin1. Furthermore, inhibition of Plk1 activity by expression of a dominant negative form of Plk1 or by transfection of small interfering RNA targeted to Plk1 enhances the ubiquitination of Pin1 and subsequently reduces the amount of Pin1 in human cancer cells. Since previous reports suggested that Plk1 is a substrate of Pin1, our work adds a new dimension to this interaction of two important mitotic regulators.     

Phosphorylation of Myosin II-interacting Guanine Nucleotide Exchange Factor (MyoGEF) at Threonine 544 by Aurora B Kinase Promotes the Binding of Polo-like Kinase 1 to MyoGEF

We previously reported that phosphorylation of myosin II-interacting guanine nucleotide exchange factor (MyoGEF) by polo-like kinase 1 (Plk1) promotes the localization of MyoGEF to the central spindle and increases MyoGEF activity toward RhoA during mitosis. In this study we report that aurora B-mediated phosphorylation of MyoGEF at Thr-544 creates a docking site for Plk1, leading to the localization and activation of MyoGEF at the central spindle. In vitro kinase assays show that aurora B can phosphorylate MyoGEF. T544A mutation drastically decreases aurora B-mediated phosphorylation of MyoGEF in vitro and in transfected HeLa cells. Coimmunoprecipitation and in vitro pulldown assays reveal that phosphorylation of MyoGEF at Thr-544 enhances the binding of Plk1 to MyoGEF. Immunofluorescence analysis shows that aurora B colocalizes with MyoGEF at the central spindle and midbody during cytokinesis. Suppression of aurora B activity by an aurora B inhibitor disrupts the localization of MyoGEF to the central spindle. In addition, T544A mutation interferes with the localization of MyoGEF to the cleavage furrow and decreases MyoGEF activity toward RhoA during mitosis. Taken together, our results suggest that aurora B coordinates with Plk1 to regulate MyoGEF activation and localization, thus contributing to the regulation of cytokinesis.     

Phosphorylation of threonine 210 and the role of serine 137 in the regulation of mammalian polo-like kinase

The mammalian polo-like kinase (Plk) plays a critical role in M-phase progression. Plk is phosphorylated and activated by an upstream kinase(s), which has not yet been identified in mammalian cells. Phosphopeptide mapping and phosphoamino acid analyses of Plk labeled in vivo and phosphorylated in vitro by Xenopus polo-like kinase kinase-1 (xPlkk1) or by lymphocyte-oriented kinase, its most closely related mammalian enzyme, indicate that Thr-210 is a major phosphorylation site in activated Plk from mitotic HeLa cells. Although the amino acid sequence surrounding Ser-137 is similar to that at Thr-210 and is conserved in Plk family members, Ser-137 is not detectably phosphorylated in mitotic mammalian cells or by xPlkk1 in vitro. Nevertheless, the substitution of either Thr-210 or Ser-137 with Asp (T210D or S137D) elevates the kinase activity of Plk. The kinase activity of the double mutant S137D/T210D is not significantly different from that of T210D or S137D, demonstrating that substitution of both residues does not have an additive effect on Plk activity. Expression of the S137D mutant construct arrested HeLa cells in early S-phase with slightly separated centrosomes, whereas cells expressing wild type and T210D were arrested or delayed in M-phase. These data indicate that the Ser-137 may have an unexpected and novel role in the function of Plk.     

Proteomic identification of Hsp70 as a new Plk1 substrate in arsenic trioxide-induced mitotically arrested cells

We previously demonstrated that when arsenic trioxide (ATO)-induced mitotically arrested HeLa S3 cells (AIMACs) were treated with staurosporine (SSP) the cells rapidly exited mitosis. To better define the cellular targets and the underlying mechanisms of AIMACs, we applied 2-D DIGE followed by LC-MS/MS analysis and showed that SSP induced a significant change in the phosphoproteome of AIMACs. Among the proteins whose phosphorylation was modulated by SSP, we identified Hsp70, Rad 23B, and eukaryotic translation initiation factor 4B as potentially new substrates of polo-like kinase 1 (Plk1), an essential serine/threonine kinase with versatile mitotic functions. Since Hsp70 is a stress protein responsible for ATO treatment, we further identified Thr(13) , Ser(362) , Ser(631) , and Ser(633) on Hsp70 intracellularly phosphorylated in AIMACs by combining TiO(2) phospho-peptides enrichment and MS/MS analysis. Using antibody specifically against phosph-Ser(631) Hsp70 and further aided by expression of kinase-dead Plk1 and pharmacological inhibition of Plk1, we concluded that Ser(631) on Hsp70 is phosphorylated by Plk1 in AIMACs. By immnuofluorescent staining, we found the colocalization of Hsp70 and Plk1 in AIMACs but not in interphase cells. In addition, Plk1-mediated phosphorylation of Hsp70 prevented AIMACs from mitotic death. Our results reveal that Hsp70 is a novel substrate of Plk1 and that its phosphorylation contributes to attenuation of ATO-induced mitotic abnormalities.     

Identification of β-catenin as a novel substrate of polo-like kinase 1

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays an important role in M phase progression by regulating various downstream substrates via phosphorylation. Here, we identified β-catenin as a novel substrate of Plk1 and determined that Ser-718 is a phosphorylation site for Plk1 by using a phospho-specific antibody that cross-reacts with Plk1-dependent phosphorylation sites. Ser-718 of β-catenin was directly phosphorylated by recombinant Plk1 in vitro, with the phosphorylation signal in cells increasing with overexpression of Plk1 and decreasing when endogenous Plk1 was depleted by small interfering RNA. The phosphorylation at Ser-718 was correlated with the cell cycle-dependent expression of Plk1 which reached a maximum in M phase. We also confirmed that there is a physical interaction between β-catenin and Plk1 using coimmunoprecipitation and a GST pull-down assay. These results demonstrate that β-catenin is a physiological substrate of Plk1 in cells, which may provide a novel insight into the role of β-catenin in M phase.     

Activation of Polo-like kinase 3 by hypoxic stresses

Hypoxia/reoxygenation stress induces the activation of specific signaling proteins and activator protein 1 (AP-1) to regulate cell cycle regression and apoptosis. In the present study, we report that hypoxia/reoxygenation stress activates AP-1 by increasing c-Jun phosphorylation and DNA binding activity through activation of Polo-like-kinase 3 (Plk3) resulting in apoptosis. The specific effect of hypoxia/reoxygenation stress on Plk3 activation resulting in c-Jun phosphorylation was the opposite of UV irradiation-induced responses that are meanly independent on activation of the stress-induced JNK signaling pathway in human corneal epithelial (HCE) cells. The effect of hypoxia/reoxygenation stress-induced Plk3 activation on increased c-Jun phosphorylation and apoptosis was also mimicked by exposure of cells to CoCl(2). Hypoxia/reoxygenation activated Plk3 in HCE cells to directly phosphorylate c-Jun proteins at phosphorylation sites Ser-63 and Ser-73, and to increase DNA binding activity of c-Jun, detected by EMSA. Further evidence demonstrated that Plk3 and phospho-c-Jun were immunocolocalized in the nuclear compartment of hypoxia/reoxygenation stress-induced cells. Increased Plk3 activity by overexpression of wild-type and dominantly positive Plk3 enhanced the effect of hypoxia/reoxygenation on c-Jun phosphorylation and cell death. In contrast, knocking-down Plk3 mRNA suppressed hypoxia-induced c-Jun phosphorylation. Our results provide a new mechanism indicating that hypoxia/reoxygenation induces Plk3 activation instead of the JNK effect to directly phosphorylate and activate c-Jun, subsequently contributing to apoptosis in HCE cells.     

Polo-Like Kinase 2-Dependent Phosphorylation of NPM/B23 on Serine 4 Triggers Centriole Duplication

Duplication of the centrosome is well controlled during faithful cell division while deregulation of this process leads to supernumary centrosomes, chromosome missegregation and aneuploidy, a hallmark of many cancer cells. We previously reported that Polo-like kinase 2 (Plk2) is activated near the G1/S phase transition, and regulates the reproduction of centrosomes. In search for Plk2 interacting proteins we have identified NPM/B23 (Nucleophosmin) as a novel Plk2 binding partner. We find that Plk2 and NPM/B23 interact in vitro in a Polo-box dependent manner. An association between both proteins was also observed in vivo. Moreover, we show that Plk2 phosphorylates NPM/B23 on serine 4 in vivo in S-phase. Notably, expression of a non-phosphorylatable NPM/B23 S4A mutant interferes with centriole reduplication in S-phase arrested cells and leads to a dilution of centriole numbers in unperturbed U2OS cells. The corresponding phospho-mimicking mutants have the opposite effect and their expression leads to the accumulation of centrioles. These findings suggest that NPM/B23 is a direct target of Plk2 in the regulation of centriole duplication and that phosphorylation on serine 4 can trigger this process.     

Uncoupling anaphase-promoting complex/cyclosome activity from spindle assembly checkpoint control by deregulating polo-like kinase 1

Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic phenotypes. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from spindle assembly checkpoint control.     

Plk1 phosphorylation of TRF1 is essential for its binding to telomeres

In a search for Polo-like kinase 1 (Plk1) interaction proteins, we have identified TRF1 (telomeric repeat binding factor 1) as a potential Plk1 target. In this communication we report further characterization of the interaction. We show that Plk1 associates with TRF1, and Plk1 phosphorylates TRF1 at Ser-435 in vivo. Moreover, Cdk1, serving as a priming kinase, phosphorylates TRF1 to generate a docking site for Plk1 toward TRF1. In the presence of nocodazole, ectopic expression of wild type TRF1 but not TRF1 with alanine mutation in the Plk1 phosphorylation site induces apoptosis in cells containing short telomeres but not in cells containing long telomeres. Unexpectedly, down-regulation of TRF1 by RNA interference affects cell proliferation and results in obvious apoptosis in cells with short telomeres but not in cells with long telomeres. Importantly, we observe that telomeric DNA binding ability of TRF1 is cell cycle-regulated and reaches a peak during mitosis. Upon phosphorylation by Plk1 in vivo and in vitro, the ability of TRF1 to bind telomeric DNA is dramatically increased. These results demonstrate that Plk1 interacts with and phosphorylates TRF1 and suggest that Plk1-mediated phosphorylation is involved in both TRF1 overexpression-induced apoptosis and its telomeric DNA binding ability.     

Cell cycle-regulated phosphorylation of the Xenopus polo-like kinase Plx1

Polo-like kinases (Plks) control multiple important events during M phase progression, but little is known about their activation during the cell cycle. The activities of both mammalian Plk1 and Xenopus Plx1 peak during M phase, and this activation has been attributed to phosphorylation. However, no phosphorylation sites have previously been identified in any member of the Plk family. Here we have combined tryptic phosphopeptide mapping with mass spectrometry to identify four major phosphorylation sites in Xenopus Plx1. All four sites appear to be phosphorylated in a cell cycle-dependent manner. Phosphorylations at two sites (Ser-260 and Ser-326) most likely represent autophosphorylation events, whereas two other sites (Thr-201 and Ser-340) are targeted by upstream kinases. Several recombinant kinases were tested for their ability to phosphorylate Plx1 in vitro. Whereas xPlkk1 phosphorylated primarily Thr-10, Thr-201 was readily phosphorylated by protein kinase A, and Cdk1/cyclin B was identified as a likely kinase acting on Ser-340. Phosphorylation of Ser-340 was shown to be responsible for the retarded electrophoretic mobility of Plx1 during M phase, and phosphorylation of Thr-201 was identified as a major activating event.     

Phosphorylation of p27Kip1 at Thr-157 interferes with its association with importin alpha during G1 and prevents nuclear re-entry

We have studied mechanisms of Akt-mediated phosphorylation and regulation of cellular localization of p27. Akt phosphorylates Thr-157 in p27 and retains it in the cytosol. In cells arrested in G(1) and then synchronized to enter into S phase, Akt-mediated phosphorylation of Thr-157 p27 occurred in the cytosol during G(1) phase of the cell cycle. Both T157A and S10A p27 mutants localized in the nucleus in all phases of the cell cycle regardless of the expression of active Akt. Thr-157 phosphorylation was undetectable in S10A-p27, suggesting that Ser-10 phosphorylation is required for p27 localization in the cytosol and subsequent phosphorylation at Thr-157. Phosphorylation at Thr-157 interrupted the association of p27 with importin alpha. A T157A-p27 mutant protein exhibited higher association with importin alpha than wild-type-p27. Treatment of transfected and endogenous p27 with alkaline phosphatase rescued its association with importin alpha. Leptomycin B inhibited cytosolic Thr-157 P-p27 staining, implying that CRM1-dependent nuclear export is required for Akt-mediated Thr-157 phosphorylation. Heterokaryon shuttling assays with NIH3T3 (mouse) cells transfected with FLAG-p27 and HeLa (human) cells revealed that both wild type and T157A-p27 shuttled from NIH3T3 to HeLa cell nuclei with similar frequencies. However, S10A-p27 was found only in the NIH3T3 nuclei of NIH3T3-HeLa cell fusions. These results suggest that 1) Ser-10 phosphorylation is required for nuclear export of p27, 2) subsequent Akt-mediated phosphorylation at Thr-157 during G(1) phase corrals p27 in the cytosol, and 3) Thr-157 phosphorylation inhibits the association of p27 with importin alpha thus preventing its re-entry into the nucleus.     

Phosphorylation of MyoGEF on Thr-574 by Plk1 promotes MyoGEF localization to the central spindle

We reported previously that a guanine nucleotide exchange factor, MyoGEF, localizes to the central spindle, activates RhoA, and is required for cytokinesis. In this study, we have found that Plk1 (polo-like kinase 1) can phosphorylate MyoGEF, thereby recruiting MyoGEF to the central spindle as well as enhancing MyoGEF activity toward RhoA. The in vitro kinase assay shows that Plk1 can phosphorylate MyoGEF on threonine 574. Immunoprecipitation/immunoblot analysis demonstrates that mutation of threonine 574 to alanine dramatically decreases threonine phosphorylation of MyoGEF in transfected HeLa cells, suggesting that threonine 574 is phosphorylated in vivo. Consistent with these observations, immunofluorescence shows that Plk1 and MyoGEF colocalize at the spindle pole and central spindle during mitosis and cytokinesis. Importantly, RNA interference-mediated depletion of Plk1 interferes with the localization of MyoGEF at the spindle pole and central spindle. Moreover, mutation of threonine 574 to alanine in MyoGEF or depletion of Plk1 by RNA interference leads to a decrease in MyoGEF activity toward RhoA in HeLa cells. Therefore, our results suggest that Plk1 can regulate MyoGEF activity and localization, contributing to the regulation of cytokinesis.     

Phosphorylation of right open reading frame 2 (Rio2) protein kinase by polo-like kinase 1 regulates mitotic progression

Polo-like kinase 1 (Plk1) plays essential roles during multiple stages of mitosis by phosphorylating a number of substrates. Here, we report that the atypical protein kinase Rio2 is a novel substrate of Plk1 and can be phosphorylated by Plk1 at Ser-335, Ser-380, and Ser-548. Overexpression of Rio2 causes a prolonged mitotic exit whereas knockdown of Rio2 accelerates mitotic progression, suggesting that Rio2 is required for the proper mitotic progression. Overexpression of phospho-mimicking mutant Rio2 S3D but not the nonphosphorylatable mutant Rio2 S3A displays a profile similar to that of wild-type Rio2. These results indicate that the phosphorylation status of Rio2 correlates with its function in mitosis. Furthermore, time-lapse imaging data show that overexpression of Rio2 but not Rio2 S3A results in a slowed metaphase-anaphase transition. Collectively, these findings strongly indicate that the Plk1-mediated phosphorylation of Rio2 regulates metaphase-anaphase transition during mitotic progression.     

C-terminal phosphorylation of LKB1 is not required for regulation of AMP-activated protein kinase, BRSK1, BRSK2, or cell cycle arrest

The tumor suppressor protein kinase LKB1 exerts its effects by phosphorylating and activating AMP-activated protein kinase (AMPK) and members of the AMPK-related kinase family, such as the brain-specific kinases BRSK1/BRSK2 (SAD-B/SAD-A). LKB1 contains a conserved serine residue near the C terminus (Ser-431 in mouse LKB1) that is phosphorylated by cyclic AMP-dependent protein kinase and p90-RSK. Although some studies suggest that LKB1 is constitutively active and is not rate-limiting for activation of AMPK, others have suggested that phosphorylation of Ser-431 is necessary to allow LKB1 to phosphorylate and activate AMPK and other downstream kinases. Prompted by our discovery of an LKB1 splice variant (LKB1S) that lacks Ser-431, we have reinvestigated this question. In HeLa cells (which lack endogenous LKB1), co-expression with STRADalpha and MO25alpha of wild type LKB1, the S431A or S431E mutants of LKB1, or LKB1(S) gave equal levels of activation of endogenous AMPK. Similarly, recombinant STRADalpha.MO25alpha complexes containing these LKB1 variants were equally effective at phosphorylating and activating AMPK, BRSK1, and BRSK2 in cell-free assays. Finally, all four LKB1 variants and a truncated LKB1 lacking the C-terminal region altogether were equally effective at causing cell cycle arrest when co-expressed with STRADalpha and MO25alpha in the G361 melanoma cell line. Our results do not support the idea that phosphorylation of Ser-431 increases the ability of LKB1 to phosphorylate downstream targets.     

Protein kinase A site-specific phosphorylation regulates ATP-binding cassette A1 (ABCA1)-mediated phospholipid efflux

ATP-binding cassette A1 (ABCA1) is a key mediator of cholesterol and phospholipid efflux to apolipoprotein particles. We show that ABCA1 is a constitutively phosphorylated protein in both RAW macrophages and in a human embryonic kidney cell line expressing ABCA1. Furthermore, we demonstrate that phosphorylation of ABCA1 is mediated by protein kinase A (PKA) or a PKA-like kinase in vivo. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assays, we show that Ser-1042 and Ser-2054, located in the nucleotide binding domains of ABCA1, are major phosphorylation sites for PKA. ApoA-I-dependent phospholipid efflux was decreased significantly by mutation of Ser-2054 alone and Ser-1042/Ser-2054 but was not significantly impaired with Ser-1042 alone. The mechanism by which ABCA1 phosphorylation affected ApoA-I-dependent phospholipid efflux did not involve either alterations in ApoA-I binding or changes in ABCA1 protein stability. These studies demonstrate a novel serine (Ser-2054) on the ABCA1 protein crucial for PKA phosphorylation and for regulation of ABCA1 transporter activity.     

Functional independency between catalytic activity and subcellular targeting of Polo-like kinase-1: Phenotypes of ectopic overexpression of various mutants

The mammalian polo-like kinase-1 (Plk1) plays key role in the progression of the M-phase. As cells enter the M-phase, Plk1 is phosphorylated by an upstream kinase on Thr-210 in N-terminal region, which is essential for Plk1 activation. In addition to catalytic activation, the subcellular localization is also an important task for Plk1, and the C-terminal polo-boxes are responsible for Plk1 targeting. Although both catalytic activation and targeting might be essential and work concurrently, it is still unclear which process precedes the other in mitotic progression, and which is more essential for the function of Plk1 through the M-phase. In order to investigate this mechanism, this study introduced several point mutations in the catalytic and polo-box domains, and overexpressed these constructs in HeLa cells. Although the cellular effects by ectopic expression were different in the cells containing various Plk1 constructs, phosphorylation itself, not the catalytic activity, was found to be important for the progression of the M-phase. Plk1 phosphorylation was also responsible for targeting by polo-box, and compensated for the polo-box mutation. These results suggested that Plk1 is phosphorylated in the starting M-phase, and this phosphorylation induces polo-box targeting through a possible conformational change. In this situation, catalytic activation does not appear to be a critical factor for the function of Plk1.     

ATR enforces the topoisomerase II-dependent G2 checkpoint through inhibition of Plk1 kinase

An ATR-dependent G(2) checkpoint responds to inhibition of topoisomerase II and delays entry into mitosis by sustaining nuclear exclusion of cyclin B1-Cdk1 complexes. Here we report that induction of this checkpoint with ICRF-193, a topoisomerase II catalytic inhibitor that does not cause DNA damage, was associated with an ATR-dependent inhibition of polo-like kinase 1 (Plk1) kinase activity and a decrease in cyclin B1 phosphorylation. Expression of constitutively active Plk1 but not wild type Plk1 reversed ICRF-193-induced mitotic delay in HeLa cells, suggesting that Plk1 kinase activity is important for the checkpoint response to ICRF-193. G(2)/M synchronized normal human fibroblasts, when treated with ICRF-193, showed a decrease in cyclin B1 phosphorylation and Plk1 kinase activity despite high cyclin B1-Cdk1 kinase activity. G(2) fibroblasts that were treated with caffeine to override the checkpoint response to ICRF-193 displayed a high incidence of chromosomal aberrations. Taken together, these results suggest that ATR-dependent inhibition of Plk1 kinase activity may be one mechanism to regulate cyclin B1 phosphorylation and sustain nuclear exclusion during the G(2) checkpoint response to topoisomerase II inhibition. Moreover, the results demonstrate an important role for the topoisomerase II-dependent G(2) checkpoint in the preservation of human genomic stability.     

Phosphorylation- and polo-box-dependent binding of Plk1 to Bub1 is required for the kinetochore localization of Plk1

Polo-like kinase 1 (Plk1) is required for the generation of the tension-sensing 3F3/2 kinetochore epitope and facilitates kinetochore localization of Mad2 and other spindle checkpoint proteins. Here, we investigate the mechanism by which Plk1 itself is recruited to kinetochores. We show that Plk1 binds to budding uninhibited by benzimidazole 1 (Bub1) in mitotic human cells. The Plk1-Bub1 interaction requires the polo-box domain (PBD) of Plk1 and is enhanced by cyclin-dependent kinase 1 (Cdk1)-mediated phosphorylation of Bub1 at T609. The PBD-dependent binding of Plk1 to Bub1 facilitates phosphorylation of Bub1 by Plk1 in vitro. Depletion of Bub1 in HeLa cells by RNA interference (RNAi) diminishes the kinetochore localization of Plk1. Ectopic expression of the wild-type Bub1, but not the Bub1-T609A mutant, in Bub1-RNAi cells restores the kinetochore localization of Plk1. Our results suggest that phosphorylation of Bub1 at T609 by Cdk1 creates a docking site for the PBD of Plk1 and facilitates the kinetochore recruitment of Plk1.