Related Experiment Videos
Phosphorylation and inactivation of rat hepatocyte glycogen synthase by phorbol esters and mezerein
Abstract:
Incubation of rat hepatocytes with active phorbol esters and mezerein provoked a decrease in glycogen synthase activity. After the incubation of [3 2 P] phosphate-labeled cells with these tumor promoters, an increase in the amount of 3 2 P bound to the immunoprecipitated enzyme was observed. The decrease in activity highly correlated with the phosphorylation in the smaller CNBr fragment (CB-1) and only at high concentration of the phorbol ester the increase in the phosphorylation of the larger CNBr fragment (CB-2) became significative. Tryptic degradation of CB-1 showed two phosphopeptides after isoelectro focusing analysis (pI 3.9 and pI 3.4) and only one of them (pI 3.9) increased its phosphorylation state after treatment of the cells. These results indicate that the decrease in activity of glycogen synthase by phorbol esters and mezerein is a result of the phosphorylation of the enzyme and that a single site located in CB-1 is preferentially phosphorylated by these agents.
Insights
Phorbol esters and mezerein decrease glycogen synthase activity by increasing enzyme phosphorylation. This effect is primarily due to increased phosphorylation at a specific site within the CB-1 fragment of the enzyme.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Glycogen synthase is a key enzyme in glycogen synthesis.
- Tumor promoters like phorbol esters can modulate cellular signaling pathways.
- The precise mechanisms by which tumor promoters affect enzyme activity are not fully understood.
Purpose of the Study:
- To investigate the effect of phorbol esters and mezerein on glycogen synthase activity in rat hepatocytes.
- To determine if changes in enzyme activity correlate with alterations in enzyme phosphorylation.
- To identify the specific sites of phosphorylation affected by these tumor promoters.
Main Methods:
- Incubation of rat hepatocytes with phorbol esters and mezerein.
- Measurement of glycogen synthase activity.
- Phosphorylation analysis of [32P] phosphate-labeled glycogen synthase using immunoprecipitation and CNBr fragmentation.
- Isoelectro focusing of tryptic phosphopeptides.
Main Results:
- Phorbol esters and mezerein decreased glycogen synthase activity.
- Tumor promoter treatment increased 32P incorporation into glycogen synthase.
- The decrease in activity correlated with increased phosphorylation in the smaller CNBr fragment (CB-1).
- A single phosphopeptide within CB-1 showed increased phosphorylation after treatment.
Conclusions:
- The reduction in glycogen synthase activity by phorbol esters and mezerein is mediated by increased enzyme phosphorylation.
- A specific site within the CB-1 fragment is preferentially phosphorylated by these tumor promoters, leading to enzyme inactivation.