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Human p59fyn(T) regulates OKT3-induced calcium influx by a mechanism distinct from PIP2 hydrolysis in Jurkat T cells
K Rigley1, P Slocombe, K Proudfoot
1Cantab Pharmaceuticals Research Limited, Cambridge, UK.
Abstract:
The earliest biochemical event after cross-linking of TCR is the tyrosine phosphorylation of a variety of substrates. At least three nonreceptor tyrosine kinases have been implicated in this signaling cascade: p59fyn(T), p56lck, and ZAP-70. Recently, PLC gamma 1 has been shown to be tyrosine phosphorylated in T cells after receptor activation. This increase in tyrosine phosphorylation correlates with the increased activity of the enzyme. The substrate for PLC gamma 1, phosphatidylinositol 4,5-bisphosphate (PIP2), is hydrolyzed to the protein kinase C activator diacylglycerol and inositol 1,4,5-triphosphate (IP3), which promotes calcium release from the endoplasmic reticulum. These results lend support to the notion that calcium mobilization after TCR cross-linking is mediated by increased levels of IP3. In this study we have cloned and transfected a human p59fyn(T) cDNA in the anti-sense configuration into the human T cell line, Jurkat, resulting in decreased expression of the protein. We find that cell lines expressing significantly reduced levels of p59fyn(T) exhibit significantly lower calcium influx following OKT3 activation. However, the level of IP3 production was unchanged and IP1 and IP2 levels were elevated. These data indicate that p59fyn(T) can regulate calcium influx by a mechanism distinct from PIP2 hydrolysis.
Insights
The T cell receptor (TCR) initiates signaling cascades. This study shows that p59fyn(T) kinase regulates calcium influx via a pathway independent of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis.
Area of Science:
- Immunology
- Cell Signaling
- Molecular Biology
Background:
- T cell receptor (TCR) activation triggers early biochemical events, including tyrosine phosphorylation of substrates.
- Key nonreceptor tyrosine kinases like p59fyn(T), p56lck, and ZAP-70 are involved in TCR signaling.
- Phospholipase C gamma 1 (PLC gamma 1) is tyrosine phosphorylated upon T cell activation, increasing its activity and leading to calcium mobilization.
Purpose of the Study:
- To investigate the role of p59fyn(T) in T cell activation and calcium influx.
- To determine if p59fyn(T) regulates calcium mobilization through phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis.
Main Methods:
- Cloning and transfection of human p59fyn(T) cDNA in anti-sense configuration into Jurkat T cells.
- Generation of cell lines with reduced p59fyn(T) expression.
- Measurement of calcium influx and inositol phosphate levels (IP3, IP1, IP2) following OKT3 activation.
Main Results:
- Cell lines with reduced p59fyn(T) expression showed significantly lower calcium influx after OKT3 activation.
- Inositol 1,4,5-triphosphate (IP3) production levels remained unchanged in these cells.
- Levels of inositol 1,4,5-trisphosphate (IP3) breakdown products, IP1 and IP2, were elevated.
Conclusions:
- p59fyn(T) plays a crucial role in regulating calcium influx in T cells following TCR activation.
- This regulation occurs through a mechanism that is distinct from the canonical pathway involving PIP2 hydrolysis and IP3 production.