Heterogeneity of adenovirus type 5 E1A proteins: multiple serine phosphorylations induce slow-migrating electrophoretic variants but do not affect E1A-induced transcriptional activation or transformation
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J D Richter1, J M Slavicek, J F Schneider
1Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Abstract:
The 289-amino-acid product encoded by the adenovirus E1A 13S mRNA has several pleiotropic activities, including transcriptional activation, transcriptional repression, and when acting in concert with certain oncogene products, cell transformation. In all cell types in which E1A has been introduced (except bacteria), E1A protein is extensively posttranslationally modified to yield several isoelectric and molecular weight variants. The most striking variant is one that has a retarded mobility, by about Mr = 2,000, in sodium dodecyl sulfate gels. We have investigated the nature of this modification and have assessed its importance for E1A activity. Phosphorylation is responsible for the altered mobility of E1A, since acid phosphatase treatment eliminates the higher apparent molecular weight products. By using several E1A deletion mutants, we show that at least two seryl residues, residing between residues 86 and 120 and 224 and 289, are the sites of phosphorylation and that each phosphorylation can independently induce the mobility shift. However, E1A mutants lacking these seryl residues transcriptionally activate the adenovirus E3 and E2A promoters and transform baby rat kidney cells to near wild-type levels.
Insights
Adenovirus E1A protein undergoes phosphorylation, a post-translational modification, leading to altered molecular weight variants. This modification, however, is not essential for E1A
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Adenovirus E1A protein exhibits pleiotropic activities, including transcriptional regulation and cell transformation.
- E1A protein undergoes extensive post-translational modifications, resulting in various molecular weight and isoelectric variants.
- A prominent variant shows retarded mobility in SDS-PAGE, suggesting a significant modification.
Purpose of the Study:
- To investigate the nature of the retarded mobility modification of adenovirus E1A protein.
- To determine the functional significance of this modification for E1A's biological activities.
Main Methods:
- Utilized acid phosphatase treatment to assess the role of phosphorylation in E1A mobility shift.
- Employed deletion mutants of E1A to map the phosphorylation sites.
- Assessed transcriptional activation of adenovirus E3 and E2A promoters and cell transformation capabilities of E1A mutants.
Main Results:
- Phosphorylation was identified as the cause of the altered mobility of E1A protein.
- Specific seryl residues between amino acid positions 86-120 and 224-289 were identified as phosphorylation sites.
- Each phosphorylation site independently induced the mobility shift.
- E1A mutants lacking these phosphorylation sites retained significant transcriptional activation and cell transformation activities.
Conclusions:
- Phosphorylation is a key post-translational modification of adenovirus E1A protein, affecting its apparent molecular weight.
- Despite the mobility shift, phosphorylation at these identified sites is not critical for the primary functions of E1A, such as transcriptional activation and cell transformation.
- Further research is needed to elucidate the precise role of these phosphorylation events in the complex functions of E1A protein.