The role of salt-inducible Kinases in maintaining and regulating the Circadian architecture

Thesis

The behavioural rhythmicity of sleep can be effectively explained by the “Two Process Model”, where “Process S” characterizes the homeostatic drive for sleep (sleep pressure and sleep need) and “Process C” reflects the circadian clock. However, the dynamic molecular interactions of Process S and C remain a mystery. Recent studies highlight the role of kinases and phosphorylation of synaptic proteins as potential key drivers for sleep need. Salt-inducible kinases (SIKs) are AMP-activated protein kinases (AMPK) and consist of three members – SIK1, 2 and 3. Notably, previous studies have proposed that SIK1 phosphorylates CREB-regulated co-activators (CRTCs) and is involved in phase shifting and photoentrainment. SIK3 was shown to phosphorylate a hub of sleep-need index phosphoproteins (SNIPPs) related to sleep need and pressure. Therefore, SIKs are proposed as master regulators of both rhythmic transcriptional pathways and post-translational modification of sleep-associated proteins.

In this DPhil thesis, we investigated the isoform-specific roles, molecular substrates, and phosphorylation sites of SIKs in sleep and circadian regulation through behavioural, transcriptomics and phosphoproteomics approaches. We showed that all SIK isoforms are expressed in the suprachiasmatic nucleus (SCN), while only SIK2 and SIK3 are expressed in the cortical layers, which are associated with distinct circadian and sleep phenotypes that we characterized through wheel running and EEG/EMG recordings. We optimized the tissue-specific protein kinase assay linked phosphoproteomics (ProKALIP) method to detect the direct substrates of SIKs, which could be applied to substrate profiling for all applicable kinases. The direct substrates of SIKs proposed a network that facilitates synaptic transmission, calcium signaling, vesicle trafficking, and neuropeptide signaling. SIK3 phosphorylates synaptic proteins that overlapped with SNIPPs, supporting its function in sleep homeostasis. Furthermore, we identified and validated connexin-43 (CX43) S373 as a direct astrocytic phosphosite of SIK1 using in vitro assays. Isoform-dependent and overlapping molecular substrates were analyzed to delineate distinct roles and signaling pathways. This thesis proposes a phosphorylationdependent signaling network mediated by SIKs and provides insights into understanding the molecular markers underlying the sleep-wake cycle.

Authors: Ru, Y

• DOI: 10.5287/ora-2r2nvpzag
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Circadian rhythms; sleep
• Subjects: Pharmacology; Neuroscience