Adv Imm 1996 lect 3 and 4



Signaling in the Immune System III

G.R. Crabtree, Feb 20, 2002

hf.grc@forsythe.stanford.edu

File name: Lymphocyte Signaling III

Signal transduction pathways used by cytokine growth factors.

In most cases the cytokines and lymphokines have been shown to induce either dimerization or heterodimerization of their receptors. For example, with the IL-2 receptor, the a and b chains are responsible for high affinity binding. Both must be present to give maximial affinity. However signaling requires the dimerization of the β and the γ chains (Nature 369, 330-3, 1994). Based on the observation that these ligands induce homodimerization, one would expect that the ligands would be homodimers, but in fact they are almost always monomers. (IL-5 is in fact a homodimer). Apparently, the monomeric ligand binds to different regions of the same receptor. This is best illustrated with growth hormone where the entire X-ray structure of the complex is known. Here, different regions of growth hormone interact with different regions of the receptor to induce homodimerization (PNAS 1996 93:1-6).

Dimerization appears to produce a platform upon which the signaling molecules assemble. This is nicely illustrated with the Jak-Stat pathway.

A. The Jak-Stat Pathway. This pathway was initially identified by studies of α and β interferon stimulation of target genes by James Darnell and George Stark. Both investigators identified a responsive element in the nucleus and then used either somatic cell genetics to identify genes necessary for the function of the element (Stark) or biochemistry to identify proteins that bound to the responsive element (Darnell). Their work has resulted in the idenification of a broad family of transcription factors and and kinases that directly carry information from the cytokine receptors to the nucleus in species as diverse as flys and humans. .Mutant IL-2, IL-4 and IL-6 receptors that are unable to bind and activate stats are nevertheless able to induce proliferation suggesting that the Stats may be dedicated to functional rather than proliferative responses.

Proposed Mechanism of JAK Activation (Figure 1)

Ligand binding to the receptor induces dimerization which in turn leads to the formation of a binding site for the Jak kinase. This binding site is usually called the "conserved box 1 motif". The JAK then phosphorylates itself and also the receptor. STATS then bind by their SH2 groups to the tyrosine phosphorylated receptor.

Specificity appears to arise from STAT activation, thereby conveying the specificity of the receptor to the target genes. However recent experiments in which receptors have been exchanged have called this conclusion into question and indicate that spcificity is more complex than simply the use of a particular stat.

Proposed Mechanism of STAT activation (Fig 1). While the STAT is attached to the receptor, it is tyrosine phosphorylated by the JAK. This phosphorylation causes its release from the receptor, its translocation to the nucleus where it dimerizes by a mechanism that requires its SH2 domain and tyrosine phosphorylation. As a homodimer or a heterodimer, it then binds to DNA and activates transcription of the genes that are direct targets of the particular cytokines.

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Thus far four different members of the JAK kinase family have been identified. All are expressed ubiquitiously except for JAK3 which is expressed largely in hematopoetic tissues. Their published interactions with cytokine receptors are outlined in Table I.

The mouse JAK3 ko is associated with a severe combined immunodeficiency and failure of maturation of both T and B cells and a signaling defect in T and B cells (Science 270,800,1995) and a similar defect is seen in humans with a mutations in Jak3 (Nature 376,65, 1995). Note in Table I below that Jak3 interacts with the γ family of signaling subunits of cytokine receptors. A deficiency of the γ subunit is very much like the deficiency of JAK3 and results in a severe combined immunodeficiency (Science, 1993, 262:1877-80). However, a deficiency of the IL-2α chain results in excess proliferation, but no develomental abnormality Thus the phenotype of the ko correlates nicely with the signaling pathway engaged by the particular subunit of the receptor. The fact that null mutations for the IL-2 receptor alpha chain give excessive proliferation probably relates to over compensation at other levels of the signaling pathway for the loss of the alpha subunit.

Table I: Activation of JAK kinases by various ligands

___

JAKs ___

Ligands tyk2 Jak1 Jak2 Jak3

___

IFN family

IFN-α/β + + - - - -

IFN-γ + + +

IL-10 + ? ? - -

GP130 family*

IL-6 + + + ?

IL-11 ? + ? ?

OnM ? + + ?

LIF ? + + ?

CNTF -/+ + + ?

G-CSF ? + ? ?

IL-12*** + - - + - -

γ-C family__________

IL-2 - - + - - +

IL-4 - - + - - +

IL-7*** - - + - - +

IL-9*** - - + - - +

IL-13*** - - + ? ?

IL-15*** ? + ? +

gp140 Family_________

IL-3*** - - - - + - -

IL-5*** - - - - + - -

GM-CSF*** - - - - + - -

Growth hormone

family______________

EPO ? - - + - -

GH ? - - + - -

PRL ? +/- + - -

Receptor tyrosine

kinases______________

EGF ? + + ?

PDGF ? + + ?

CSF-1 ? + +

from Schindler and Darnell (Ann. Rev. Biochem. 1995)

STAT proteins bind and are activated by the combination of a specific receptor and a specific JAK. STAT6 appears to be largely dedicated to IL-4 signaling and genetic defects in its gene are associated with defects in IL-4 signaling (Nature 380 627, 1996).

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Table II: Activation of STAT proteins by various ligands

__

STATs_______________

Ligands 1 2 3 4 5 6 STF

__

IFN family_______

IFN-α + + + _

IFN-γ + _ _ _

IL-10 +/_ _ + _

gp130 family*_____

IL-6 +/_ _ + _

IL-11 +/_ _ + _

OnM +/_ _ + _

LIF +/_ _ + _

CNTF +/_ _ + _

G-CSF +/_ _ + _

IL-12 + +

γ-C family________

IL-2** +/_ + + +a

IL-4 + +b

IL-7 + +a

IL-9 + +a

IL-13 ? +b

IL-15 + +a

gp140 family_______

IL-3 + +c,d

IL-5 + +c

GM-CSF + +c

Growth hormone family

EPO _ _ _ _ ? +e

GH +/_ _ + _ +

PRL +/_ _ _ _ +

Receptor tyrosine

kinases_____________

EGF + _ + _

PDGF + _ + _

CSF-1 + +

_____

Table adapted from Schindler and Darnell (Ann. Rev. Biochem. 1995)

1. Suppressors of Cytokine Signaling (SOCS) In vitro studies have also implicated a family of SH2-containing proteins, the suppressors of cytokine signaling (SOCS) proteins, in the negative regulation of cytokine

signal transduction. Of the eight SOCS proteins (SOCS1 to 7 and CIS), SOCS1 and SOCS3 appear to be the most potent inhibitors of cytokine signaling (Nicholson et al.., EMBO J. 18, 375–385, 1999). SOCS1 was initially identified in a functional screen for proteins capable of inhibiting IL-6 signaling (Starr et al., Nature 387, 917–921, 1997 ). Subsequent studies have shown that STAT activation in response to many cytokines results in increased transcription of the SOCS1 and SOCS3 genes and that, when overexpressed, SOCS1 and SOCS3 inhibit the biological effect of cytokines, including IFN, that act through the JAK/STAT pathway. Consistent with the observation that SOCS1 can inhibit the action of a broad range of cytokines, SOCS1 appears to bind to all four members of the JAK family and inhibit their catalytic activity. These studies have led to the view that SOCS1 may be part of a general negative feedback loop regulating cytokine action. Gene knock outs of the SOS have lead consistently to a phenotype of excessive cytokine senstivity. For example excessive interferon γ sensitivity in SOCS1 mice (Cell 1999 98: 597) SOCS3 deletion results in an embryonic lethality at 12–16 days associated with marked erythrocytosis.(Cell 98, 617–627, September 1999)

B. The Rapamycin-Senstive TOR pathway.

The second phase of T cell activation, the proliferative phase is blocked by a drug called rapamycin at subnanamolar concentrations (The low concentrations necessary for inhibition and the unusal mechanism of action of this drug indicates that rapamycin probably only has one target). Indeed cytokine induced proliferation of most cell types is inhibited by this drug. Rapamycin forms an inhibitory complex with FKBP (the same one that binds FK506) which then binds and inactivates a kinase, FRAP, related to the ATM gene. How FRAP functions in signaling is not known, but it blocks the activity of p70S6kinase (Nature 358, 70 1992) and a molecule that regulates the association of PHAS-1 and eIF-4E, which are involved in protein synthesis (Embo Journal, 1995 14,5701-9; in response to mitogens (Science, 1994 266, 653-6; Nature, 1994, 371,762-7). The block of protein synthesis appears to be very specific in that the synthesis of most proteins are not effected by rapamycin.

The p85 subunit of PI-3 kinase binds to subunits of several cytokine receptors. PI3 kinase has been associated with activation of cell growth by many studies. Although a direct connection has not yet been made, PI3 kinase is likely to be upstream of TOR and FRAP since its activation is inhibited by Wortmanin, an inhibitor of PI3 kinase.

The current evidence suggest the following cascade by which cytokines might control synthesis of specific proteins necessary for cell proliferation.

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Recent studies in yeast indicate that rapamycin might function very much like FK506 and block the nuclear entry of a transcription factor necessary for G1-to-S progress (Nature. 1999 Dec 9;402(6762):689-92). Thus the rapamycin sensitive pathway in yeast may be quite similar to NFAT signaling in mammalian cells (Nature 1991; 352:803-7.).

C. Other Signaling Pathways used by cytokine receptors:

Phosphatase in cytokine signaling

Hematopoietic cell phosphatase plays a negative role perhaps by dephosphoryating JAKs.

Syph, SH, PTP2 or PTP-1D appears to play an activating role like CD45. They bind to certain receptors and perhaps relieve inhibition by dephosphorylating inhibitory phosphotyrosines, such as those on the src-like tyrosine kinases.

Selected Reading.

The first report of programed cell death or apoptosis:

Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972 Aug; 26(4):239-57.

The Cell Death Pathways

Vaux and Korsmeyer Cel 96, 245, 1999

S. Nagata Cell 88,355,1997

Discovery of NFkB signaling (Cell. 1986 Dec 26;47(6):921-8).

Discovery of NFAT signaling (Science. 1988 Jul 8;241(4862):202-5.)

Discovery of STAT/JAK Signaling.

The realization that an interferon inducible nuclear protein bound a common regulatory sequence: Levy et al Genes and Development 2:383 1988

The definition of the strategy for a somatic cell genetic approach to the isolation of genes in the pathway begining with a responsive gene.

Pellegrini et Mol. Cell. Biol 9,4605,1989.

Reviews of the stat-jak pathway

Schindler and Darnell Annu. Rev. Biochem. 1995, 64,621-651

Darnell, Kerr and Stark Science 264, 1415, 1994

The rapamycin sensitive TOR pathway:

Keith CT; Schreiber SL. PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints.

Science, 1995 Oct 6, 270(5233):50-1.

Map Kinase signaling Review

Nature. 2001 Mar 1;410(6824):37-40.

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