Regulation of suppressor of cytokine signalling-3 expression by cyclic-AMP in pro-myeloid cells

Mullen, Laura Catherine (2006) Regulation of suppressor of cytokine signalling-3 expression by cyclic-AMP in pro-myeloid cells. MSc(R) thesis, University of Glasgow.

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Abstract

Suppressors of cytokine signalling (SOCS) protein induction via the "Janus kinase- signal transducer and activator of transcription" (JAK-STAT) pathway has been shown to be a critical negative feedback mechanism that prevents inappropriately sustained signalling from activated cytokine receptors. It also provides a mechanism by which other signalling modules could potentially regulate the JAK-STAT pathway. In this study, I have demonstrated that elevation of the prototypical second messenger cyclic AMP (cAMP) is capable of promoting the time- and concentration-dependent accumulation of the SOCS-3 isoform in U937 human promyeloid cells and HL60 human promyoblast cells. Experiments with MG132 demonstrated that cAMP specifically promoted SOCS-3 synthesis rather than blocking its degradation by the proteasome. The accumulation of SOCS-3 in U937 cells correlated with a reduced ability of the granulocyte-colony stimulating factor (G-CSF) receptor, which is a bona fide target for SOCS-3 in vivo, to promote the Tyr phosphorylation of STAT3 and the dual Thr/Tyr phosphorylation of ERK, suggesting that the cAMP-mediated accumulation of SOCS-3 is sufficient to suppress JAK-STAT pathway activation by this receptor. Further characterisation of the response demonstrated that SOCS-3 induction could not be blocked by H89, an inhibitor of cAMP-dependent protein kinase (PKA), suggesting that a PKA independent mechanism was responsible. Consistent with this hypothesis, selective activation of PKA with the selective cAMP analogue 6Be-cAMP failed to promote SOCS-3 accumulation. Surprisingly, selective activation of "Exchange protein activated by cAMP" (Epac), a recently identified PKA-independent intracellular sensor of cAMP, using the cAMP analogue 8pCPT- 20Me-cAMP also failed to promote SOCS-3 accumulation. Moreover, the inability of both 6Be-cAMP and 8pCPT-20Me-cAMP to promote SOCS-3 accumulation was not due to a lack of biological activity, since both were able to stimulate the phosphorylation of ERK in U937 cells at the concentrations used to assess SOCS-3 induction. Finally, preliminary experiments employing inhibitors of various signalling pathways revealed that cAMP-mediated SOCS-3 accumulation occurred via a JAK-, p38- and ERK-independent mechanism. Thus, it is proposed that cAMP elevation may promote the accumulation of SOCS-3 in U937 cells via a novel pathway leading to increased SOCS-3 synthesis that is independent of the known cAMP sensors PKA and Epac, and which may thus involve a currently unknown intracellular sensor of cAMP.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Keywords: Immunology.
Subjects: Q Science > QR Microbiology > QR180 Immunology
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Supervisor, not known
Date of Award: 2006
Depositing User: Enlighten Team
Unique ID: glathesis:2006-9058
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 25 Jul 2018 13:20
Last Modified: 25 Jul 2018 13:31
URI: http://theses.gla.ac.uk/id/eprint/9058

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