CBER Research Projects

Project Title

Molecular Mechanism of Lymphocyte Activation

Principal Investigator

Ezio Bonvini

Laboratory

Laboratory of Immunobiology; Division of Monoclonal Antibodies; Office of Therapeutics Research and Review

Project Summary

Perturbation of the T- or B-cell antigen receptor (TCR or BCR) by antigen or by anti-receptor antibodies induces protein tyrosine kinase activation, the phosphorylation and activation of phospholipase Cgamma-1 (PLCg1), and the activation of small G-proteins, such as Ras. The activation of PLCg1, in particular, mediates phosphoinositide (PI) hydrolysis which in turn controls Ca2+ mobilization and protein kinase C activation, obligatory events in the activation of T- or B-lymphocytes. The laboratory is concerned with establishing the mechanisms by which immune receptors regulate PLCg1. Mutational analysis demonstrated that PLCg1 amino terminal SH2 (SH2N) domain was required and sufficient for TCR- or BCR-induced tyrosine phosphorylation of PLCg1, and for the association of PLCg1 with the T cell adapter, LAT, or the B cell adapter, BLNK. PLCg1 membrane translocation required the SH2N domain and was decreased by mutation of the SH3 domain. BCR-induced PI hydrolysis was abrogated by mutation of either the SH2N or the carboxyl-terminal SH2 (SH2C) domain and was decreased by mutation of the SH3 domain. A role for lipid rafts, specialized membrane microdomains, in phosphorylation and activation of PLCg1 was also demonstrated. The cellular proto-oncogene, Cbl, is an adapter that associates with numerous signaling proteins, including PLCg1, involved in signal transduction by distinct receptors. Cbl is a major target of tyrosine phosphorylation after TCR engagement. We have found that over-expression of Cbl reduced TCR-induced AP1 (a Ras-dependent transcription factor) and NF-AT (a Ca2+-dependent transcription factor) reporter activities, suggesting that Cbl could act at a point shared by both the Ras and PLCg1/Ca2+ pathways. Cbl over-expression decreased TCR-induced PI-hydrolysis, but had no effect on TCR-induced tyrosine phosphorylation of PLCg1. In contrast, over-expression of an oncogenic Cbl mutant, 70Z/3, enhanced PI-hydrolysis. These data support a sequential model of immune receptor-induced PLCg1 activation where the earliest event is the engagement of the SH2N domain by a phosphoprotein that recruits PLCg1 in an activation complex within the membrane with ensuing phosphorylation, while the SH3 domain contributes additional membrane anchoring. Up-regulation of PLCg1 activity requires translocation and phosphorylation and is abrogated or reduced by disruption of the SH2N or SH3 domains, respectively. The SH2C domain is exclusively required for receptor-induced PLCg1 activation. Cbl regulates receptor-induced PLCg1 activity by affecting, in an SH3 domain-dependent manner, a point downstream of or independent of PLCg1 phosphorylation.

Publications

• FEBS Lett 2000 Mar 31;470(3):273-280
  Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cgamma-1 activity.
  Graham LJ, DeBell KE, Veri M, Stoica B, Mostowski H, Bonvini E, Rellahan B
  Pub Med

• Front Biosci 2001 Nov 1;6:A25-A32
  Specific regions of the extracellular domain of dlk, AN EGF-like homeotic protein involved in differentiation, participate in intramolecular interactions.
  Baladron V, Ruiz-Hidalgo MJ, Gubina E, Bonvini E, Laborda J
  Pub Med

• Mol Cell Biol 2001 Oct;21(20):6939-50
  Membrane raft-dependent regulation of phospholipase cgamma-1 activation in t lymphocytes.
  Veri MC, DeBell KE, Seminario MC, DiBaldassarre A, Reischl I, Rawat R, Graham L, Noviello C, Rellahan BL, Miscia S, Wange RL, Bonvini E
  Pub Med

• J Biol Chem 2001 May 4;276(18):15554-60
  Epidermal growth factor (EGF) receptor kinase-independent signaling by EGF.
  Deb TB, Su L, Wong L, Bonvini E, Wells A, David M, Johnson GR
  Pub Med

Last Updated: 4/1/2002

Back to Top     Back to Index

Date created: September 25, 2003

CDER Home Page | CDER Site Info | Contact CDER | What's New @ CDER
FDA Home Page | Search FDA Site | FDA A-Z Index | Contact FDA | HHS Home Page

FDA/Center for Drug Evaluation and Research