Glucose Deprivation Triggers β-catenin Degradation via PKC

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(Last Updated On: April 4, 2020)
Different signaling pathways within a single cell
The complex network of different signaling pathways within a single cell. Image: Roadnottaken via Common Wikimedia

Glucose deprivation leads to the autophagy

Autophagy is a catabolic process by which a cell maintains the homeostasis by degrading unnecessary cell organelles and defective components in the lysosome. The autophagic end products are recycled for the biosynthesis of new biomolecules, proteins, and cell membranes. There are three types of autophagy in the cell.

  • Microautophagy; the direct engulfment of the cytoplasmic components.
  • Chaperon-mediated autophagy; the selective degradation of proteins containing KFERQ like sequence.
  • Macroautophagy; the cytoplasmic components are enclosed in a double membrane forming an autophagosome that infuses with the lysosome to form an autolysosome.

Recently, it has been found that the β-catenin interacts with a microtubule-associated protein 1A/B-light chain-3 (LC3) and leads to autophagic degradation during the nutritionally stressed conditions.

In the absence of Wnt stimulation, a destruction complex is formed which is composed of casein kinase-1α, glycogen synthase kinase-3β (GSK3β), axin and adenomatous polyposis coli (ACP). The casein kinase-1α and GSK3β phosphorylate the cytoplasmic Ser/Thr-rich sequence of the β-catenin at its N-terminal. The phosphorylation of this sequence generates a recognition site for the ubiquitin ligase (E3), β-transducin repeat-containing protein (β-TrCP) and ultimately degraded by the 26S proteasome.

However, in the presence of Wnt stimulation, the destruction complex dissociates into its components inhibiting the β-catenin degradation. Then, β-catenin is accumulated in the cytoplasm and translocates into the nucleus. In the nucleus, β-catenin displaces the Groucho family of transcription repressors from the lymphoid enhancer-binding factor and T-cell specific transcription factors (TCF) and acts as a transcriptional co-activator for the target genes.

Researchers have studied the relationship between glucose deprivation-mediated autophagy and β-catenin protein stability. They found that glucose deprivation induces the autophagy by enhancing the proteasomal degradation of the β-catenin while they also found that the proteasomal degradation of the β-catenin is GSK3β independent and follows the cyclin-dependent protein kinase (PKCα) dependent pathway.

Methodologies used for the glucose deprivation-induced degradation of β-catenin

For this study, researchers took human embryonic kidney cells (HEK293) and human foreskin fibroblast cells (HFF-1). These cell lines were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin at 37°C.

Cells were washed with phosphate-buffered saline (PBS) to evaluate the effect of glucose deprivation and then cultured in the same medium but lacking the glucose. The researchers used different inhibitors for the kinases such as lithium chloride for GSK3β, Go6976 for PKCα, and MLN-4924 for NEED8 activity enzyme.

The researchers used plasmids with wild-type β-catenin, mutant and a construct of GSK3β. The transfected cells were cultured in the glucose-free medium and after 24 hours of glucose deprivation, inhibitors were added.

The activity of β-catenin and T-cell specific transcription factors (TCF) was measured via the luciferase activity assay. During which mutant lymphoid enhancer-binding factors/TCF binding sites were used as negative control while β-galactosidase was used as an internal control.

Nuclear proteins and cytoplasmic proteins were extracted and analyzed by using a 10% SDS-PAGE and immune-blotting procedure. The result reveals the time-dependent reduction in the cell number of both the HEK293 and HFF-1 along with the abnormal cellular phenotype under the glucose deprivation condition.

Observation of the glucose deprivation-induced degradation of the β-catenin

Researchers have analyzed the effect of glucose deprivation on the cell cycle and found that the glucose deprivation leads to the accumulation of the sub-G1 positive cells in the HEK293 cells while the HFF-1 cells showed a drastic shift to the sub-G1 cell population. However, researchers did not find any changes in the apoptosis-related proteins in both cell lines under the glucose deprivation condition, but they observed that glucose deprivation leads to the cell cycle arrest by downregulating the phospho-Rb, CDK2/4, and cyclin B and upregulating the P21 and P27 proteins.

Observation of the molecular markers during the glucose deprivation reveals the elevated levels of the phospho-ERK1/2, phospho-GSK3β, and phospho-AMP-activated protein kinase while the level of phospho-ASK was dropped. The level of LC3-II, an essential protein for autophagy was found to be increased significantly in the glucose deprivation. All these results reveal that the glucose deprivation induces cell cycle arrest and leads to autophagy in the HEK293 cells.

During the investigation, researchers found that the glucose deprivation significantly downregulates the β-catenin proteins but doesn’t affect the mRNA expression level of the β-catenin and a nuclear β-catenin binding protein TCF4 in the HEK293 cells. Therefore, researchers examined the nuclear and cytoplasmic distribution of the β-catenin under the glucose deprivation and they found that the decreased level of the β-catenin proteins in both compartments.

Glucose deprivation leads to the inhibition of β-catenin

To understand the mechanism of how the glucose deprivation leads to the inhibition of the β-catenin signaling, researchers used TOPFlash having eight copies of the lymphoid enhancer-binding factors/TCF binding sites.

Researchers further analyzed whether the administration of the glucose will bring the level of β-catenin and LC3 back to their normal concentration. After supplementation of the glucose in the medium, researchers found that the level of β-catenin and LC3-II proteins were completely recovered to their normal concentration.

Glucose deprivation-induced degradation of β-catenin occurs in proteasome

To examine the glucose deprivation leads to the proteasomal degradation of the β-catenin, researchers used MG132, cell-permeable proteasome inhibitors where the researchers found that the glucose deprivation leads to the increased in the level of the β-catenin ubiquitination that can be blocked by the use of SCF complex inhibitor MLN4924.

MLN4924 is an E3 ubiquitin ligase complex that is very much important for the protein ubiquitination involved in the cell cycle and also marks various cytosolic proteins for the degradation.

Degradation of β-catenin follows the PKCα signaling pathway, not the GSK3β pathway

To examine the GSK3β independent degradation of the β-catenin, researchers treated the glucose-deprived HEK293 cells with lithium chloride, a direct inhibitor of the GSK3β. They found the increased level of the phospho-GSK3β (an inactive form of the GSK3β) and still the level of β-catenin was low indicating the glucose deprivation-mediated degradation of the β-catenin is not associated with the GSK3β.

To understand which kinase affects the glucose deprivation-mediated β-catenin degradation, researchers used different inhibitors for the protein kinases including cyclin-dependent protein kinase (PKCα), Ca++/calmodulin dependent protein kinase II, mitogen-activated protein kinase kinase ½ (MEK1/2), P38 and c-Jun N-terminal kinase (JNK). They found that β-catenin degradation was significantly blocked and the level of LC3-II protein was also decreased significantly by the inhibition of the PKCα using Go6976.

These results indicate that the glucose deprivation induces the proteasomal degradation of the β-catenin follows the PKCα pathway. In conclusion, the glucose deprivation leads to autophagy by GSK3β independent proteasomal degradation of the β-catenin via PKCα pathway.

Reference: Journal of Biological Chemistry (Glucose Deprivation Triggers Protein Kinase C-dependent β-Catenin Proteasomal Degradation)

Article DOI: 10.1074/jbc.M114.606756

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