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Expression of v-src in T cells correlates with nuclear expression of NF-kappa B
D M Eicher1, T H Tan, N R Rice
1Leukocyte Cell Biology Section, NCI-FCRDC, Frederick, MD 21702-1201.
Abstract:
NF-kappa B is a rapidly inducible transcriptional activator that responds to a variety of signals and influences the expression of many genes involved in the immune response. Protein tyrosine kinases transmit signals from cytokine and immune receptors. Very little information exists linking these two important classes of signaling molecules. We now demonstrate that v-src expression correlates with nuclear expression of a kappa B binding complex similar to that induced by phorbol ester and ionomycin, as detected by electrophoretic mobility shift assay using a variety of kappa B sites. This complex was blocked by the tyrosine kinase inhibitor, herbimycin A. The v-src-induced complex comprised the p50 and p65 components of NF-kappa B, as determined by supershift and immunoblot analysis. As a functional correlate of this finding, transient co-transfection of HIV-1 LTR reporter constructs in a different T cell line demonstrated that v-src activated this promoter in a kappa B-dependent manner. We found that transactivation of the HIV-1 LTR by v-src was more sensitive to mutations of the proximal, rather than the distal, kappa B element. The implications for T cell receptor signaling and HIV-1 gene expression are considered.
Insights
The study shows that v-src signaling activates Nuclear Factor-kappa B (NF-kappa B) binding complexes, impacting immune response genes. This activation, blocked by tyrosine kinase inhibitors, has implications for T cell receptor signaling and HIV-1 gene expression.
Area of Science:
- Molecular Biology
- Immunology
- Virology
Background:
- Nuclear Factor-kappa B (NF-kappa B) is a key transcriptional activator in immune responses.
- Protein tyrosine kinases are crucial for signal transduction from cytokine and immune receptors.
- The interplay between NF-kappa B and tyrosine kinases, particularly v-src, is not well understood.
Purpose of the Study:
- To investigate the link between v-src expression and NF-kappa B activation.
- To determine the functional consequences of v-src-induced NF-kappa B activation on gene expression, specifically the HIV-1 LTR.
- To elucidate the role of tyrosine kinase signaling in NF-kappa B complex formation.
Main Methods:
- Electrophoretic mobility shift assay (EMSA) to detect NF-kappa B binding complexes.
- Supershift and immunoblot analysis to identify NF-kappa B components (p50, p65).
- Transient co-transfection assays with HIV-1 LTR reporter constructs to assess promoter activity.
Main Results:
- v-src expression correlated with nuclear NF-kappa B binding complex formation, similar to phorbol ester/ionomycin induction.
- This v-src-induced complex formation was inhibited by the tyrosine kinase inhibitor herbimycin A.
- The v-src-induced complex contained p50 and p65 subunits of NF-kappa B.
- v-src activated the HIV-1 LTR promoter in a NF-kappa B-dependent manner, with greater sensitivity to mutations in the proximal kappa B element.
Conclusions:
- v-src signaling activates NF-kappa B, influencing the expression of target genes.
- Tyrosine kinase activity is essential for v-src-mediated NF-kappa B activation.
- Findings suggest a mechanism linking T cell receptor signaling pathways to NF-kappa B activation and potential implications for HIV-1 pathogenesis.