Embryonic stem cells (ESC); proliferate while maintain the ability to differentiate into several cell types (self-renewal). For the efficient use of ESC in cell therapy it is necessary to characterize the specific signaling pathways of differentiation and self-renewal of ESC. The family of serine / threonine kinase protein kinase C (PKC) has been identified as key enzymes for the processes of proliferation and differentiation of ESCs, however, the exact function of each isoform of this family remains unclear. In the recent years our laboratory has characterized the role of different isozymes of PKCs in undifferentiated ESC. Our data suggest that among the PKCs expressed in ESC, lower molecular weight forms of the PKCBI isoform are expressed in the nucleus of ESCs. Probably, the lack of the pseudo-substrate site that is responsible for self-inhibition of the enzyme generates catalytically active forms of PKCBI. Furthermore, studies of phosphoproteomics indicate that most substrates of PKCBI in undifferentiated ESC are nuclear proteins that regulate transcription of proteins involved in proliferation/differentiation. We also realized that during differentiation there is a shift in the sub-cellular localization of PKCBI, which in some cells is now expressed in the cytoplasm, while other cells all together seize to express PKCBI. Together, these data contribute to the hypothesis that PKCBI may be involved in important processes of undifferentiated ESCs, such as the maintenance of their undifferentiated state. Thus, in this project we propose to investigate the nuclear functions of PKCBI and the molecular mechanisms by which PKCBI translocates to the nucleus using molecular biology techniques, confocal microscopy and mass spectrometry. Besides that, we intend identify nuclear proteins that interact directly/indirectly to PKCBI in undifferentiated ESCs. Understanding the mechanisms that lead to PKCBI translocation to the nucleus of undifferentiated ESC and identifying the proteins that interact with nuclear PKCBI, will aid us to better understand the signaling pathways of both proliferation and self renewal in the ESC.
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