P56lck, ZAP-70, SLP-76, and Calcium-regulated Effectors Are Involved in NF-κB Activation by Bisperoxovanadium Phosphotyrosyl Phosphatase Inhibitors in Human T Cells

Michel Ouellet,Benoit Barbeau,Michel J Tremblay

doi:10.1074/jbc.274.49.35029

Michel Ouellet, Benoit Barbeau + Show 1 more

Open Access

https://doi.org/10.1074/jbc.274.49.35029

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Abstract

This study investigates the second messengers involved in NF-kappaB activation by the bisperoxovanadium (bpV) phosphotyrosyl phosphatase inhibitors. We first initiated a time course analysis of bpV-mediated activation of the human immunodeficiency virus type-1 long terminal repeat- and NF-kappaB-driven reporter gene. Our results showed a slower and more transient activation of both kappaB-regulated luciferase-encoding vectors by bpV compounds when compared with the action of tumor necrosis factor-alpha (TNF). Time course analyses of NF-kappaB translocation by shift assay experiments further confirmed these results, hence implying distinct pathways of NF-kappaB activation for bpV compounds and TNF. Attempts to characterize the bpV-dependent signaling cascade revealed that the src family protein tyrosine kinase p56(lck) was critical for NF-kappaB induction by bpV. Furthermore, p56(lck) interaction with the intracytoplasmic tail of CD4 markedly enhanced such induction. Optimal activation of NF-kappaB following bpV treatment necessitated downstream effectors of p56(lck) such as the syk family protein tyrosine kinase ZAP-70 and the molecular adaptor SLP-76. Importantly, reduced NF-kappaB activation was observed when capacitative calcium entry was deficient but also upon pharmacological inhibition of calmodulin and calcineurin. Altogether, these results suggest that induction of NF-kappaB by phosphotyrosyl phosphatase bpV inhibitors necessitates both proximal and distal effectors of T cell activation.

Highlights

Mechanisms underlying cellular activation involve a complex interplay of many cellular enzymes, adaptors, and receptors, each having a defined role in the fate of the cell
Different Kinetics of nuclear factor-␬B (NF-␬B) Activation by bpV Compounds and tumor necrosis factor-␣ (TNF)—In order to characterize the NF-␬B signaling pathway initiated by the bpV PTP inhibitors, kinetic analyses of NF-␬B activation were carried out using bpV and other well known NF-␬B-inducing agents
In stably transfected J␬B cells expressing luciferase under the control of five consensus binding sites for NF-␬B, the induction kinetics were different since PMA, PHA, PMA/PHA, and TNF all showed a peak of activity after a 6-h treatment (Fig. 1, panel C), whereas that of cells stimulated with bpV appeared later, between 8 and 12 h (Fig. 1, panel D, and data not shown)

Summary

Introduction

Mechanisms underlying cellular activation involve a complex interplay of many cellular enzymes, adaptors, and receptors, each having a defined role in the fate of the cell. These new bisperoxovanadium compounds are highly purified by 51V NMR and contain an ancillary ligand in the inner coordination sphere of the vanadium molecule whose function is to increase the stability of the compound as well as to provide a certain degree of specificity [16] Their mode of action is thought to be the same as pervanadate which can enter the active site of the enzyme due to its high similarity with phosphate groups and oxidize the conserved catalytic cysteine residue located at the bottom of the active site [17].

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