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Tumor target binding induces phosphorylation of two M(r) 65,000 lymphokine-activated killer proteins
M J Frederick1, L V Rodriguez, D A Johnston
1Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Abstract:
Effector-target cell conjugate formation is an essential step during lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Protein phosphorylation changes in human LAKs after contact with NK-resistant (LAK-sensitive) tumors were examined by two-dimensional SDS-PAGE. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets led to increased phosphorylation of two M(r) 65,000 LAK proteins, pp65a and pp65b, with isoelectric points of 5.1 and 5.2, respectively. Phosphorylation of both substrates was initiated between 1 and 5 min after coincubation with tumor targets. Contact between LAKs and targets was required for p65 phosphorylation because soluble tumor factors failed to induce phosphorylation. Normal peripheral blood lymphocyte targets, which are bound very poorly by LAKs and are resistant to killing, failed to induce LAK p65 phosphorylation. The broad protein kinase inhibitor staurosporine inhibited phosphorylation of pp65a and pp65b, supporting the hypothesis that activation of a LAK protein kinase leads to p65 phosphorylation. Cross-linking of CD16 (Fc gamma RIIIA), which mediates antibody-dependent cellular cytotoxicity in LAKs, also led to increased pp65a and pp65b phosphorylation. Collectively, these data provide correlative evidence that p65 phosphorylation may be involved in the cytolytic function of LAKs.
Insights
Lymphokine-activated killer (LAK) cell contact with tumor cells triggers phosphorylation of specific LAK proteins (pp65a/b). This phosphorylation, dependent on cell contact and inhibited by staurosporine, suggests a role in LAK cell-mediated cytotoxicity.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Effector-target cell conjugate formation is crucial for lymphokine-activated killer (LAK) cell cytotoxicity.
- Understanding molecular events during LAK cell interaction with target cells is key to elucidating cytotoxic mechanisms.
Purpose of the Study:
- To investigate protein phosphorylation changes in human LAK cells upon engagement with NK-resistant tumor targets.
- To identify specific LAK proteins involved in the phosphorylation cascade triggered by target cell interaction.
Main Methods:
- Two-dimensional SDS-PAGE was employed to analyze protein phosphorylation patterns in LAK cells.
- LAK cells were co-incubated with tumor cell lines (SK-Mel-1, Raji) and normal lymphocytes.
- The effect of staurosporine (protein kinase inhibitor) and CD16 cross-linking on phosphorylation was assessed.
Main Results:
- Co-incubation with tumor targets induced increased phosphorylation of two M(r) 65,000 LAK proteins, pp65a and pp65b.
- Phosphorylation occurred rapidly (1-5 min) and required direct cell-to-cell contact.
- Staurosporine inhibited pp65 phosphorylation, and CD16 cross-linking also enhanced it.
Conclusions:
- The phosphorylation of pp65a and pp65b is a specific event triggered by LAK cell interaction with susceptible tumor targets.
- These findings support the hypothesis that LAK protein kinase activation and subsequent p65 phosphorylation are involved in LAK cell-mediated cytotoxicity.