ABSTRACT
Drosophila tumor suppressor WARTS (Wts) is an evolutionally conserved serine / threonine kinase and participates in a signaling complex that regulates both proliferation and apoptosis to ensure the proper size and shape of the fly. Human counterparts of this complex have been found to be frequently downregulated or mutated in cancers. WARTS, a human homolog of Wts, is also known as tumor suppressor and mitotic regulator, but its molecular implications in tumorigenesis are still obscure. Here, we show that WARTS binds via its C-terminus to the PDZ domain of a proapoptotic serine protease Omi / HtrA2. Depletion of WARTS inhibited Omi / HtrA2-mediated cell death, whereas overexpression of WARTS promoted this process. Furthermore, WARTS can enhance the protease activity of Omi / HtrA2 both in vivo and in vitro. Activation of Omi / HtrA2-mediated cell death is thus a potential mechanism for the tumor suppressive activity of WARTS.
Subject(s)
Apoptosis/physiology , Protein Serine-Threonine Kinases/physiology , Serine Endopeptidases/metabolism , Tumor Suppressor Proteins/physiology , Cells, Cultured , Cytosol/metabolism , High-Temperature Requirement A Serine Peptidase 2 , Humans , Mitochondria/metabolism , Mitochondrial Proteins , Protein Serine-Threonine Kinases/metabolism , Transfection , Tumor Suppressor Proteins/metabolismABSTRACT
Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB. We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were over-expressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis.
Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Cyclin B/metabolism , Polyadenylation , Protein Kinases/metabolism , RNA, Messenger/metabolism , Transcription Factors/metabolism , Xenopus Proteins/metabolism , mRNA Cleavage and Polyadenylation Factors/metabolism , Amino Acid Sequence , Animals , Aurora Kinase A , Aurora Kinases , CDC2 Protein Kinase/genetics , Cell Cycle Proteins/genetics , Cells, Cultured , Cyclin B/genetics , Cyclin B1 , Homeodomain Proteins/metabolism , Humans , LIM Domain Proteins , Molecular Sequence Data , Phosphorylation , Protein Kinases/genetics , Protein Serine-Threonine Kinases , RNA, Messenger/genetics , Rats , Xenopus Proteins/geneticsABSTRACT
Aurora-A, a serine/threonine mitotic kinase, was reported to be overexpressed in various human cancers, and its overexpression induces aneuploidy, centrosome amplification and tumorigenic transformation in cultured human and rodent cells. However, the underlying mechanisms and pathological settings by which Aurora-A promotes tumorigenesis are largely unknown. Here, we created a transgenic mouse model to investigate the involvement of Aurora-A overexpression in the development of mammary glands and tumorigenesis using a Cre-loxP system. The conditional expression of Aurora-A resulted in significantly increased binucleated cell formation and apoptosis in the mammary epithelium. The surviving mammary epithelial cells composed hyperplastic areas after a short latency. Induction of Aurora-A overexpression in mouse embryonic fibroblasts prepared from the transgenic mice also led to aberrant mitosis and binucleated cell formation followed by apoptosis. The levels of p53 protein were remarkably increased in these Aurora-A-overexpressing cells, and the apoptosis was significantly suppressed by deletion of p53. Given that no malignant tumor formation was found in the Aurora-A-overexpressing mouse model after a long latency, additional factors, such as p53 inactivation, are required for the tumorigenesis of Aurora-A-overexpressing mammary epithelium. Our findings indicated that this mouse model is a useful system to study the physiological roles of Aurora-A and the genetic pathways of Aurora-A-induced carcinogenesis.
Subject(s)
Mammary Glands, Animal/pathology , Mitosis/physiology , Protein Serine-Threonine Kinases/metabolism , Recombinases/physiology , Animals , Apoptosis/physiology , Aurora Kinase A , Aurora Kinases , Base Sequence , Cell Transformation, Neoplastic , Fluorescent Antibody Technique , Hyperplasia , Mammary Neoplasms, Experimental/enzymology , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Transgenic , Models, Animal , Molecular Sequence Data , Protein Serine-Threonine Kinases/genetics , Recombinases/metabolism , Tumor Suppressor Protein p53/physiologyABSTRACT
Radiologic findings of a case with scalp plexiform schwannoma-an unusual variant of the benign, solitary schwannoma in the skin-are reported. T2-weighted MR imaging exhibited the most specific features: a multinodular pattern and hypointense capsule that separated the tumor from surrounding soft tissue. A surgical specimen was histologically confirmed as schwannoma. The MR imaging findings reported herein may aid in the preoperative diagnosis of this relatively rare scalp tumor.
Subject(s)
Magnetic Resonance Imaging , Neurilemmoma/diagnosis , Scalp , Skin Neoplasms/diagnosis , Tomography, X-Ray Computed , Adult , Biomarkers, Tumor/analysis , Diagnosis, Differential , Humans , Male , Neurilemmoma/pathology , Neurilemmoma/surgery , S100 Proteins/analysis , Scalp/pathology , Scalp/surgery , Skin Neoplasms/pathology , Skin Neoplasms/surgeryABSTRACT
The Aurora (Ipl1)-related kinases are universal regulators of mitosis. We now show that Aurora-A, in addition to Aurora-B, regulates kinetochore function in human cells. A two-hybrid screen identified the kinetochore component CENP-A as a protein that interacts with Aurora-A. Aurora-A phosphorylated CENP-A in vitro on Ser-7, a residue also known to be targeted by Aurora-B. Depletion of Aurora-A or Aurora-B by RNA interference revealed that CENP-A is initially phosphorylated in prophase in a manner dependent on Aurora-A, and that this reaction appears to be required for the subsequent Aurora-B-dependent phosphorylation of CENP-A as well as for the restriction of Aurora-B to the inner centromere in prometaphase. Prevention of CENP-A phosphorylation also led to chromosome misalignment during mitosis as a result of a defect in kinetochore attachment to microtubules. Our observations suggest that phosphorylation of CENP-A on Ser-7 by Aurora-A in prophase is essential for kinetochore function.
Subject(s)
Autoantigens , Chromosomal Proteins, Non-Histone/metabolism , Kinetochores/enzymology , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Aurora Kinase B , Aurora Kinases , Cell Nucleus/metabolism , Centromere Protein A , Chromosomal Proteins, Non-Histone/genetics , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Kidney/cytology , Metaphase/physiology , Microtubules/physiology , Mitosis/physiology , Molecular Sequence Data , Nuclear Proteins/genetics , Phosphorylation , Prophase/physiology , Proteins , RNA, Small Interfering , Serine/metabolismABSTRACT
Aurora family kinases contribute to regulation of mitosis. Using RNA interference in synchronized HeLa cells, we now show that Aurora-A is required for mitotic entry. We found that initial activation of Aurora-A in late G2 phase of the cell cycle is essential for recruitment of the cyclin B1-Cdk1 complex to centrosomes, where it becomes activated and commits cells to mitosis. A two-hybrid screen identified the LIM protein Ajuba as an Aurora-A binding protein. Ajuba and Aurora-A interact in mitotic cells and become phosphorylated as they do so. In vitro analyses revealed that Ajuba induces the autophosphorylation and consequent activation of Aurora-A. Depletion of Ajuba prevented activation of Aurora-A at centrosomes in late G2 phase and inhibited mitotic entry. Overall, our data suggest that Ajuba is an essential activator of Aurora-A in mitotic commitment.
Subject(s)
Homeodomain Proteins/physiology , Mitosis , Protein Kinases/physiology , Animals , Aurora Kinase A , Aurora Kinases , CDC2 Protein Kinase/metabolism , COS Cells , Cell Cycle , Cell Cycle Proteins , Cell Line , Centrosome/ultrastructure , Cyclin B/metabolism , Cyclin B1 , G2 Phase , Gene Deletion , Glutathione Transferase/metabolism , HeLa Cells , Homeodomain Proteins/metabolism , Humans , LIM Domain Proteins , Microscopy, Fluorescence , Models, Biological , Molecular Sequence Data , Phosphorylation , Precipitin Tests , Protein Binding , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , RNA Interference , Recombinant Proteins/metabolism , Temperature , Time Factors , Two-Hybrid System Techniques , Xenopus ProteinsABSTRACT
BACKGROUND: Various mitotic events are controlled by Cdc2-cyclin B and other mitotic kinases. Aurora/Ipl1-related mitotic kinases were proved to play key roles in mitotic progression in diverse lower organisms. Aurora-A is a mammalian counterpart of aurora/Ipl1-related kinases and is thought to be a potential oncogene. However, the regulation of aurora-A activation and the commitment of aurora-A in the progression of G2-M phase are largely unknown in mammalian cells. RESULTS: We demonstrated that aurora-A is activated depending on the activation of Cdc2-cyclin B in mammalian cells. Since Cdc2-cyclin B does not directly phosphorylate aurora-A, indirect pathways such as the inhibition of PP1 by Cdc2-cyclin B may act for the activation of aurora-A kinase. Microinjection of anti-aurora-A antibodies into HeLa cells at late G2 phase caused a significant delay in mitotic entry. Furthermore, aurora-A activation at G2-M transition was inhibited by DNA damage, and the over-expression of aurora-A induced the abrogation of the DNA damage-induced G2 checkpoint. CONCLUSIONS: Aurora-A is activated downstream of Cdc2-cyclin B and plays crucial roles in proper mitotic entry and G2 checkpoint control. Dysregulation of aurora-A induces abnormal G2-M transition in mammalian cells and may lead to chromosome instability, which results in the development and progression of malignant tumours.
