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Characterization of pp85, a target of oncogenes and growth factor receptors
1Department of Biochemistry, Tufts University Health Sciences Campus, Boston, Massachusetts 02111.
Abstract:
An 85,000-molecular-weight polypeptide (85K polypeptide) has previously been identified as a common substrate for tyrosine phosphorylation upon polyomavirus middle T transformation or upon platelet-derived growth factor stimulation of 3T3 cells. In each case, pp85 has an associated phosphatidylinositol kinase activity. The tissue distribution of pp85 was determined by middle T blotting experiments; the highest levels were found in brain, lung, and spleen tissues. High-resolution examination of 85K by isoelectric focusing demonstrated that there are at least 10 different forms. These were resolved into two families, 85K and 86K; the ratio of the two families changed in different cells. Similar forms were found for pp85 associated with pp60v-src. Individual species within each family differed by phosphorylation. Analysis of pp85 and pp86 by immunoprecipitation with anti-phosphotyrosine antibody showed increasing phosphorylation in response to middle T or pp60v-src transformation. The association of middle T with pp85 and pp60c-src was examined in pulse-chase experiments. Association of middle T with pp60c-src was slow and was accompanied by progressive modification of middle T. pp85 formed a dissociable complex with middle T within 2.5 min.
Insights
An 85,000-molecular-weight polypeptide (85K polypeptide) with phosphatidylinositol kinase activity is a common substrate for tyrosine phosphorylation. Its varied forms and phosphorylation levels change in response to viral transformation and growth factor stimulation.
Area of Science:
- Cellular biology
- Molecular oncology
- Biochemistry
Background:
- An 85,000-molecular-weight polypeptide (85K polypeptide) is a known substrate for tyrosine phosphorylation.
- This phosphorylation occurs during polyomavirus middle T antigen transformation and platelet-derived growth factor stimulation.
- The 85K polypeptide, also known as pp85, exhibits associated phosphatidylinositol kinase activity.
Purpose of the Study:
- To investigate the tissue distribution of pp85.
- To characterize the different forms of pp85 using high-resolution techniques.
- To analyze the phosphorylation status and complex formation of pp85 in response to specific cellular stimuli.
Main Methods:
- Middle T blotting experiments were used to determine tissue distribution.
- Isoelectric focusing was employed for high-resolution examination of 85K polypeptide forms.
- Immunoprecipitation with anti-phosphotyrosine antibody and pulse-chase experiments were utilized to study phosphorylation and complex formation.
Main Results:
- Highest levels of pp85 were detected in brain, lung, and spleen tissues.
- Isoelectric focusing revealed at least 10 forms of 85K, resolving into 85K and 86K families with varying ratios in different cells.
- pp85 and pp86 showed increased phosphorylation upon middle T or pp60v-src transformation, and pp85 formed a dissociable complex with middle T rapidly.
Conclusions:
- The 85K polypeptide (pp85) is a widely distributed protein with complex isoforms and dynamic phosphorylation.
- Its interaction with middle T antigen and pp60c-src is crucial in viral transformation pathways.
- pp85's enzymatic activity and regulatory modifications highlight its significance in cellular signaling.