Characterisation of cell cycle functions of the protein tyrosine phosphatase TCPTP.
Creators: name: Hauser, Christine
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: Tyrosine phosphorylation is an integral mechanism for the regulation of fundamental cellular processes such as the survival, proliferation and cell cycle progression. Hyperactivation of protein tyrosine kinases (PTKs) such as lanus-activated kinases (JAKs) and Src family kinases (SFKs), and the elevated tyrosine phosphorylation of substrates such as signal transducer and activator of transcription (STAT) 3, can contribute to the development of many human tumors. Tyrosine phosphorylation is regulated by the opposing actions ofPTKs and protein tyrosine phosphatases (PTPs) but few PTPs have been implicated in cell cycle processes. In this project the contribution of the PTP known as TCPTP to the regulation of cell cycle progression was investigated. TCPTP is an intracellular non-transmembrane tyrosine-specific phosphatase that is ubiquitously expressed as two splice variants -a 48 kDa form (TC48), localised to the endoplasmic reticulum, and a 45 kDa form (TC45), predominantly localised to the nucleus, but which can also shuttle into the cytoplasm. TCPTP was originally cloned from a T-cell library and plays important roles in inflammation. However, previous studies have also implicated TCPTP in the regulation ofthe mammalian cell cycle. TCPTP mRNA levels were found to fluctuate during the cell cycle and TCPTP dissociates from chromatin during mitosis. Furthermore TCPTP was found to be phosphorylated by cyclin dependent kinases during mitosis. In this study we describe that TCPTP protein levels do not change during the cell cycle and confirm that endogenous TCPTP is regulated by post-translational modifications and cell cycle dependent changes in sub-nuclear localisation. Two different approaches were employed to extensively characterise TCPTP's cell cycle functions using either immortalised mouse embryonic fibroblasts (MEFs) lacking TCPTP (-/-) versus those reconstituted with physiological levels ofTC45 or human HeLa cells where TCPTP expression was knocked down by RNA-interference. By these approaches, TCPTP was identified as a negative regulator of SFK, JAKI and STAT3 signalling during the cell cycle. A comprehensive analysis of the different cell cycle phases revealed that TCPTP can regulate G/S and mitotic progression in a cell type specific manner. Regulation of G/S-progression by TCPTP was dependent on SFK activity. While the contribution of SFK activity to regulation of mitosis remains to be determined, several SFK substrates were hyperphosphorylated in TCPTP deficient cells in mitosis, one of which was identified as cortactin. Sand G2-phase progression were not affected by TCPTP deficiency. However TCPTP was found to localise to replication sites during S-phase and the replication checkpoint response was abrogated in TCPTP deficient cells. TCPTP was found to attenuate SFK, JAKI and STAT3 signaling to eyelin D and that way contribute to the replication checkpoint. Taken together, the results presented in this thesis suggest that TCPTP acts as a specific negative regulator of SFK, JAKI and STAT3 signalling to control cell cyele progression and replication checkpoint maintenance. These findings may also have implications for cancer development as increased oncogenic kinase activity and checkpoint bypass contribute to genomic instability and thus the onset and progression of cancer.