Systematic identification of regulatory proteins critical for T-cell activation.
BACKGROUND
The activation of T cells, mediated by the T-cell receptor (TCR), activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation.


RESULTS
In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 x 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLC gamma 1 and SHP-1 (PTP1C) as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1, IL-10R alpha and integrin alpha2), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using truncated Lck, PLC gamma 1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes.


CONCLUSIONS
This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.
Chu
Peter
P
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
Pardo
Jorge
J
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
Zhao
Haoran
H
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
Li
Connie C
CC
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.|Current address: Exelixis Inc., 170 Harbor Way, South San Francisco, CA 94083, USA.
Pali
Erlina
E
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
Shen
Mary M
MM
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
Qu
Kunbin
K
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
Yu
Simon X
SX
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
Huang
Betty Cb
BC
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
Yu
Peiwen
P
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.|Current address: Exelixis Inc., 170 Harbor Way, South San Francisco, CA 94083, USA.
Masuda
Esteban S
ES
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
Molineaux
Susan M
SM
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
Kolbinger
Frank
F
Novartis Pharma AG, S-386.6.25, CH-4002 Basel, Switzerland.
Aversa
Gregorio
G
Novartis Forschungsinstitut GmbH, Brunner Strasse 59, A-1235 Vienna, Austria.
de Vries
Jan
J
Novartis Forschungsinstitut GmbH, Brunner Strasse 59, A-1235 Vienna, Austria.
Payan
Donald G
DG
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
Liao
X Charlene
XC
Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.|Current address: Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
D015703:Antigens, CD / Q000502:physiology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000502:physiology; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D005796:Genes / Q000502:physiology; D006801:Humans; D019169:Jurkat Cells / Q000737:chemistry / Q000378:metabolism; D037181:Lectins, C-Type; D008213:Lymphocyte Activation / Q000502:physiology*; D011506:Proteins / Q000502:physiology*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D012107:Research Design; D015398:Signal Transduction / Q000502:physiology; D013601:T-Lymphocytes / Q000502:physiology; D015854:Up-Regulation / Q000502:physiology; D000099192:CD69 Antigens
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D037181:Lectins, C-Type; D011506:Proteins; D011948:Receptors, Antigen, T-Cell; D000099192:CD69 Antigens