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Pyruvate kinase type M2 is phosphorylated at tyrosine residues in cells transformed by Rous sarcoma virus
P Presek1, M Reinacher, E Eigenbrodt
1Rudolf Buchheim-Institut für Pharmakologie, Justus Liebig-Universität Giessen, FRG.
Abstract:
Chicken embryo cells (CECs) contain pyruvate kinase (PK) type M2 (M2-PK). Transformation of CECs by Rous sarcoma virus (RSV) leads to a reduction in the affinity of PK for the substrate phosphoenolpyruvate. In vitro, M2-PK can be phosphorylated at tyrosine residues by pp60v-src, the transforming protein of RSV. To study tyrosine phosphorylation of M2-PK in intact RSV-transformed cells, the protein was immunoprecipitated from 32P-labeled normal and RSV-SR-A-transformed CECs. Phosphoamino acid analysis of immunoprecipitated M2-PK revealed that M2-PK of both normal and transformed CECs contained phosphoserine and small amounts of phosphothreonine. Only M2-PK of transformed CECs contained phosphotyrosine in addition. For enzyme kinetic studies M2-PK was partially purified by chromatography upon DEAE-Sephacel and hydroxyapatite. A decreased affinity for phosphoenolpyruvate was observed 3 h after the onset of transformation using the temperature-sensitive mutant of RSV, ts-NY 68. The kinetic changes were correlated with tyrosine phosphorylation of M2-PK, but there is no direct evidence that they are caused by post-translational modification of the enzyme.
Insights
Rous sarcoma virus (RSV) transformation of chicken embryo cells (CECs) causes pyruvate kinase M2 (M2-PK) to undergo tyrosine phosphorylation. This modification correlates with reduced substrate affinity, impacting cellular metabolism during viral transformation.
Area of Science:
- Biochemistry
- Molecular Biology
- Virology
Background:
- Chicken embryo cells (CECs) possess pyruvate kinase (PK) type M2 (M2-PK).
- Rous sarcoma virus (RSV) transformation alters cellular metabolism.
- M2-PK is a key enzyme in glycolysis.
Purpose of the Study:
- To investigate the tyrosine phosphorylation of M2-PK in intact RSV-transformed CECs.
- To correlate kinetic changes in M2-PK with its post-translational modifications.
- To understand the role of pp60v-src in M2-PK regulation.
Main Methods:
- Immunoprecipitation of M2-PK from 32P-labeled normal and RSV-transformed CECs.
- Phosphoamino acid analysis to identify phosphorylated residues.
- Enzyme kinetic studies using partially purified M2-PK.
Main Results:
- RSV-transformed CECs showed M2-PK with phosphotyrosine, in addition to phosphoserine and phosphothreonine found in normal CECs.
- A decreased affinity for phosphoenolpyruvate was observed in M2-PK from transformed cells.
- Tyrosine phosphorylation of M2-PK was correlated with observed kinetic changes.
Conclusions:
- RSV transformation induces tyrosine phosphorylation of M2-PK in CECs.
- This post-translational modification is linked to altered M2-PK kinetics.
- Further studies are needed to establish a direct causal link between tyrosine phosphorylation and kinetic changes.