TIPE2: A Candidate for Targeting Antitumor Immunotherapy.

TNF-α-induced protein 8-like 2 (TIPE2 or TNFAIP8L2) is a recently discovered negative regulator of innate and adaptive immunity. TIPE2 is expressed in a wide range of tissues, both immune and nonimmune, and is implicated in the maintenance of immune homeostasis within the immune system. Furthermore, TIPE2 has been shown to play a pivotal role in the regulation of inflammation and the development of tumor. This review focuses on the structural characteristics, expression patterns, and functional roles of TIPE proteins, with a particular emphasis on the role and underlying mechanisms of TIPE2 in immune regulation and its involvement in different diseases. However, the current body of evidence is still limited in providing a comprehensive understanding of the complex role of TIPE2 in the human body, warranting further investigation to elucidate the possible mechanisms and functions of TIPE2 in diverse disease contexts.

GAPDH facilitates homologous recombination repair by stabilizing RAD51 in an HDAC1-dependent manner.

Homologous recombination (HR), a form of error-free DNA double-strand break (DSB) repair, is important for the maintenance of genomic integrity. Here, we identify a moonlighting protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a regulator of HR repair, which is mediated through HDAC1-dependent regulation of RAD51 stability. Mechanistically, in response to DSBs, Src signaling is activated and mediates GAPDH nuclear translocation. Then, GAPDH directly binds with HDAC1, releasing it from its suppressor. Subsequently, activated HDAC1 deacetylates RAD51 and prevents it from undergoing proteasomal degradation. GAPDH knockdown decreases RAD51 protein levels and inhibits HR, which is re-established by overexpression of HDAC1 but not SIRT1. Notably, K40 is an important acetylation site of RAD51, which facilitates stability maintenance. Collectively, our findings provide new insights into the importance of GAPDH in HR repair, in addition to its glycolytic activity, and they show that GAPDH stabilizes RAD51 by interacting with HDAC1 and promoting HDAC1 deacetylation of RAD51.

G-Quadruplex-Mediated Transcriptional Regulation of SYT7: Implications for Tumor Progression and Therapeutic Strategies.

Synaptotagmin 7 (SYT7), a member of the synaptotagmin family, exhibits high expression in various tumors and is closely associated with patient prognosis. The tight regulation of SYT7 expression assumes paramount significance in the progression of tumorigenesis. In this study, we detected a high GC content in the first 1000 bp of the promoter region of SYT7, suggesting a potential role of the G-quadruplex in its transcriptional regulation. Circular dichroism spectroscopy results showed that -187 to -172 bp sequence can form a typical parallel G-quadruplex structure, and site mutation revealed the critical role of the ninth guanine in its formation. Then, treatment of two ligands of G-quadruplex (TMPyP4 and Pyridostatin) reduced both the expression of SYT7 and subsequent tumor proliferation, demonstrating the potential of the G-quadruplex as a targeted therapy for tumors. By shedding light on the pivotal role of the G-quadruplex in regulating SYT7 transcription, our study not only advances our comprehension of this intricate regulatory mechanism but also emphasizes the significance of SYT7 in tumor proliferation. These findings collectively contribute to a more comprehensive understanding of the interplay between G-quadruplex regulation and SYT7 function in tumor development.

Excessive iron inhibits insulin secretion via perturbing transcriptional regulation of SYT7 by OGG1.

Although iron overload is closely related to the occurrence of type 2 diabetes mellitus (T2DM), the specific mechanism is unclear. Here, we found that excessive iron inhibited the secretion of insulin (INS) and impaired islet ß cell function through downregulating Synaptotagmin 7 (SYT7) in iron overload model in vivo and in vitro. Our results further demonstrated that 8-oxoguanine DNA glycosylase (OGG1), a key protein in the DNA base excision repair, was an upstream regulator of SYT7. Interestingly, such regulation could be suppressed by excessive iron. Ogg1-null mice, iron overload mice and db/db mice exhibit reduced INS secretion, weakened ß cell function and subsequently impaired glucose tolerance. Notably, SYT7 overexpression could rescue these phenotypes. Our data revealed an intrinsic mechanism by which excessive iron inhibits INS secretion through perturbing the transcriptional regulation of SYT7 by OGG1, which suggested that SYT7 was a potential target in clinical therapy for T2DM.

[Genetic analysis of a fetus with with 45,X/46,X,idic(Y)(q11.2) mosaicism].

OBJECTIVE: To explore the genetic characteristics of a fetus with sex chromosome abnormality indicated by non-invasive prenatal testing (NIPT) at 25+ gestational weeks. METHODS: A pregnant woman who was admitted to the Taizhou Hospital for abnormal NIPT result on January 6, 2023 was selected as the study subject. Relevant clinical data was collected. The fetus was subjected to chromosomal karyotyping analysis, copy number variation sequencing (CNV-seq), fluorescence in situ hybridization (FISH), and multiplex PCR assays. RESULTS: NIPT had suggested monosomy of X chromosome. The fetus was found to have a chromosomal karyotype of 45,X[59]/46,X,del(Y)(q11.2)[17] at 30+ weeks of gestational age. CNV-seq suggested the presence a 7.98 Mb deletion at Yq11.222q12 and a mosaicism 16.92 Mb deletion. FISH suggested that the fetus harbored two SRY genes and a mosaicism sex chromosomal abnormality, and multiplex PCR revealed that its AZF b+c region was completely deleted. C-banded karyotyping showed darkly stained dense mitotic granules at both ends of the Y chromosome. The fetus was ultimately determined as a 45,X/46,X,idic(Y)(q11.2) mosaicism. Following elected abortion, testing of the fetal tissue confirmed the presence of 45,X/46,XY mosaicism, and CNV-seq result of the placental tissue was compatible with that of NIPT. CNV-seq analysis of the couple revealed no obvious abnormality. CONCLUSION: With combined NIPT, karyotyping, CNV-seq, FISH and multiplex PCR assays, the fetus was diagnosed as a 45,X/46,X,idic(Y)(q11.2) mosaicism with deletion of the AZF b+c region. Above finding has enabled prenatal diagnosis for the fetus.

Short-Term Starvation Weakens the Efficacy of Cell Cycle Specific Chemotherapy Drugs through G1 Arrest.

Short-term starvation (STS) during chemotherapy can block the nutrient supply to tumors and make tumor cells much more sensitive to chemotherapeutic drugs than normal cells. However, because of the diversity of starvation methods and the heterogeneity of tumors, this method's specific effects and mechanisms for chemotherapy are still poorly understood. In this study, we used HeLa cells as a model for short-term starvation and etoposide (ETO) combined treatment, and we also mimicked the short-term starvation effect by knocking down the glycolytic enzyme GAPDH to explore the exact molecular mechanism. In addition, our study demonstrated that short-term starvation protects cancer cells against the chemotherapeutic agent ETO by reducing DNA damage and apoptosis due to the STS-induced cell cycle G1 phase block and S phase reduction, thereby diminishing the effect of ETO. Furthermore, these results suggest that starvation therapy in combination with cell cycle-specific chemotherapeutic agents must be carefully considered.

FEN1 inhibitor synergizes with low-dose camptothecin to induce increased cell killing via the mitochondria mediated apoptotic pathway.

Camptothecin has been used in tumor therapy for a long time but its antitumor effect is rather limited due to the side effect and the drug resistance. FEN1, a major component of DNA repair systems, plays important roles in maintaining genomic stability via DNA replication and repair. Here we found that FEN1 inhibitor greatly sensitizes cancer cells to low-dose camptothecin. The combinative treatment of FEN1 inhibitor and 1 nM camptothecin induced a synthetic lethal effect, which synergistically suppressed cancer cell proliferation and significantly mediated apoptosis both in vitro and in vivo. Our study suggested that targeting FEN1 could be a potent strategy for tumor-targeting cancer therapy.

Polß modulates the expression of type I interferon via STING pathway.

DNA Polymerase ß (Polß) is a key enzyme in base excision repair (BER), which is very important in maintaining the stability and integrity of the genome. Mutant Polß is closely associated with carcinogenesis. However, Polß is highly expressed in most cancers, but the underlying mechanism is not well understood. Here, we found that breast cancer cells MCF-7 with Polß knockdown exhibited high levels of type I interferon and were easily eliminated by natural killer (NK) cells.Similarly, Polß-mutant (R137Q) mice exhibited chronic inflammation symptoms in multiple organs and upregulated type I interferon levels. Further results showed that Polß deficiency caused more DNA damage accumulation in cells and triggered the leakage of damaged DNA into the cytoplasm, which activated the STING/IRF3 pathway, promoted phosphorylated IRF3 translocating into the nucleus and enhanced the expression of type I interferon and proinflammatory cytokines. In addition, this effect could be eliminated by Polß overexpression, STING inhibitor or STING knockdown. Taken together, our findings provide mechanistic insight into the role of Polß in cancers by linking DNA repair and the inflammatory STING pathway.

Src-mediated phosphorylation of GAPDH regulates its nuclear localization and cellular response to DNA damage.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme involved in energy metabolism. Recently, GAPDH has been suggested to have extraglycolytic functions in DNA repair, but the underlying mechanism for the GAPDH response to DNA damage remains unclear. Here, we demonstrate that the tyrosine kinase Src is activated under DNA damage stress and phosphorylates GAPDH at Tyr41. This phosphorylation of GAPDH is essential for its nuclear translocation and DNA repair function. Blocking the nuclear import of GAPDH by suppressing Src signaling or through a GAPDH Tyr41 mutation impairs its response to DNA damage. Nuclear GAPDH is recruited to DNA lesions and associates with DNA polymerase ß (Pol ß) to function in DNA repair. Nuclear GAPDH promotes Pol ß polymerase activity and increases base excision repair (BER) efficiency. Furthermore, GAPDH knockdown dramatically decreases BER efficiency and sensitizes cells to DNA damaging agents. Importantly, the knockdown of GAPDH in colon cancer SW480 cells and xenograft models effectively enhances their sensitivity to the chemotherapeutic drug 5-FU. In summary, our findings provide mechanistic insight into the new function of GAPDH in DNA repair and suggest a potential therapeutic target in chemotherapy.

[Cytogenetic analysis of an amniotic sample with X chromosome abnormality signaled by non-invasive prenatal testing].

OBJECTIVE: To determine the chromosomal karyotype of a fetus with copy number variation (CNV) of the X chromosome signaled by non-invasive prenatal testing (NIPT). METHODS: NIPT was performed on the peripheral blood sample taken from the pregnant women. Amniotic fluid and cord blood samples were subjected to conventional G banded karyotyping, and were further analyzed by high-throughput sequencing for chromosome microdeletion/microduplication. The results were then verified by fluorescence in situ hybridization (FISH) on metaphase cells. RESULTS: The NIPT test of pregnant women suggested low risk for 21-trisomy, 18-trisomy, and 13-trisomy, whilst indicated the number of chromosome X to be low. The G banded karyotype of the amniotic fluid and cord blood cells was 46,XX. The result of high-throughput sequencing chromosome microdeletion/microduplication detection was seq[hg19](X)× 1, (Y)× 2. FISH showed a clear red signal at each end of a whole chromosome, and a green signal on the other chromosome, with a karyotype of 46,X,ish idic(Y) (q11.23) (SRY++, DXZ1+). C banding showed that there is a dense and a slightly loose centromere at both ends of the Y chromosome, and the parachromatin region was missing. The karyotype of amniotic fluid and cord blood cells was finally determined to be 46,X, pus idic(Y) (q11.23). CONCLUSION: For chromosome anomalies suggested by auxiliary report of NIPT, conventional karyotyping combined with high-throughput sequencing for chromosome microdeletion/microduplication should be adopted for the prevention and reduction of the rate of chromosome microdeletion/microduplication syndromes.

Two-way crosstalk between BER and c-NHEJ repair pathway is mediated by Pol-ß and Ku70.

Multiple DNA repair pathways may be involved in the removal of the same DNA lesion caused by endogenous or exogenous agents. Although distinct DNA repair machinery fulfill overlapping roles in the repair of DNA lesions, the mechanisms coordinating different pathways have not been investigated in detail. Here, we show that Ku70, a core protein of nonhomologous end-joining (NHEJ) repair pathway, can directly interact with DNA polymerase-ß (Pol-ß), a central player in the DNA base excision repair (BER), and this physical complex not only promotes the polymerase activity of Pol-ß and BER efficiency but also enhances the classic NHEJ repair. Moreover, we find that DNA damages caused by methyl methanesulfonate (MMS) or etoposide promote the formation of Ku70-Pol-ß complexes at the repair foci. Furthermore, suppression of endogenous Ku70 expression by small interfering RNA reduces BER efficiency and leads to higher sensitivity to MMS and accumulation of the DNA strand breaks. Similarly, Pol-ß knockdown impairs total-NHEJ capacity but only has a slight influence on alternative NHEJ. These results suggest that Pol-ß and Ku70 coordinate 2-way crosstalk between the BER and NHEJ pathways.-Xia, W., Ci, S., Li, M., Wang, M., Dianov, G. L., Ma, Z., Li, L., Hua, K., Alagamuthu, K. K., Qing, L., Luo, L., Edick, A. M., Liu, L., Hu, Z., He, L., Pan, F., Guo, Z. Two-way crosstalk between BER and c-NHEJ repair pathway is mediated by Pol-ß and Ku70.

NHERF1 and NHERF2 regulation of SR-B1 stability via ubiquitination and proteasome degradation.

Scavenger receptor class B type 1 (SR-B1), an HDL receptor plays a crucial role in cholesterol metabolism in the liver, steroidogenic tissues, and vascular cells including macrophages. SR-B1 is subject to regulation at the transcription, posttranscription and posttranslational levels. We previously provided evidence that PDZ domain containing NHERF1 and NHERF2 regulate SR-B1 protein levels post-transcriptionally, although the underlying mechanism(s) by which NHERF1 and NHERF2 regulate SR-B1 protein levels is not well understood. In this study, we demonstrate that SR-B1 is degraded intracellularly via ubiquitin-proteasome pathway and that SR-B1 can be ubiquitinated at K500 and K508 residues. Overexpression of NHERF1 or NHERF2 enhanced SR-B1 ubiquitination and degradation. NHERF1 and NHERF2 promote SR-B1 ubiquitination at sites K508 and K500, respectively. These results suggest that NHERF1 and NHERF2 down-regulated SR-B1 at least in part via the ubiquitin/proteasome pathway.

His-87 ligand in mitoNEET is crucial for the transfer of iron sulfur clusters from mitochondria to cytosolic aconitase.

MitoNEET is the first identified iron sulfur protein that located in the mitochondrial outer membrane. We showed that knockdown of mitoNEET did not affect the iron sulfur protein expression in mitochondria and cytoplasm, but significantly reduced the cytosolic aconitase activity. The reduction of aconitase activity was rescued by transfection of wild type mitoNEET, but not by mitoNEET mutants H87C and H87S. Our results confirm the observation that mitoNEET is important in transferring the iron sulfur clusters to the cytosolic aconitase in living cells and the His-87 ligand in mitoNEET plays important role in this process.

The alteration of protein prenylation induces cardiomyocyte hypertrophy through Rheb-mTORC1 signalling and leads to chronic heart failure.

G protein-regulated cell function is crucial for cardiomyocytes, and any deregulation of its gene expression or protein modification can lead to pathological cardiac hypertrophy. Herein, we report that protein prenylation, a lipidic modification of G proteins that facilitates their association with the cell membrane, might control the process of cardiomyocyte hypertrophy. We found that geranylgeranyl diphosphate synthase (GGPPS), a key enzyme involved in protein prenylation, played a critical role in postnatal heart growth by regulating cardiomyocyte size. Cardiac-specific knockout of GGPPS in mice led to spontaneous cardiac hypertrophy, beginning from week 4, accompanied by the persistent enlargement of cardiomyocytes. This hypertrophic effect occurred by altered prenylation of G proteins. Evaluation of the prenylation, membrane association and hydrophobicity showed that Rheb was hyperactivated and increased mTORC1 signalling pathway after GGPPS deletion. Protein farnesylation or mTORC1 inhibition blocked GGPPS knockdown-induced mTORC1 activation and suppressed the larger neonatal rat ventricle myocyte size and cardiomyocyte hypertrophy in vivo, demonstrating a central role of the FPP-Rheb-mTORC1 axis for GGPPS deficiency-induced cardiomyocyte hypertrophy. The sustained cardiomyocyte hypertrophy progressively provoked cardiac decompensation and dysfunction, ultimately causing heart failure and adult death. Importantly, GGPPS was down-regulated in the hypertrophic hearts of mice subjected to transverse aortic constriction (TAC) and in failing human hearts. Moreover, HPLC-MS/MS detection revealed that the myocardial farnesyl diphosphate (FPP):geranylgeranyl diphosphate (GGPP) ratio was enhanced after pressure overload. Our observations conclude that the alteration of protein prenylation promotes cardiomyocyte hypertrophic growth, which acts as a potential cause for pathogenesis of heart failure and may provide a new molecular target for hypertrophic heart disease clinical therapy.

Egr-1 decreases adipocyte insulin sensitivity by tilting PI3K/Akt and MAPK signal balance in mice.

It is well known that insulin can activate both PI3K/Akt pathway, which is responsible for glucose uptake, and MAPK pathway, which is crucial for insulin resistance formation. But, it is unclear exactly how the two pathways coordinate to regulate insulin sensitivity upon hyperinsulinism stress of type 2 diabetes mellitus (T2DM). Here, we show that an early response transcription factor Egr-1 could tilt the signalling balance by blocking PI3K/Akt signalling through PTEN and augmenting Erk/MAPK signalling through GGPPS, resulting in insulin resistance in adipocytes. Egr-1, PTEN and GGPPS are upregulated in the fat tissue of T2DM patients and db/db mice. Egr-1 overexpression in epididymal fat induced systematic insulin resistance in wild-type mice, and loss of Egr-1 function improved whole-body insulin sensitivity in diabetic mice, which is mediated by Egr-1 controlled PI3K/Akt and Erk/MAPK signalling balance. Therefore, we have revealed, for the first time, the mechanism by which Egr-1 induces insulin resistance under hyperinsulinism stress, which provides an ideal pharmacological target since inhibiting Egr-1 can simultaneously block MAPK and augment PI3K/Akt activation during insulin stimulation.

Selection and characterization of human PCSK9 antibody from phage displayed antibody library.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), which involves in low-density lipoprotein cholesterol (LDL-C) metabolism by interacting with the LDL receptor, is considered as a potent therapeutic target for treating hypercholesterolemia. Here, a fab antibody phage display library was constructed and employed for bio-panning against recombinant PCSK9. A Fab fragment (designated PA4) bound with high affinity to PCSK9 was isolated after four rounds of panning. The fully human antibody IgG1-PA4 bound specifically to PCSK9 with nanomolar affinity. In vitro, IgG1-PA4 inhibited PCSK9 binding to LDLR and attenuated PCSK9-mediated degradation of LDLR on the HepG2 cell surface. In C57BL/6 mice, administration of IgG1-PA4 at 30 mg/kg increased hepatic LDLR protein levels by as much as 3 fold when compared with control. Taken together, these results suggested that the IgG1-PA4 can be served as a potential candidate for the treatment of hypercholesterolemia by inhibiting PCSK9-mediated degradation of cell surface LDLRs.

APE1 inhibition enhances ferroptotic cell death and contributes to hepatocellular carcinoma therapy.

Ferroptosis, a regulated form of cell death triggered by iron-dependent lipid peroxidation, has emerged as a promising therapeutic strategy for cancer treatment, particularly in hepatocellular carcinoma (HCC). However, the mechanisms underlying the regulation of ferroptosis in HCC remain to be unclear. In this study, we have identified a novel regulatory pathway of ferroptosis involving the inhibition of Apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme with dual functions in DNA repair and redox regulation. Our findings demonstrate that inhibition of APE1 leads to the accumulation of lipid peroxidation and enhances ferroptosis in HCC. At the molecular level, the inhibition of APE1 enhances ferroptosis which relies on the redox activity of APE1 through the regulation of the NRF2/SLC7A11/GPX4 axis. We have identified that both genetic and chemical inhibition of APE1 increases AKT oxidation, resulting in an impairment of AKT phosphorylation and activation, which leads to the dephosphorylation and activation of GSK3ß, facilitating the subsequent ubiquitin-proteasome-dependent degradation of NRF2. Consequently, the downregulation of NRF2 suppresses SLC7A11 and GPX4 expression, triggering ferroptosis in HCC cells and providing a potential therapeutic approach for ferroptosis-based therapy in HCC. Overall, our study uncovers a novel role and mechanism of APE1 in the regulation of ferroptosis and highlights the potential of targeting APE1 as a promising therapeutic strategy for HCC and other cancers.

Effect of prone positioning on survival in adult patients receiving venovenous extracorporeal membrane oxygenation for acute respiratory distress syndrome: a prospective multicenter randomized controlled study.

Background: Current data supporting the use of prone positioning (PP) during venovenous extracorporeal membrane oxygenation (VV-ECMO) in patients with acute respiratory distress syndrome (ARDS) are limited. This prospective randomized controlled study aimed to determine whether PP implemented within 24 hours of ECMO can improve survival in these patients. Methods: From June 2021 to July 2023, 97 adult patients receiving VV-ECMO for ARDS in three centers were enrolled and 1:1 randomized into PP (n=49) and control groups (n=48). Patients in the PP group receiving prone positioning, while the control group were maintained in the supine position. The primary outcome was 30-day survival, and secondary outcomes included in-hospital survival and other clinical outcomes. Results: All 97 patients were included for analysis. Patient characteristics did not significantly differ between the two groups. The median duration of PP was 81 hours, and the median number of PP sessions was 5 times. PP improved oxygenation and ventilator parameters. The incidence of complications during PP was low, with pressure sores being the most frequent (10.2%). The 30-day survival was significantly higher in the PP group (67.3% vs. 45.8%; P=0.033), as was in-hospital survival (61.2% vs. 39.6%; P=0.033). In the PP group, the successful ECMO weaning rate was significantly higher (77.5% vs. 50.0%; P=0.005), and the duration of ECMO support was significantly shorter {10 [8-11] vs. 10 [8-14] days; P=0.038}. However, in subgroup analysis of COVID patients the 30-day survival, in-hospital survival, successful ECMO weaning rate and the duration of ECMO support did not differ between the groups. The duration of mechanical ventilation, length of intensive care unit stay, and length of hospital stay did not significantly differ between the groups. Conclusions: When initiated within 24 hours of ECMO, PP can improve 30-day survival in patients with ARDS receiving VV-ECMO. In addition, it may improve the successful ECMO weaning rate and reduce the duration of ECMO support. However, considering the limitations, more strictly designed, large sample prospective randomized controlled trials are proposed. Trial Registration: Chinese Clinical Trial Registry ChiCTR2300075326.

The 8-oxoguanine DNA glycosylase-synaptotagmin 7 pathway increases extracellular vesicle release and promotes tumour metastasis during oxidative stress.

Reactive oxygen species (ROS)-induced oxidative DNA damages have been considered the main cause of mutations in genes, which are highly related to carcinogenesis and tumour progression. Extracellular vesicles play an important role in cancer metastasis. However, the precise role of DNA oxidative damage in extracellular vesicles (EVs)-mediated cancer cell migration and invasion remains unclear. Here, we reveal that ROS-mediated DNA oxidative damage signalling promotes tumour metastasis through increasing EVs release. Mechanistically, 8-oxoguanine DNA glycosylase (OGG1) recognises and binds to its substrate 8-oxo-7,8-dihydroguanine (8-oxoG), recruiting NF-κB to the synaptotagmin 7 (SYT7) promoter and thereby triggering SYT7 transcription. The upregulation of SYT7 expression leads to increased release of E-cadherin-loaded EVs, which depletes intracellular E-cadherin, thereby inducing epithelial-mesenchymal transition (EMT). Notably, Th5487, the inhibitor of DNA binding activity of OGG1, blocks the recognition and transmission of oxidative signals, alleviates SYT7 expression and suppresses EVs release, thereby preventing tumour progression in vitro and in vivo. Collectively, our study illuminates the significance of 8-oxoG/OGG1/SYT7 axis-driven EVs release in oxidative stress-induced tumour metastasis. These findings provide a deeper understanding of the molecular basis of cancer progression and offer potential avenues for therapeutic intervention.

Calmodulin activation of polo-like kinase 1 is required during mitotic entry.

Polo-like kinase 1 (Plk1) is a conserved key regulator of the G2/M transition, but its upstream spatiotemporal regulators remain unknown. With the help of immunofluorescence, co-immunoprecipitation, and glutathione S-transferase (GST) pull-down assay, we found that calmodulin (CaM) is one such regulatory molecule that associates with Plk1 from G2 to metaphase. More importantly, this interaction results in considerable stimulation of Plk1 kinase activity leading to hyperphosphorylation of Cdc25C. Our results provide new insight into the role of CaM as an upstream regulator of Plk1 activation during mitotic entry.