Prof. Noah Isakov

Prof. Noah Isakov Profile

Professor Emeritus
PhD. 1981

Department : Shraga Segal Department of Microbiology, Immunology and Genetics
Room : 129
בנין מעבדות מחקר רפואה ע"ש דייכמן - פלאם - M6
Phone : 972-8-6477269
972-8-6477267
Email : noah@bgu.ac.il
Office Hours :  
Immunology, Signal transduction, Biochemistry, Molecular and Cell Biology

Education

  • Ph.D., The Weizmann Institute of Science (1981)

Research Interests

The general aim of our laboratory is to decipher signal transduction mechanisms that regulate T lymphocyte activation, function and differentiation into effector cells, as well as the characterisation of proto-oncogen products that are involved in cell transformation.

Research Abstract

Immunophilins control T lymphocyte adhesion and migration by regulating C3G binding to CrkII. Crk adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII, the two alternative-spliced forms of CRK, possess an N-terminal SH2 domain, followed by an SH3 domain, while CrkII possesses in addition a C-terminal linker region plus an SH3 domain, which operate as regulatory moieties. Here, we investigated the ability of immunophilins, that function as peptidyl-prolyl isomerases (PPIases), to regulate Crk proteins in human T lymphocytes. We found that endogenous CrkII, but not CrkI, associates with the immunophilins, cyclophilin A (CypA) and FKBP12, in resting human Jurkat T cells. In addition, CypA increased C3G binding to CrkII, while inhibitors of immunophilins, such as cyclosporine A (CsA) and FK506, inhibited CrkII, but not CrkI association with C3G. Expression in Jurkat T cells of PICCHUx, a plasmid encoding the human CrkII1-236 sandwiched between CFP and YFP, demonstrated a basal level of FRET, which increased in response to cell treatment with CsA and FK506, reflecting increased trans-to-cis conversion of CrkII. Crk-C3G complexes are known to play an important role in integrin-mediated cell adhesion and migration. We found that overexpression of CrkI or CrkII increased adhesion and migration of Jurkat T cells. However, immunophilin inhibitors suppressed the ability of CrkII-, but not CrkI-overexpressing cells to adhere to fibronectin-coated surfaces and migrate towards the SDF1? chemokine. The present data demonstrate that immunophilins regulate CrkII, but not CrkI activity in T cells and suggest that CsA and FK506 inhibit selected effector T cell functions via a CrkII-dependent mechanism.EndFragment

A novel motif in the V3 domain of PKC-theta determines its immunological synapse localization and functions in T cells via association with CD28. Protein kinase C-theta (PKC-theta) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. We found that the PKC-theta V3 domain is necessary and sufficient for IS localization mediated by Lck-dependent association with CD28. We identified a conserved proline-rich motif in V3 required for CD28 association and IS localization. CD28 association was essential for PKC-theta-mediated downstream signaling and TH2 and TH17, but not TH1, differentiation. Ectopic V3 expression sequestered PKC-theta from the IS and interfered with its functions. These results identify a unique mode of CD28 signaling, establish a molecular basis for the IS localization of PKC-theta, and implicate V3-based “decoys” as therapeutic modalities for T cell-mediated inflammatory diseases.

Development of unique antibodies directed against each of the six different phosphotyrosine residues within the T cell receptor CD3zeta chain. Signal transduction from the T cell antigen receptor (TCR)/CD3 complex involves six different immunoreceptor tyrosine-based activation motifs (ITAM) located within the cytoplasmic tails of the CD3 chains. Each ITAM possesses two conserved tyrosine residues that can undergo phosphorylation upon TCR/CD3 crosslinking and become a docking site for SH2-containing effector molecules. Specificity of the SH2 domains is determined by their ability to bind a phosphorylated tyrosine in the context of a longer peptide motif within the target protein. As a result, phosphorylation of different tyrosines within the CD3 cytoplasmic tails creates docking sites for distinct SH2-containing signaling proteins that differentially impact on the quality of the T cell response. In the present study, we prepared antibodies specific for each of the six different phosphotyrosines of the mouse CD3zeta chain. The antibodies were characterized with respect to their cross-reactivity, ability to recognize the phosphorylated versus non-phosphorylated forms of tyrosine-containing motifs, and cross-reactivity with the homologous phospho-motifs on the human CD3zeta protein. The antibodies were found to be specific and selective for phospho-CD3zeta. They can serve as useful tools for distinguishing between the six potential tyrosine phosphorylation sites on the CD3zeta chain, and for correlating the phosphorylation of specific CD3zeta tyrosine residues with activation of signaling pathways that dictate T cell differentiation into responding, anergic, or apoptotic cells.

Hodgkin's lymphoma cells exhibit high expression levels of the PICOT protein. PICOT was originally discovered as a protein kinase C (PKC) binding protein in human Jurkat T-lymphocytes in which it was found to modulate PKCtheta-dependent functions. In addition, RT-PCR analysis suggested the expression of PICOT in a wide range of organs and cell types, including cells that are devoid of PKCtheta. We aimed at analyzing the expression of the PICOT protein in mouse lymphoid organs, and to compare them with those of Jurkat T-lymphocytes and other cell lines. We also analyzed whether PICOT expression in T-lymphocytes is dependent on the presence of PKCtheta, and whether it correlates with cell growth rate. Western blot analyses demonstrated PICOT expression in all lymphoid organs and cell lines tested. In addition, similar expression levels were observed in lymphoid organs of wild-type and PKCtheta-null mice, suggesting that PICOT expression in T-lymphocytes is independent of PKCtheta. However, PICOT expression levels were higher in Jurkat T-lymphocytes and other lymphoma cell lines compared to freshly isolated lymphocytes, while T-lymphocyte mitogens, such as concanavalin A, increased PICOT expression concomitantly with the induction of a faster T-lymphocyte growth rate. Finally, immunohistochemistry of freshly-isolated lymph nodes from Hodgkin's lymphoma patients revealed significantly higher levels of PICOT in Hodgkin's cells, compared to the normal surrounding lymphocytes. The present results show a direct correlation between PICOT expression levels and increased cell growth, both in vitro and in vivo, and suggest that immunostaining of PICOT might be useful for in situ identification of transformed cells, such as those of Hodgkin's lymphoma.

Research Topics

  • Signal Transduction Mechanisms in T lymphocytes 
  • Molecular and cellular analysis of PKC-theta-mediated regulation of T cell functions
  • Deciphering the role of PICOT in growth regulation of normal and transformed cells
  • PICOT as a dual regulator of cardiomyocyte hypertrophy and isotropy
  • The role of human ZAP-70 in early stages of T cell activation    
  • Immunophilin-mediated regulation of Crk-dependnet signalling events
  • Regulation of immune cell functions by Pin1       
  • The role of Crk adaptor proteins in T cell activation, adhesion and migration

Major expertise and techniques in the lab

  • Immunology, biochemistry, cellular and molecular biology

Publications and funding summary / representative publications and grants

Publications

Books

Regulation of immune system cell functions by protein kinase C. Frontiers in Immunology. 2012. Noah Isakov and Amnon Altman, Guest Editors.

Signal Transduction Mechanisms in T lymphocytes, 2012. A Special Issue of Journal of Clinical & Cellular Immunology. N. Isakov, Editor.

Manuscripts

Nath, R.P., Dong, G., Braiman, A. and Isakov, N. 2014. Immunophilins Control T Lymphocyte Adhesion and Migration by Regulating CrkII Binding to C3G. J. Immunol. 193:3966-3977. DOI: 10.4049/jimmunol.1303485.

Nath, P. R. and Isakov, N. 2014. PKC-theta-regulated signaling in health and disease. Biochem. Soc. Trans. 42:1484-1489. DOI: 10.1042/BST20140180.

Gelkop, S., Weisman, B., Pulak, R.N., Zharhary, D. and Isakov, N. 2012. Development of unique antibodies directed against each of the six different phosphotyrosine residues within the T cell receptor CD3z chain. J. Immunol. Methods. 375:129-37. doi:10.1016/j.jim.2011.10.001

Kong, K-F., Yokosuka, T., Canonigo-Balancio, A. J., Isakov, N., Saito, T. and Altman, A. 2011. A novel motif in the V3 domain of protein kinase C-? (PKC?) determines its immunological synapse localization and functions in T cells via association with CD28. Nat. Immunol. 12:1105-1112. doi: 10.1038/ni.2120.

Grant

2014-2018 US-Israel Binational Science Foundation: 'Regulation of PKC-theta and PKC-theta-directed signaling events in activated T cells’. 

Existing collaborations

  • Amnon Altman - Regulation of PKC-theta and PKC-theta-directed signaling events in activated T cells
  • Yoram Etzion - PICOT involvement in atrial fibrillation
  • Alex Braiman - Regulation of CrkII conformation and function by immunophilins