1
Lymphocyte Signaling II
Advanced Immunology, Feb 15th , 2002
Gerald R. Crabtree
file name: Lymphocyte Signaling II
hf.grc@forsythe.stanford.edu
The Complexities of Antigen Receptor Signaling.
The large number of subunits of the antigen receptor and the generalization that allosteric mechanisms are often
employed by multimeric molecules has given rise to a two-sided controversy about the mechanism of action of the T
cell receptor and perhaps also the B cell receptor. One school feels that the antigen receptor (and not other receptors)
has a certain intelligence and somehow senses the type of antigen and then transmits a signal that is related to the type
of antigen. The other school feels that the antigen receptor has no more brains than a pop-up toaster and simply
behaves as all other receptors and that the multiple chains are necessary only to contact different signaling molecules
and facilitate oligmerization. I will try to avoid this controversy, however, my bias is that the antigen receptor is no
different than the PDGF, EGF, TGF, FGF, EPO, Insulin, IL-2, and other well studied receptors and that the different
responses seen with antigen simply reflect the diversity of antigens, and the relative binding affinity of antigen/MHC
complexes as well as their kinetic constants. Several new pieces of structural evidence have emerged that indicate that
the antigen receptor does not undergo any substantial allosteric change on antigen binding, and crystallization of TCRantigen-MHC complexes with agonist and antigonist peptides (see work by Chris Garcia, Don Wiley and coworkers).
This data indicates that the major difference between different related antigenic peptides may be the duration of the
occupancy of the receptor by antigen-MHC complexes. But how is the duration of T cell receptor occupancy
transmitted to a specific transcriptional reglatory program that will produce activation, negative or positive selection?
Sequence of Events after antigen binding.
Current Model for Signal Transduction in
T Lymphocyte Activation
A ntigen Preseenting C ell
MHC
MHC
 
C D4
C D4
MHC
 
C D4
 
A ntigen
C D3
P03
C D3
T Lymphocyte
lck
lckinactive due to
phosphorylation by
? csk
C D3
lck
Physical
approximation of
lckt o TCR
D ephosphorylat ion
and activat ion of
lckby ?CD 45
P03
P03 P0
3
lckact ive
lck
Tyrosine Phosphorylation
of CD 3 TA M's by ?lck or ?fyn
Zap 70 att aches
to CD 3
phospho- TA Ms
P0 3
P03 P0
3
zap 70
lckact ive
fyn active
zap 70 act ive
syk active
Tryosine Phosphorylation
of subst rates including PLC 
Ras Acti vati on,
El evati on of
Intracell ular Ca++
The mechanism illustrated in the figure is reasonably consistent with the observations made by many groups. It
describes the events that lead to tyrosine phosphorylation of TCR and  as well as substrates such as PLC SOS and
ZAP70. These events occur within seconds and lead to the activation of ZAP70 kinase, src kinases and the
phosphorylation of the vav exchange factor for rho like GTPases. Rac was recently shown to be activated by the VAV
protein after it is tyrosine phosphorylated (Nature 385, 169, 1997). Within the past year it has become clear that
activation of rac and vav lead to a second event necessary to initiate signaling. This second event requires actin
polymerization (Penninger and Crabtree, Cell 96, 9, 1999) and leads to the formation of the CAP. Without VAV the
cap does not form (Curr Biol. 1998 May 7;8(10):563-72.. WASP the gene affected in Wiscott Aldrich Syndrome (an
actin regulatory protein) is also required for cap formation. The cap is a localized grouping of signaling molecules that
was first defined in the 70’s by Raff and colleagues (Nature 242, 257, 1972) and includes actin, and a group of cell
2
surface and signaling molecules that collect on one part of the membrane in response to PHA, anti CD3, and
antigen/MHC interactions. The cap has received more attention recently and has been renamed the SMAC (Super
Molecular Activation Cluster) or even “synapse” to refer more specifically to the structure present at the interface of
the antigen presenting cell and the T cell. The purpose of the cap is probably to maintain a stable aggregate of
signaling molecules at the cell surface. In essence the cap probably regulates the effective molarity of signaling
molecules and may serve as a scaffold on which a precise geometry of signaling molecules can occur. The signaling
pathway below is thought to lead to the formation of CAP and SMACs, however the details of this are uncertain and
the role for the WASP protein is not yet clear, but likely relates to its role in actin polymerization probably by
regulating the ARP2/3 complex (Cell. 1999 97:221-31).
Most workers in this field agree that aggregation is probably essential for antigen receptor
signaling. This has been suspected since Peter Nowell first discovered lymphocyte activation using plant
lectins that aggregate receptors on the cell surface. (Cancer Res. p 462,1960). Indeed a seemingly
physiologically normal signal can be generated by simply oligmerizing the intracellular 100 aa of the zeta
chain (Spencer et al. Science. 1993 Nov 12; 262, 1024). The outcome of aggregation is the phosphorylation
of TCR zeta and episolon chains by the src like typrosine kinases lck and fyn. These kinases are kept in an
inactive form by phosphorylation probably by the c-src kinase, csk. The conversion to the active form
requires the tyrosine phosphatase, CD45. CD45 is highly abundant and may be simply passively brought
into the complex or it may have a negative ligand (Science. 1998 Jan 2; 279(5347): 88-91).
Cell Membrane Molecules Involved in T-cell Activation
A. The antigen receptor and its associated molecules, the CD3 or T3 molecules.
The intracellular domains of the variable chains of the antigen receptor are small and probably not capable of
transmitting signals to the interior of the cell. Present evidence indicates that binding of antigen/MHC to the
antigen receptor somehow transmits a signal to the CD3 molecule. This most likely involves aggregation of
the CD3 chains which can be induced with simple dimeric synthetic ligands that penetrate the cell membrane
and aggregate receptors. (Spencer et al. Science 262, 1019, 1993). The CD3 complex made up of 5 invariant
chains (gamma, delta, epsilon that can pair with either two zetas or a zeta and an eta chain. Zeta and eta have
an intracellular domain that is tyrosine phosphorylated after T cell activation.
The intracellular domain of zeta or epsilon are able to transmit signals since aggregating the intracellular
domain alone is sufficient for activation (Cell 1991,64,891 and Science 262, 1019,1993). The internal region
of the zeta chain contains a repeated motif (TAM) that is tyrosine phosphorylated shortly after antigen
presentation and interacts with the SH2 domain of the ZAP70 tyrosine kinase. This kinase is felt to be
largely responsible for activating substrates necessary for phosphotydilinositol breakdown, leading to an
influx of Ca++ and activation of PKC and ras (Cell 71 649,1992). A deficiency in this kinase leads to a
developmental failure to produce CD8 cells and a SCID syndrome with defects in both T and B cells that
result from a failure of T cell signal transduction.
Critical Cell Membrane Signaling Molecules

3
CD4 and CD8
These coreceptor molecules function in signal transduction by an interaction with MHC on the outside of the
cell and lck on the inside of the cell. They appear to bring lck to the antigen receptor CD3 chains after
antigen binding. They will be covered in the lectures by Dr. Parnes.
Lck and Fyn and other tyrosine kinases
These src like tyrosine kinases are essential for T cell activation as shown by a number of studies, most
notably the knock out of the genes and the fact that cell lines lacking them do not signal the activation of ras
and the influx of Ca++. Fyn mutant mice show a defect in the ability of thymocytes to signal properly, but
these cells go through development normally, while the defect in lck mutant mice is an early developmental
arrest. They are normally membrane bound by virtue of myristoylation with an inhibitory phosphorylation
that is removed with antigen receptor activation. The most likely candidate phosphatase is CD45.
CD45 (Leukocyte Common Antigen)
This is a transmembrane tyrosine phosphatase that is required for signal transduction by the antigen receptor.
Presumably it modulates the function of p56lck and fyn by removing inhibitory phosphorylation in either
molecule. These phosphorylations are felt to result from the actions of the csk (c-src kinase) present in T
cells and many cell types. When cross-linked artificially by using the extracellular domain of another
receptor, it blocks T cell activation, suggesting that it has a negative role in controlling antigen receptor
signalling. However, its mode of action is unknown. Interestingly, it is also the "memory antigen" for Tcells, and appears in an alternatively spliced form after antigen activation. The alternatively spliced CD45
may remain on the surface of the cell for the lifetime of the individual. Recent data indicate that it might be
crossed linked to inhibit T cell activation (Cell. 2000 Dec 22;103(7):1059-70). However the autoimmune
phenotype of these mice was apparently due to an inbreed gene in the line in which the knock in was made
and is not solely due to the CD45 mutation. (Art Weiss, personal communication).
csk or the c src kinase. This appears to be the kinase that adds the inhibitory phosphorylation to lck and fyn.
CD28 Provides a costimulatory function in lymphocyte activation. If sub-optimal levels of antigenic
stimulation are given, there is a requirement for CD28. Its receptor is the B7 molecule on B lymphocytes.
CTLA4 This transmembrane receptor appears to function as a negative signal since null mutants show excessive
activation. However, null mutants of the IL-2, IL2 receptor, Jnk, NFAtc2 and a number of other positive signaling
molecules also show paradoxical activation. Nevertheless CTLA4 is very likely to be a negative regulator of the
antigen receptor signaling pathway acting by competing for B7 which is the ligand for CD28.
Accessory Molecules These include CD2, T11, TAP, Ly 6 and Tp44 and others. However, several lines of evidence
indicate that these molecules require both the antigen receptor and the CD3 molecule to activate T-cells. Although you
will occasionally see these pathways referred to as "alternative pathways" of T-cell activation, most likely signals
initiated by these accessory molecules are mediated through CD3 by lateral interactions in the cell membrane (which
could still be functionally important).
Linking and Adaptor Proteins Involved in T cell Activation
Zap 70 or syk- A non-receptor tyrosine kinase that binds to the TCR zeta chains and phosphorylates a variety of
substrates that lead to the activation of ras and influx of Ca2+.
Linker of Activated T cells (LAT) is a 38 kDa protein that is rapidly phosphorylated by Zap 70 after T cell
activation and essential for T cell development (Cell 92, 83, 1998). This protein is palmitoylated on C26 and
C29, a modification that is required for its presence in glycolipid micro domains in the membrane or Rafts.
These modifications appear to be essential for the function of LAT since mutations at these sites interfere
with the function of the protein. LAT appears to be required for both activation of PLC and Ca2+ influx as
well as activation of the ras pathway.
SLP-76 is a 76 kDa protein that binds to GRB2 by it SH2-domain and is tyrosine phosphorylated. It is
recruited to the antigen receptor complex after lymphocyte activation and is required for thymic development
past the DP stage and probably for signaling by the antigen receptor (Cell 94, 229, 1998). SLP-76 deficient
cells show a selective defect in the activation of PLC but normal induced tyrosine phosphorylation of other
proteins.
SLAP- Src-like adaptor protein This is a 130 kDa protein that binds to the TCR complex including SL-76
after activation and appears to play a negative role in TCR signaling. It exact mechanism is not understood.
4
SHIP, SH2 containing inositol polyphosphate 5-phosphatase. This is an inhibitor of T Cell activation by virtue of
its ability to remove the 5’ phosphate from IP3 and PIP3. PIP3 is thought to recruit the Tek kinases such as Itk to the
membrane where they probably play an important role in the activation of PLC.
Cbl- This molecule binds to the phosphotyrosines of signaling molecules by its SH2 domain. It is a E3 ubiquitin
ligase that leads to the rapid degradation of activated signaling molelcules in lymphocytes and many other cell types.
For example it binds to phospho ZAP70 leading to its degradation. It is one of a group of proteins that inhibit T cell
activation.
I.
Signaling Pathways Initiated by the Antigen Receptor
A. The Ca++/Calcineurin/NF-AT Pathway
This is actually the only T cell signaling pathway to be objectively identified. This was done by starting in the nucleus
and working backward biochemically from the regulatory regions of the IL-2 gene to the cell membrane. The other
pathways such as Map kinase, Jnk, NFkB etc were defined by a candidate gene approach.
Calcineurin was recognized to be critical for T cell activation when it was found to be the in vitro and in vivo target of
the drug cyclosporin A (Nature. 1992 Jun 25;357(6380):695-7; Cell. 1991 Aug 23;66(4):807-15). This is the single
most effective immunosuppressant known and was responsible for the revolution in transplant therapy about 15 years
ago. Today many millions of people owe their lives to this drug. The increase in intracellular calcium seen after
activating many receptors is thought to be the result of the activation of PLC or PLC (PLC is generally activated by
G protein coupled receptors) and the release of DAG and IP3 from membrane lipids. IP3 interact with its receptor
(IP3R) on the ER and causes the release of intracellular stores of calcium leading to the depletion of ER stores. This is
thought to be somehow sensed by highly selective Ca++-Release Activated Ca++ channels (CRAC channels) leading to
++
their opening and the influx of Ca . You will see this mechanism referred to as capacitive Calcium entry and Rich
Lewis will discuss it in later lectures. Although this activation mechanism is essential for a wide variety of processes in
many cell types it is poorly understood and the CRAC channel has yet to be molecularly characterized. The Calcium
concentrations produced by this channel exceed the critical 400 nM levels essential to activate Calcineurin and if
sustained leads to the activation of Calcineurin phosphatase activity.
The major role of Calcineurin and probably calcium in T cell activation is the activation of NF-ATc family
members. The NF-AT-1 transcription complex was defined as a protein complex binding to the Antigen receptor
response elements in the Il-2 gene
and other immune response genes
(Science 241, 202, 1988) and
consist of two components
(Nature 1991, 352, 6258) one of
which is cytoplasmic and the other
newly induced in the nucleus.
The cytoplasmic family members
have the name “NF-ATc” and are
normally present in the cytoplasm
and the subscript “c” was
originally used to denote that they
are Cytoplasmic, Cyclosporinsensitive, and Calcineurindependent in their actions (Nature
1991, 352, 6258; Immunology
Today 13, 136). Since the
original discovery of the two
components the nomenclature has
become confused and a table
below offers some clarification.
The Ca2+. Calcineurin, NF-AT signaling pathway is used in several different tissues. Remarkably, in neurons
in the hippocampus normal synaptic activity leads to the activation of calcineurin and nuclear localization of NF-ATc4
(Nature 401, 703-8, 1999). Thus, one could say that if you think about it, NF-ATc4 goes into the nucleus. NFAT
signaling is also used to pattern the mammalian vascular system (Cell. 2001 Jun 29;105(7):863-75; Nature. 1998 Mar
12;392(6672):182-6). Another role of this signaling pathway is in the guidance of axons to their targets in the brain
and the periphery during the development of the nervous system. (manuscript in perparation). In the later case it
appears to mediate two different types of signals at the two extensions of sensory axons.
One critical characteristic of the NF-ATc family of transcription factors is that they discriminate between
different types of calcium stimuli (Nature 383, 6603, 1996). NF-ATc requires the CRAC channel to maintain it in the
5
nucleus, since it is opposed by a powerful export kinase that oppose the actions of calcineurin and leads to the rapid
export of the protein form the nucleus. This export is mediated by a group of priming kinases that seem to be either
PKA, CK1, JNK, or MEKK. This priming kinase is followed by a more specific phosphorylation that is essential for
export. Purification of the kinase that phosphorylates the residues essential for export gave GSK3 (Science 275, 5308,
1997), Interestingly GSK3 is negatively regulated by T cell activation stimuli indicating that both import and export of
NF-ATc proteins are controlled by the antigen receptor. This characterisitic makes NF_ATc a good candidate to
monitor the duration of antigen receptor interactions as the cell surface.
NF-Atc family members have an inadequate DNA binding protein which makes them dependent on other proteins and
hence provides a structural basis for their abilility to integrate signaling pathways.
Characteristics of NF-AT family members
NFATC1
(NF-ATc)
EXPRESSION
T and B Lymphocytes
Embryonic Heart Valve Precurors
KNOCK OUT
Essential for Heart Valve development
B and T cell proliferation
Cytokine Production
T cell proliferation
NFATC2
(NF-ATp)
NFATC3
(NF-ATx,
NF-AT4)
NFATC4
(NF-AT3)
T and B cells, Mast Cells,
Brain, Cartilage, Neural tube
Thymus, T cells and dorsal somites
Hippocampus, Several embryonic patterns
Brain, Hippocampus and Cerebellum, neural
Crest. Widely expressed in early embryo
?
NFATC5
TonEBP
Probably ubiquitious
?
T cell proliferation
Cytokine Production
The NF-ATc1/c2 double knock out is deficient in T cell activation and makes no cytokines (Immunity 2001
Jan;14(1):13-20) while the NF-ATC2/C3 mouse gives extreme lymphoid hyperplasia and the highest levels of IgE
production ever recorded. In addition the C2/c3 double knock has a biased TH1 phenotype. The C3/C4 double knock
out shows a failure to pattern the embryonic vascular system and specific defects in the patterning of the central
nervous system. Because of extensive redundancy with other family members and the fact that all four family members
are expressed in lymphocytes it has been difficult to assess the role of these proteins in lymphocytes were the pathway
was first defined.
The NFATc family members are redundant in their function and early knock out studies gave conflicting
results. In addition this family of proteins has essential functions in the early development of the cardiovascular and
nervous systems making it difficult to study their roles in development. More recently, double and triple knock out
have revealed that NF-ATc1 and NF-ATc2 are essential for activation of essentially all cytokine genes in T cells
Calcineurin, the Achilles Heal of T cell signal transduction.
2+
The Ca -dependent activation of calcineurin seems to be a particularly critical step in T cell activation since
efforts over the past twenty years to find molecules that block the mixed lymphocyte reaction have repeatedly found
calcineurin inhibitors. A number of different inhibitors such as cyclosporin and FK506 specifically block the
phosphatase activity of calcineurin (Cell 66, 807,1991, Nature 357,695,1992) and are effective immunosuppressants.
Remarkably only 50% or less of the activity of calcineurin need be blocked to stop signal transduction and produce
effective immunosuppression (Blood 84, 3974-9 ;1995). This indicates that the immune response is extremely sensitive
to inhibition at this level. Both FK506 and Cyclosporin A use a very unusual mechanism to inhibit calcineurin; they
6
bind to intracellular proteins (FKBP and cyclophilin respectively). This complex then inhibits calcineurin by binding
at subnanamolar affinity to calcineurin. The specificity of these drugs results from this ususal mechanism.
The ras/ERK/Fos Pathway
From the TCR to Ras
The physiologic activation of ras was first reported in T cells (Downward et al. Nature 346, 719,1990). The mode of
coupling to ras from the TCR- CD3 complex is reasonably well understood. The phospho TAM's in TCR bind the
dual SH2 domains of ZAP70 (Nature 376, 32, 1995). ZAP70 is a tyrosine kinase that appears to mediate all of the
actions of the T cell antigen receptor. The critical event in controlling the kinase activity of ZAP70 is its recruitment to
the membrane (EMBO 16, 5618, 1997). In B cells the homologue Syk appears to play a similar role. ZAP70 is thought
to activate PLC by tyrosine phosphorylation and to lead to the generation of IP3 and the influx of Ca2+ from the
exterior. In addition, ZAP70 links to SOS through LAT (Cell 92, 83, 1998) and perhaps the SLP 76 protein (Cell 1998
94, 229). SLP76 binds the phosphotryosines in ZAP70 and was initially identified as a lymphocyte-specific protein that
could bind the adapter, GRB2. Grb-2, has the organization: SH3-SH2-SH3, and interacts with the SH3 binding regions
of mSOS, a quanine nucleotide exchange factor for ras. SOS appears to be activated by membrane localization in the
proximity of ras (PNAS 92,9810,1995).
PMA and ionomycin can act as pharmacologic mimics of receptor signaling for many cell types and have
given many fundamental insights into the mechanisms underlying signal transduction primarily by allowing
biochemical approaches to the signaling pathways. In lymphocytes, PMA directly activates PKC, which in turn can
activate ras in T cells by promoting the activity of ras GAP. In many cell lines PMA is essential to obtain full
activation when T cells are triggered by antibodies to the TCR. PMA here probably functions in several ways
including: 1) activation of rasGAP; 2) Activation of NFkB dependent transcription and 3) substituting for the
costimulatory requirement by
bypassing the CD28-generated signals.
Effectors of the actions of Ras in
T cells: The Map and related
kinases.
This well known signaling pathway is
common to many developmental
pathways from the fly eye to the worm
vulva, to mammalian T cells. The
MAPkinase cascade has the general
form of GTPase-to-raf (MAPKKK)-toMEK-to-MAKP. Several transcription
factors have been shown to be the
targets of the different MAPkinases.
Most notably SRF, and Elk-1. (See for
example: Cell 73, 381-93, 1993). For
review of the different MAP kinase
pathways see (Cell 80,179,1995). This
pathway may play a role in the activation of IL-2 receptor and perhaps other early activation genes in lymphocytes.
Knock outs of the different genes involved in the mapkinase pathway have generally been either lethal or had little
effect on the T cell activation pathways.
Fos-Jun, AP-1. The formation of the AP-1 complex is controlled at many levels with at least 8 members taken two at
a time to make the AP-1 complex. Fos is primarily transcriptionally controlled by the serum response factor, while jun
and related family members are controlled mostly post transciptionally. Until recently Jnk was felt to be the most likely
regulator of Jun, however recent evidence in which a mutant having alanines at the perported JNK phosphorylation
sites has demonstrated that Jnk phosphorylation is not necessary for the essential functions of jun. Proc Natl Acad Sci
U S A. 200;98:1769-7.
C. The rac/JNK/Jun Pathway
One consequence of antigen receptor stimulation is the activation of the AP-1 transcription factors, which are
thought to be involved in the activation of genes such as IL-2, IL-4 and others necessary for an immune response. AP1 is made up of two components: a fos and a jun family member that heterodimerize. Recent evidence indicates that
the GTPase, rac is involved in activating the cJun transcription factor (Cell 81 1147 1995), by activating the cJun Nterminal kinase or JNK. This kinase binds tightly to the N terminus of jun and phosphorylates it at sites that were at
first thought to be physiologic(Cell 76, 1025,1994), but now are known from gene replacement studies to be nonessential for most of the functions of Jun (I. Wagner personal communication). For this reason the function of JNK
and it role in lymphocyte activation is unclear, however the three JNK or Stress activated kinases are all regulated by
7
various forms of cellular stress. Jnk is activated by many things including UV light and many growth factors and
inflammatory stimuli including CD28, IL-1, IL-6 and others. The other component of AP-1 are the fos family
members. These fos proteins are regulated through the ras-MAP kinase pathway described above by the SRF
transcription factor. This transcription factors was discovered by Richard Treisman and served as the terminus that
allowed the dissection of the map kinae pathway (Cell 46, 567, 1986).
One troublesome observation is that none of the knockouts of AP-1 components have shown much of an
immunologic phenotype. Neither have knockouts of JNK1-2 or the activator of JNK, SEK1 (MKK4) (there is a Nature
paper that indicates that the SEK1 ko had an immunologic phenotype, however this was an artifact of the blastocyst
reconstitution, Fred Alt, personal communication). Since AP-1 was identified as a possible activator of immune
response genes by the error-prone candidate-gene approach (i.e. investigators saw a sequence and used high
concentrations of AP-1 to force binding) and not by an unbiased purification or an unbiased genetic screen, its role will
require additional studies and confirmation. Recently a triple knock out of JNK1-3 showed no defect in T cell
activation but a bias in T cell differentiation. However, in other studies by other groups a minor defect in T cell
activation was noted but no defect in T cell differentiation was observed in Jnk mutants.
The Co-stimulatory Requirement
CD28-B7
An additional signaling pathway that supplements the antigen receptor pathway is the stimulus provided by
the interaction of CD28 on T cells with B7 on the surface of activated B cells. (Mol Cell Biol 7, 4472-81,1987 )
Knockouts of these genes have resulted in partial compromise of the immune responses and soluble inhibitors of the
CD28/B7 interaction induce anergy in vitro and are immunosuppressive in mice. However, clinical trials of CD28
fusion proteins have not been encouraging. The signaling pathway used by CD28 is not clear. Most reports now
indicate that that NF-AT is the target of costimulation through a pathway that includes PI3 kinase, AKT and perhaps
GSK3 which is the export kinase for NFATc family members (J Biol Chem. 2002;277, 912-21). One function of the
Cd28 is to enhance the aggregation of the antigen receptor as shown in recent results from Mark Davis’ lab showing
that it is involved in the movement of antigen receptors to the CAP and aids in the transmission of antigen receptor
signals (Science. 1998 Dec 18;282(5397):2266-9).
A recent analysis of the genes induced by costimulation indicates that few if any new genes are activated by
costimlulation verus CD3 alone. These studies strongly argue that no new signaling pathway is used for CD28
signaling.
NFkB Signaling. Early studies demonstrated
that signaling by the antigen receptor alone was
not sufficient for full activation of T cells
(Rosenstreich and Mizel J. Immunol. 123:1749
(1979)). Soluble factors from macrophages were
found to provide an additional stimulus that was
later shown to be IL-1/IL-6 and/or TNF. These
play critical roles in a wide variety of
inflammatory and immune processes. There are
now at least four homologues including c-rel,
p50, and p65 that are activated in response to a
wide variety of stimuli, but most notably by
TNF, Fas signaling, IL-6, and IL-1. These
proteins are homologous to the fly maternal effect gene dorsal. Like NFkB the major level of control of the protein is
by nuclear localization. In flys nuclear localization of dorsal, the NFkB homologue, is controlled by cactus, which is
homologous to IkB (the inhibitor of kappa B); pelle, which is a serine kinase; and tube which is not similar to anything.
Nuclear localization occures after degradation of IkB. Degradation of IkB is brought about by phosphorylation by the
IKKinase. This kinase can be activated by dimerization of TRAFT a protein that binds to the complex associated with
the TNF receptor.
The cell death pathway: Signaling by Fas, TNF and related molecules
Fas is activated by a cell-cell interaction that is believed to induce clustering (possibly trimerization of the fas
receptor. The receptor forms a scaffold for the binding of TAFF which in turn binds procaspase 8.
Activation of this caspase is aided by cytochrome c from mitochrondria, Apaf-1 and caspase 9 to lead to
8
activation of other caspases and eventually a nuclease that cleaves DNA and results in irreversible genetic
damage and death. Bcl2 prevents oligomerization of Bax, Bak or related molecules and the release of
cytochrome c from mitochrondria thus preventing the activation of caspases. Knockout of cytochrome c
results prevents stress-induced cell death (Cell 101, 389, 2000). Agents that block caspase proteolytic
function also block cell death and hence make good reagents.
How do signaling pathways awaken genetic loci that are deeply embedded within chromatin?
Accumulating evidence indicates that a significant problem in activating genes in response to signaling is to
first make them accessible to the transcription factors controlled by the signaling pathway. Most genes are embedded
in chromatin and are inaccessible to signaling pathways. Hence an important question in signaling is “How do the
incoming signaling molecules gain access to a gene”. The remodeling of chromatin at specific loci in the nucleus
seems to precede the expression of the cell surface receptors that will activate these loci. Thus one of the important
unsolved mysteries of signaling transduction is understanding how a permissive chromatin structure is established at a
specific genetic locus, which then allows a signaling pathway access to that locus.
Resting T cells are the closest thing in mammals to a cellular spore. They have very low rates of transcription
and highly condensed chromatin with small nuclei that that seem impervious to DNA binding proteins and to probes of
DNA accessibility such as DNAse I. Signaling by the antigen receptor results in the rapid decondensation of
chromatin, a step that must precede the activation of genes such as IL-2, 4, CD40L and others that are involved in
coordinating the immune response.
Recent data indicate that signaling by the antigen receptor leads to the activation and chromatin attachment of
chromatin remodeling complexes related to the yeast SWI/SNF complex called the BAF complexes, These complexes
consist of 11 subunits each of which is encoded by a gene family. These subunits are combinatorially assembled and
result in the production of a remarkably heterogenous group of complexes (Cell 95, 625, 1998). The localization of the
BAF complex to chromatin is mediated by PIP2 and dependent on actin, which is a subunit of the complex. It is as yet
not known what targets chromatin remodeling to the correct genetic loci. A likely guess is that specific types of
transcription factors can interact with the chromatin remodeling complexes and hence lead to binding to specific loci.
Cre-lox mediated deletion of Brg1 results in complete arrest at the transition from double negative to double positive
cells indicating that it is required for effective antigen receptor signaling.
Transcription factors with interesting effects on lymphocyte development and activation.
bmi-1. A homologue of the polycomb group, required to maintain stable repression of homeotic genes. Deletion arrest
thymic development at the CD4-, CD8- stage (Genes and Devel. 8:757, 1994).
Hox 1.5. Null mutants have no thymus (Nature 350,473,1991) and also lack other structures such as the thyroid.
HNF-3 homologues- A gene called WHN is mutated in the nude mouse and may be required for the formation of the
thymus. Nature, 1994 Nov 3, 372(6501):103-7
The Ikaros gene is required for the development of all lymphoid lineages. (Cell 1994, 79, 143).
Rel B ablations result in an absence of the thymic medulla. Nature, 1995 Feb 9, 373(6514):531-6.
cRel ablations result in a lack of IL-2 production Genes Dev. 1995 Aug 15;9(16):1965-77.
Ablations of the genes for either pax-5 or E2A block the transition from pro B cells to early B cells. Nature. 1999 Oct
7;401(6753):603-6. Pax 5 appears to be the factor that commits proB cells to the B cell lineage. Nature. 1999 Oct
7;401(6753):556-62
c-myc. This gene is rapidly activated following T cell activation, however its targets are not known with certainly.
Many lines of evidence supports its role in controlling cell cycle entry.
cMyb appears to be essential for normal T cell development and hematopoetic development. Genes and Development,
1994 Apr 1, 8(7):770-82. cMyb is activated by growth factor signaling, however its activation pathway has not been
defined. This is likely to be an exciting problem since a good inhibitor, rapamycin blocks c-myb activation and will
make a great tool to get at this pathway.
c-ets and ets-related proteins. The ets family members have a number of important roles in other process and the
pattern of expression of the ets proteins are interesting. Knockout of ets-1 leads to defective survival and activtion of T
cells.
tal-1 deficient mice lack for all hematopoetic linages.
GATA-3 deficient mice lack normal T cell development and a recent and controversial Cell paper suggest that it is
necessary and sufficient for TH1 development.
9