Modulating p56Lck in T-Cells by a Chimeric Peptide Comprising Two Functionally Different Motifs of Tip from Herpesvirus saimiri.

Jean-Paul Vernot,Diego Fernando Martínez,Luis Alberto Pérez-Quintero,Ana María Perdomo-Arciniegas

doi:10.1155/2015/395371

Jean-Paul Vernot, Diego Fernando Martínez + Show 2 more

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https://doi.org/10.1155/2015/395371

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Abstract

The Lck interacting protein Tip of Herpesvirus saimiri is responsible for T-cell transformation both in vitro and in vivo. Here we designed the chimeric peptide hTip-CSKH, comprising the Lck specific interacting motif CSKH of Tip and its hydrophobic transmembrane sequence (hTip), the latter as a vector targeting lipid rafts. We found that hTip-CSKH can induce a fivefold increase in proliferation of human and Aotus sp. T-cells. Costimulation with PMA did not enhance this proliferation rate, suggesting that hTip-CSKH is sufficient and independent of further PKC stimulation. We also found that human Lck phosphorylation was increased earlier after stimulation when T-cells were incubated previously with hTip-CSKH, supporting a strong signalling and proliferative effect of the chimeric peptide. Additionally, Lck downstream signalling was evident with hTip-CSKH but not with control peptides. Importantly, hTip-CSKH could be identified in heavy lipid rafts membrane fractions, a compartment where important T-cell signalling molecules (LAT, Ras, and Lck) are present during T-cell activation. Interestingly, hTip-CSKH was inhibitory to Jurkat cells, in total agreement with the different signalling pathways and activation requirements of this leukemic cell line. These results provide the basis for the development of new compounds capable of modulating therapeutic targets present in lipid rafts.

Highlights

Selective phosphorylation of tyrosine residues in the Tcell receptor- (TCR-) associated CD3 complex and ζ chains follows TCR engagement and activation by the MHC-peptide complex [1]
Our results show that T-cells that have been previously stimulated by the strong activator PHA-P can still be further activated by the chimeric peptide hTip-CSKH, suggesting either that stimulation with PHA-P is not maximal in this condition and hTip-CSKH delivers a synergic proliferation signal or that hTip-CSKH signals through other pathway(s)
We have developed a novel chimeric peptide to modulate Lck signaling in cell membrane lipid rafts

Summary

Introduction

Selective phosphorylation of tyrosine residues in the Tcell receptor- (TCR-) associated CD3 complex and ζ chains follows TCR engagement and activation by the MHC-peptide complex [1]. Tyrosine phosphorylation is mediated by protein members of the nonreceptor Src tyrosine kinase family, mainly p56Lck (Lck) and p59Fyn (Fyn) [2]. Lck is lymphoidspecific and essential for T-cell development and function [3, 4]; it associates with surface molecules such as CD2, CD4, CD8, CD45, and IL-2 receptor [4,5,6]. Lck can be either phosphorylated by serine-threonine or tyrosine kinases but it is well known that its activity is mainly positively and negatively regulated by tyrosine phosphorylation in positions 394 and 505, respectively [7, 8]. Stimulation of T-cell lines defective in Lck expression has shown an abnormal tyrosine phosphorylation pattern of downstream protein targets [9]. Lck has acquired importance as a therapeutic target for regulating T-cell response due to this central role in T-cell function [10,11,12]

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