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Roles of human VRK1 Ser-Thr kinase in the regulation of cell proliferation
|Authors:||Moura, David S.|
|Advisor:||Lazo, Pedro A.|
|Publisher:||CSIC-USAL - Instituto de Biología Molecular y Celular del Cancer de Salamanca (IBMCC)|
|Abstract:||Cell proliferation and cell differentiation are different mechanisms occurring at different times of the cell life. However, the regulation of the switch from proliferation to differentiation and the role and fate of proliferation-related proteins are mostly unknown.
Cell division is a high-regulated mechanism, involving several kinases, namely Vaccinia-related kinase 1 (VRK1) and Aurora kinase B (AurKB). These kinases assure the correct transmission of genetic information to the newly-formed cells, DNA replication, chromosome segregation and chromatin condensation. VRK1 is the first member of the VRK family and is ubiquitously expressed in all the tissues, especially high-proliferative tissue, including liver and thymus or cancer cells. Therefore, based on VRK1 features and expression, its role has been mostly related to the regulation of cell proliferation, including the regulation of transcription factors involved in cell cycle progression and DNA damage repair pathways, modulation of p53 levels, nuclear envelope assembly, Cajal body (CB) formation and maintenance, and fragmentation of the Golgi apparatus. Apart from these, VRK1 also contributes to the mitotic chromatin condensation through histone H3 phosphorylation, mainly, in the Thr3 residue (H3T3ph). H3T3ph is especially important for the formation, localization and function of the chromosomal passenger complex (CPC) on the centromeres, during mitosis. (see group publications). In turn, AurKB is a member of the aurora kinase family, alongside with AurKA and AurKC. The activity of this family of kinases is fulfilled during mitosis, and therefore, they are mitotic kinases critical for the mitotic regulation of centromere function, chromatin condensation, spindle assembly, chromosome alignment and segregation and, cytokinesis. AurKB is a component of CPC, along with Survivin, Borealin and INCENP and contributes to chromatin condensation, through H3S10 phosphorylation.
On the other hand, the transcriptional factor (TF) Sox2, a member of the SRY-related HMG-box family of TFs is a master regulator of pluripotency, proliferation, survival and differentiation of embryonic stem cell (ESC).|
Moreover, Yamanaka’s factor Sox2 is a cancer initiating cell biomarker in poorly differentiated or undifferentiated cancers, since these types of cancers have been characterized by many phenotypic traits similar to undifferentiated embryonic stem cells. Indeed, Sox2 is overexpressed in numerous tumors like breast cancer and lung cancer or in cancer cell lines suggesting the enormous oncogenic potential of this TF. It is also important to notice, that Sox2 combines with Oct4 to control cell cycle, through cyclin D1 promoter activation. Finally, also macro-histone H2A (macroH2A) can regulate cell differentiation and cell proliferation. Macro H2A (macroH2A1.1, macroH21.2 and macroH2A2) are histones variant that can replace conventional histone H2A in the nucleosomes, repressing cell proliferation genes and favoring cell differentiation genes. These histone variants change the chromatin conformation, affecting TFs binding and creating de novo activation and repression binding sites. It was also reported that macroH2A variants could regulate VRK1 levels. Initially, we observe that VRK1 is a chromatin kinase, besides its usual nucleo-plasmatic and cytoplasmatic localization, and interacts with the histone H3 by immuprecipitation (IP) and ‘’Pull-down’’ assays in early mitosis. Moreover, VRK1 phosphorylates directly the mitotic H3T3, as observed by kinase assay with cold ATP and phospho-specific antibodies. Next and focusing on VRK1 and AurKB relationship we observed that VRK1 and AurKb inhibits the kinase activity of one another, as observed by kinase assay with radiolabeled ATP. Moreover, increasing doses of VRK1, active or inactive, affected negatively the phosphorylation of AurKB-specific residue S10 on histone H3 in kinase assay with cold ATP. Besides, also increasing doses of AurKB, active and inactive, affected negatively the phosphorylation of H3T3 and the phosphorylation of p53T18 by VRK1. This effect is consequence of the formation of a stable complex between the two protein kinases and independent of any type of phosphorylation of one kinase on another. By IP and ‘’Pull-down’’ assay we observed that transfected VRK1 and transfected AurKB interact, forming a stable complex independent of VRK1 activity. VRK1 interacts with AurKB through both amino and carboxy terminals. The interaction between endogenous VRK1 and AurKB occurs in early mitosis, after cell arrest with nocodazole. Yet, no co-localization between the two kinases was
observed by immunofluorescence (IF). Moreover, VRK1 downregulation affected the localization of AurKB in early mitosis in IF assays. VRK1 downregulation avoids centromeric accumulation of AurKB, leading to a more diffuse distribution on the chromatin. This result is due to the loss of H3T3 phosphorylation, as observed by IF and western blot (WB). Loss of VRK1-dependent H3T3 phosphorylation affects the interaction of AurKB with Histone H3 in IP assays. Regarding VRK1 and reprogramming factors, VRK1 and Sox2 distribution was analyzed by immunohistochemistry on a stratified squamous epithelium and it was observed that Sox2 and VRK1 co-localize in the undifferentiated layers of the epithelium. The same result was observed by IF in several cell lines, such as NTera-2 (NT2), MCF-7 or MDA-MB-231. Moreover, VRK1 and Sox2 interact in IP assay. The interaction occurs between transfected proteins and between endogenous proteins, and it is through both VRK1 amino and carboxy terminals. Sox2 is a phosphorylation target of VRK1 as determined by kinase assay with radiolabelled ATP. Sox2, as a regulator of cell proliferation regulates proliferative genes, including VRK1. SOX2 regulates VRK1 promoter in luciferase assays, increasing, when overexpressed, the RNA and protein levels of the kinase, as observed by qRT-PCR, WB and IF. The result was observed in NT2, MDA-MB-231 and MCF-7 cell line. In turn, VRK1 downregulation increases RNA (qRT-PCR) and protein levels (IF and WB) of Sox2 in NT2, MDA-MB-231 and MCF-7, creating a feedback loop between VRK1 and Sox2. The overexpression of VRK1 produced the opposite result. Furthermore, VRK1 and Sox2 cooperate in the activation of CCND1 gene, in luciferase assays potentiating proliferation. After induction of cell differentiation with retinoic acid, both Sox2 and VRK1 RNA and protein are downregulated and they are absent or significantly down-regulated in terminally differentiated epithelial cells. In terminally differentiated cells, macroH2A variants 1.2 and 2 repress VRK1 and CCND1 gene promoter, as determined by luciferase assay. In conclusion, VRK1 participates in the regulation of cell proliferation, through two new and independent mechanisms. One by actively regulating AurKB and the other by regulating the balance between cell proliferation and cell differentiation.
|Description:||Tesis Doctoral presentada por David da Silva Moura, elaborada en el Programa de Doctorado Biociencias: Biología y Clínica del Cáncer y Medicina Traslacional, y realizada en el Instituto de Biología Molecular y Celular del Cáncer de Salamanca (CSIC-USAL).|
|Appears in Collections:||(IBMCC) Tesis|