identification-kinase-substrate-pairs-using-high-throughput

Identification of Kinase-substrate Pairs Using High Throughput Screening


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Children's Hospital of Eastern Ontario Research Institute

Spinal muscular atrophy (SMA) is the number 1 genetic killer of young children. It is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. Although SMA is primarily a motor neuron disease, metabolism abnormalities such as metabolic acidosis, abnormal fatty acid metabolism, hyperlipidemia, and hyperglycemia have been reported in SMA patients. We thus initiated an in-depth analysis of glucose metabolism in SMA.

Glucose metabolism and pancreatic defects in spinal muscular atrophy.

Previous work in insulinoma cell lines has established that calcineurin plays a critical role in the activation of cAMP-responsive element binding protein (Creb), a key transcription factor required for β-cell function and survival, by dephosphorylating the Creb coactivator Creb-regulated transcription coactivator (Crtc)2 at 2 regulatory sites, Ser171 and Ser275. Here, we report that Crtc2 is essential both for glucose-stimulated insulin secretion and cell survival in the β-cell. Endogenous Crtc2 activation is achieved via increasing glucose levels to the physiological feeding range, indicating that Crtc2 is a sensor that couples ambient glucose concentrations to Creb activity in the β-cell. Immunosuppressant drugs such as cyclosporin A and tacrolimus that target the protein phosphatase calcineurin are commonly administered after organ transplantation. Chronic use is associated with reduced insulin secretion and new onset diabetes, suggestive of pancreatic β-cell dysfunction. Importantly, we show that overexpression of a Crtc2 mutant rendered constitutively active by introduction of nonphosphorylatable alanine residues at Ser171 and Ser275 permits Creb target gene activation under conditions when calcineurin is inhibited. Taken together, these data suggest that promoting Crtc2-Creb activity is required for β-cell function and proliferation and promoting this pathway could ameliorate symptoms of new onset diabetes after transplantation.

CRTC2 is required for β-cell function and proliferation.

Energy sensing by the AMP-activated protein kinase (AMPK) is of fundamental importance in cell biology. In the pancreatic β-cell, AMPK is a central regulator of insulin secretion. The capacity of the β-cell to increase insulin output is a critical compensatory mechanism in prediabetes, yet its molecular underpinnings are unclear. Here we delineate a complex consisting of the AMPK-related kinase SIK2, the CDK5 activator CDK5R1 (also known as p35) and the E3 ligase PJA2 essential for β-cell functional compensation. Following glucose stimulation, SIK2 phosphorylates p35 at Ser 91, to trigger its ubiquitylation by PJA2 and promote insulin secretion. Furthermore, SIK2 accumulates in β-cells in models of metabolic syndrome to permit compensatory secretion; in contrast, β-cell knockout of SIK2 leads to accumulation of p35 and impaired secretion. This work demonstrates that the SIK2-p35-PJA2 complex is essential for glucose homeostasis and provides a link between p35-CDK5 and the AMPK family in excitable cells.

Role of the SIK2-p35-PJA2 complex in pancreatic β-cell functional compensation.

Precise regulation of insulin secretion by the pancreatic beta cell is essential for the maintenance of glucose homeostasis. Insulin secretory activity is initiated by the stepwise breakdown of ambient glucose to increase cellular ATP via glycolysis and mitochondrial respiration. Knockout of Lkb1, the gene encoding liver kinase B1 (LKB1) from the beta cell in mice enhances insulin secretory activity by an undefined mechanism. Here, we sought to determine the molecular basis for how deletion of Lkb1 promotes insulin secretion.

Article	Year
Glucose metabolism and pancreatic defects in spinal muscular atrophy. Annals of neurology\| PubMed ID: 22926856	2012
CRTC2 is required for β-cell function and proliferation. Endocrinology\| PubMed ID: 23677932	2013
Role of the SIK2-p35-PJA2 complex in pancreatic β-cell functional compensation. Nature cell biology\| PubMed ID: 24561619	2014
LKB1 couples glucose metabolism to insulin secretion in mice. Diabetologia\| PubMed ID: 25874445	2015

Courtney Reeks

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