Salt-Inducible Kinase 1 and Transducer or Regulated CREB Activity 2 in the Rat Pineal Gland

  • Author / Creator
    Kanyo, Richard
  • In the rat pineal gland, the transcription of the melatonin-rhythm generating enzyme, arylalkyl-N-acetyltransferase (AA-NAT), is stimulated by norepinephrine (NE), acting through the cAMP → protein kinase A (PKA) → cAMP response element-binding protein (CREB) signalling pathway. Although the PKA-mediated phosphorylation of CREB is the main activation mechanism, the transcriptional activity of CREB can also be regulated by the salt-inducible kinase (SIK)/transducer of regulated CREB activity (TORC) pathway. Therefore, the objective of this study was to investigate the mechanisms involved in the regulation of SIK1 and TORC2 in the rat pineal gland and their roles in Aa-nat transcription. We found a marked nocturnal induction of Sik1 transcription in the rat pineal gland. In cultured pinealocytes, this induction is driven primarily by a β-adrenoreceptor/cAMP-dependent mechanism with a minor contribution from the α1-adrenoreceptor/Ca2+-pathway. Manipulating the level of Sik1 expression in pinealocytes shows that endogenous SIK1 can function as a transcription repressor of Aa-nat. Studies on TORC2 show that although the TORC2 protein is dephosphorylated in the rat pineal gland with the onset of darkness and in pinealocytes within 15 min of NE stimulation, this occurs without any changes in the Torc2 mRNA levels. The β-adrenoreceptor/cAMP-pathway and protein phosphatase 2A are involved in the NE-stimulated dephosphorylation of TORC2, which also result in its nuclear translocation. Elevating intracellular Ca2+ also induces dephosphorylation of TORC2 but has no effect on its nuclear translocation. As for the role of TORC2 on Aa-nat transcription, whereas overexpression of TORC2 has an enhancing effect on the NE-stimulated Aa-nat expression, knockdown of TORC2 only has a small inhibitory effect. Finally, knockdown of endogenous Sik1 has no effect on the phosphorylation status or cellular distribution of TORC2 under basal or stimulated conditions. We show that the β-adrenoreceptor/cAMP signalling mechanism is the dominant pathway in inducing Sik1 transcription and nuclear translocation of TORC2. However, the repressive effect of SIK1 on Aa-nat cannot be explained by regulating the cellular distribution of TORC2. Moreover, the phosphorylation status of TORC2 may not accurately reflect its activation and that TORC2 cellular distribution is not regulated solely by SIK1.

  • Subjects / Keywords
  • Graduation date
    Spring 2014
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Gosgnach, Simon (Physiology)
    • Ezzat, Shereen (Medicine)
    • Chik, Constance (Medicine)
    • Karpinski, Ed (Physiology)
    • Morrish, Don (Medicine)