Monoclonal antibody study of the subcellular localization and DNA-stimulating activity of murine sarcoma virus-activated transformation-associated proteins
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Abstract:
Previously, we reported the monoclonal antibody detection of transformation-associated proteins (TAP) in ts110 murine sarcoma virus-transformed normal rat kidney (6M2) cells (Chan et al., 1986). In this study, we used the same monoclonal antibody to investigate the subcellular localization, the fate and the mitogenic activity of TAP, as well as the correlationship between TAP synthesis and the expression of transformation properties of 6M2 cells. It was found that TAP were localized in the cytoplasm (probably the Golgi apparatus) of 6M2 cells. TAP were found as three intracellular polypeptides (mol wt of 66K, 63K, and 60K, respectively), and were rapidly released into extracellular medium. Upon release, TAP changed to two extracellular polypeptides (mol wt of 68K and 64K, respectively). Furthermore, the synthesis of TAP was temperature sensitive and correlated closely with the expression of transformation properties of the 6M2 cells. TAP have been purified by monoclonal antibody-affinity column chromatography and found to have a synergistic effect with insulin in stimulating the DNA synthesis of normal rat kidney cells.
Insights
Transformation-associated proteins (TAP) are found in the cytoplasm of 6M2 cells and secreted as modified forms. TAP synthesis correlates with cell transformation and stimulates DNA synthesis with insulin.
Area of Science:
- Cell Biology
- Virology
- Biochemistry
Background:
- Monoclonal antibodies were previously used to detect transformation-associated proteins (TAP) in ts110 murine sarcoma virus-transformed normal rat kidney (6M2) cells.
- This study further investigates the characteristics and functions of TAP.
Purpose of the Study:
- To determine the subcellular localization of TAP in 6M2 cells.
- To analyze the fate and mitogenic activity of TAP.
- To correlate TAP synthesis with the expression of cellular transformation properties.
Main Methods:
- Monoclonal antibody-affinity column chromatography for TAP purification.
- Analysis of intracellular and extracellular TAP polypeptide profiles and molecular weights.
- Assessment of TAP's effect on DNA synthesis in normal rat kidney cells.
Main Results:
- TAP were localized in the cytoplasm, likely the Golgi apparatus, of 6M2 cells.
- Three intracellular TAP polypeptides (66K, 63K, 60K) were identified and rapidly released extracellularly, transforming into two distinct polypeptides (68K, 64K).
- TAP synthesis was temperature-sensitive, closely correlating with the expression of transformation properties in 6M2 cells.
Conclusions:
- TAP are secreted and undergo modification, with their synthesis linked to cellular transformation.
- Purified TAP exhibit synergistic mitogenic activity with insulin, stimulating DNA synthesis in normal rat kidney cells.