Identification Of A Novel Cytokine Inducible Stat5 Phosphoserine Site (ps193) That Positively Regulates Its Transcriptional Activity And Is Found Constitutively Activated In Certain Hematopoietic Cancers
Date of Award
Doctor of Philosophy
Robert A. Kirken
Hematological malignancies such as leukemia and lymphoma, can develop from aberrant changes in the cell signaling molecules to drive their uncontrolled cellular proliferation and differentiation. Activation, maturation, expansion and differentiation of T cells are critically regulated by the dynamics of various transcription factors activated by a variety of cytokines. Additionally, multiple effector molecules that mediate these T cells dependent signals include the JAK (Janus Kinase)-STAT (Signal transducer and activator of transcription) cascade. These signaling proteins are activated in response to a broad array of cytokines. Constitutively activated JAKs and STATs have been described in several T cell malignancies. A growing body of evidence also suggests that dysregulated of STAT5 may be a major contributor to this event and promote the development of certain types of T cell cancers while the conserved c-terminal domain and tyrosine phosphorylation sites within STATs are important for their dimerization and transcriptional activity, serine phosphorylation has been identified in the constitutively activated STAT molecules in various cancer patient samples. Based on tandem mass-spectroscopic analysis, we have identified a novel STAT5S193 site that undergoes phosphorylation in a cytokine dependent manner. The central hypothesis of this thesis was to elucidate a cytokine dependence of this novel phosphorylation site and determine its function related to transcriptional activity and T cell responses. This site was inducibly phosphorylated in response to IL-2, IL-7, IL-9 and IL-15. Moreover the phosphor-kinetics of the STAT5pS193 were rapid and attained maximal phosphorylation within 15 min and was cytoplasmically localized in the inactive state but nuclear upon activation.
In the second objective of this research, we have investigated key regulatory pathways that control the phosphorylation and dephosphorylation state of STAT5 S193. Based on the inhibitor studies we identified that STAT5 S193 phosphorylation is an mammalian target of rapamycin (mTOR) dependent manner however protein phosphatase 2A (PP2A) negatively regulates its phosphorylation. A cell based (HEK293) reconstitution showed that STAT5pS193 was independent on JAK3 activation. However HEK-293 reconstitution system suggests that this site is not required for receptor recruitment, tyrosine phosphorylation, dimerization nor nuclear translocation of STAT5. However, EMSA and luciferase assay suggest that phosphorylation of STAT5 S193 positively regulates the transcriptional and functional activity of STAT5. Collectively we can conclude that this novel phospho-serine site is a positive regulator of the functional activity of STAT5 in an mTOR and PP2A dependent manner in human lymphocytes.
Given this data, hematopoietic tumors were evaluated for constitutively phosphorylated STAT5 S193. Indeed several established tumor cell lines and primary cancer samples were found to be phosphorylated. These data suggest that STAT5 S193 phosphorylation may be important for the oncogenic activity of STAT5 and may therefore serve as a therapeutic target for controlling those types of cancer.
Received from ProQuest
Mitra, Abhisek, "Identification Of A Novel Cytokine Inducible Stat5 Phosphoserine Site (ps193) That Positively Regulates Its Transcriptional Activity And Is Found Constitutively Activated In Certain Hematopoietic Cancers" (2010). Open Access Theses & Dissertations. 2544.