We seek to understand the molecular basis of early activation events in T-lymphocytes in response to physiologically important stimuli; the functional responses most important to our research are T-lymphocyte cytoskeletal reorganization, adhesion, migration, and gene transcription. We emphasize analysis of: 1) changes in serine/threonine phosphorylation as key signaling events which regulate these processes, and 2) the serine/threonine kinases that mediate such phosphorylation. One major project is to understand the spatial reorganization during chemokine-induced polarization of peripheral blood T cells (PBT) and to elucidate the biochemical basis for that reorganization using a combination of microscopic, biochemical and molecular genetic approaches. Of particular importance we have demonstrated that chemokines induce an acute change in the balance between serine-threonine kinases and phosphatases, which we find is required for moesin dephosphorylation, for loss of microvilli and for polarization. This promises to be a major novel signaling element in chemokine signaling; we are exploring the kinases/phosphatases involved and the signaling pathways coupling them to G-protein-coupled receptors. Phosphorylation cascades are critical both to antigen-specific activation of T cells and to chemokine-mediated stimulation. We are investigating serine/threonine phosphorylation in lymphocytes, since it is much less well understood than tyrosine phosphorylation. Mechanism-based understanding of these processes will require detailed knowledge of the kinases involved. The protein kinase C family of serine/threonine kinases has been repeatedly implicated in lymphocyte adhesion and cytoskeletal reorganization, but there is little precise understanding of mechanisms. We have undertaken a systematic structure-function analysis of PKC-theta and have begun to extend that analysis to structure and function of other serine/threonine kinases which have a close evolutionary relationship to theta and are expressed in lymphocytes. We have previously identified three regulatory phosphorylation sites on PKC-theta and now have identified two new ones, whose functional significance is under investigation. Moreover we are investigating in detail the peptide specificity of phosphorylation by different PKC isoforms. Our findings differ strikingly from the prevailing view of PKC specificity, showing greater similarity than expected between some and greater difference than expected between others. We have constructed homology models from which to predict and test hypothesis regarding the structural basis for such similarities and differences. Early results are beginning to confirm our hypothesis that exploiting this understanding will help us decipher the complex phosphorylation cascades involved in T-lymphocyte activation.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC009257-27
Application #
6762141
Study Section
(EIB)
Project Start
Project End
Budget Start
Budget End
Support Year
27
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Hao, Jian-Jiang; Wang, Guanghui; Pisitkun, Trairak et al. (2008) Enrichment of distinct microfilament-associated and GTP-binding-proteins in membrane/microvilli fractions from lymphoid cells. J Proteome Res 7:2911-27
Dong, Xiaoyun; Patino-Lopez, Genaro; Candotti, Fabio et al. (2007) Structure-function analysis of the WIP role in T cell receptor-stimulated NFAT activation: evidence that WIP-WASP dissociation is not required and that the WIP NH2 terminus is inhibitory. J Biol Chem 282:30303-10
Liu, Yin; Kruhlak, Michael J; Hao, Jian-Jiang et al. (2007) Rapid T cell receptor-mediated SHP-1 S591 phosphorylation regulates SHP-1 cellular localization and phosphatase activity. J Leukoc Biol 82:742-51
Liu, Yin; Belkina, Natalya V; Graham, Caroline et al. (2006) Independence of protein kinase C-delta activity from activation loop phosphorylation: structural basis and altered functions in cells. J Biol Chem 281:12102-11
Zhu, Guozhi; Fujii, Koichi; Liu, Yin et al. (2005) A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families. J Biol Chem 280:36372-9
Sarkar, Kakali; Kruhlak, Michael J; Erlandsen, Stanley L et al. (2005) Selective inhibition by rottlerin of macropinocytosis in monocyte-derived dendritic cells. Immunology 116:513-24
Zhu, Guozhi; Fujii, Koichi; Belkina, Natalya et al. (2005) Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases. J Biol Chem 280:10743-8
Zhu, Guozhi; Liu, Yin; Shaw, Stephen (2005) Protein kinase specificity. A strategic collaboration between kinase peptide specificity and substrate recruitment. Cell Cycle 4:52-6
Anderson, Arthur O; Shaw, Stephen (2005) Conduit for privileged communications in the lymph node. Immunity 22:3-5
Salganik, M P; Milford, E L; Hardie, D L et al. (2005) Classifying antibodies using flow cytometry data: class prediction and class discovery. Biom J 47:740-54

Showing the most recent 10 out of 16 publications