The real LAT steps forward

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The real LAT steps forward Doreen Cantrell

Antigen receptors initiate T-cell activation and determine the specificity of the immune response by activating membrane-localized protein tyrosine kinases. Signalling pathways initiated by these kinases control expression of the genes that mediate T-cell effector function. A major challenge in immunology is to work out the route taken by membrane-generated signals as they transit to the nucleus. Substrates for the ZAP70/Syk tyrosine kinases are important, but ‘missing’, links in this process. There has finally been some progress in characterizing one of these important linkers: LAT, an integral membrane protein that acts as an adaptor to couple antigen receptors to intracellular signalling cascades.

The T-cell antigen receptor complex (TCR) controls the activation of T lymphocytes and determines the specificity of T-cell differentiation and proliferation. An immediate consequence of triggering the TCR is the activation of cellular protein tyrosine kinases (PTKs), including p56Lck, p59Fyn and ZAP70. The molecular details of this activation process have been described extensively1. It is also well documented that the role of ZAP70 and Syk (the functional analogue of ZAP70 in B cells and mast cells) is to couple antigen receptors to essential signalling pathways – one mediated by the GTPases p21ras and Rac2,3 and one mediated by Ca2⫹/calcineurin and initiated by the hydrolysis of polyphosphoinositides by phospholipase C␥1 (PLC␥1). Characterization of TCR signaltransduction pathways also identified a mystery 36-kDa tyrosine-phosphorylated protein that seemed to act as a linker between the TCR-activated PTKs, Ras and PLC␥14–8. Zhang, Samelson and coworkers have now isolated and characterized this protein and have shown that it is a substrate for ZAP70/Syk and couples these PTKs to the downstream signalling pathways that regulate transcription factors involved in induction of cytokine gene expression9 (Fig. 1).

receptors/adaptors, forming protein complexes that regulate the membrane localization and catalytic activity of Sos10. Grb2 does not bind with high affinity to the tyrosine-phosphorylated TCR subunits11, and initial efforts to understand the mechanism for Ras activation in T cells proposed that Shc, an adaptor that can bind with high affinity to Grb2 when tyrosine phosphorylated, was involved in the regulation of Grb2–Sos complexes in lymphocytes. In T cells activated by cytokines such as interleukin 2 (IL-2), the formation of Shc–Grb2–Sos complexes can be demonstrated readily12,13. Similarly, in antigenreceptor-activated B cells, tyrosine kinases couple to the Grb2–Sos complex through Shc14. There were also some reports of Shc–Grb2–Sos complexes in TCRactivated cells, and tyrosine phosphorylation of Shc does indeed occur in response to TCR triggering15. Nevertheless, experiments that looked empirically at endogenous Grb2 complexes in T cells identified a 36-kDa tyrosine phosphoprotein as the predominant molecule binding to Grb2 SH2 domains in TCRactivated cells5–7,13. The formation of p36–Grb2–Sos complexes was triggered rapidly by TCR ligation but not in response to cell activation by cytokines or costimulatory receptors16. Thus, p36 seemed to be a selective substrate for antigen-receptor-activated PTKs and was a good candidate for a substrate of ZAP70 in T cells. This possibility made p36 the focus of much attention because physiological substrates for ZAP70 were unknown at the time. Interest in p36 intensified further when it was implicated as an adaptor for a number of different signalling pathways. The p36 molecule itself was identified in other TCR-activated signalling complexes, including those containing PLC␥1 and phosphoinositide 3-kinase (PI 3-K)7. In addition, Grb2 can bind to several proteins through its SH3 domains, including TCR-activated PTK substrates such as cbl and SLP76. Thus, p36–Grb2 complexes have the potential to regulate a variety of signalling molecules other than those of the Sos–Ras pathway. The Grb2–SLP 76 complex was first seen in studies of endogenous p36–Grb2 complexes from T cells derived from peripheral blood. These showed that Grb2 in lymphocytes binds to the Ras GEF Sos but also associates constitutively through its SH3 domains with a 75-kDa protein that is rapidly tyrosine phosphorylated in response to TCR triggering6. This protein was identified subsequently as SLP76, a protein isolated from T-leukaemic cells by its ability to bind to Grb2 SH3 domains17–19. It is now known that tyrosine-phosphorylated SLP76 forms a complex with Vav, a GEF for the Rho-family GTPase Rac-120,21. The formation of this complex is proposed to regulate the cellular localization of Vav, thereby promoting its interactions with Rac and/or tyrosine kinases. The p36–Grb2 complex can therefore link receptors to the Ras signalling pathway through Sos and might indirectly promote activation of Rhofamily GTPases through regulating formation of SLP76–Vav complexes.

Doreen Cantrell is at the Imperial Cancer Research Fund, 44 Lincoln’s Inn Field, London, UK WC2A 3PX. E-mail: cantrell@ icrf.icnet.uk

p36 and TCR signalling Proteins of 36–38 kDa are prominent substrates for TCR-activated PTKs4. However, the special significance of these molecules for TCR signalling was first revealed by experiments examining the mechanisms for regulating the Ras GTPase. The adaptor Grb2 has an evolutionarily conserved role in regulating the activity of Ras. The Ras GDP–GTP exchange factor (GEF) Sos associates constitutively with the Src-homology 3 (SH3) domains of Grb2, and, in activated cells, the Grb2 SH2 domain interacts with tyrosine-phosphorylated

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Cloning p36 Frustratingly, it proved very difficult to isolate p36. There was a flurry of excitement when Lnk, an trends in CELL BIOLOGY (Vol. 8) May 1998

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comment TCR

CD4

PtdIns(4,5)P2

p110 LAT p85

PLC LAT Fyn

PtdIns(4,5)P2

DAG

PKC

Lck ZAP70

IP3

Grb2 Sos Grb2 Vav SLP76

PtdIns(3,4,5)P3

Ras signalling pathways Raf-1/MEK ERK2

Rho/Rac signalling pathways

Ca2+/ calcineurin

Transcription factors and cytokine gene induction FIGURE 1 LAT couples the T-cell antigen receptor complex (TCR) to enzymes that regulate inositol lipid metabolism and integrates signals from the TCR that lead to the activation of GTPase effector pathways. The adaptor Grb2 has an evolutionarily conserved role in activation of Ras. In TCR-activated cells, the Grb2 Src-homology 2 (SH2) domain interacts with LAT, a 36-kDa protein that is rapidly tyrosine phosphorylated in antigen-receptor-activated T cells probably by ZAP70. Grb2 is complexed through its SH3 domains to at least two proteins – the Ras GDP–GTP exchange factor (GEF) Sos and SLP76. SLP76 is also a selective substrate for TCR-activated PTKs and is found in a complex with Vav (a Rac GEF). Tyrosine-phosphorylated LAT can bind to two enzymes that regulate inositol lipid metabolism in T cells: phospholipase C␥1 (PLC) and phosphoinositide 3-kinase (indicated as subunits p85 and p110) and probably plays a role in targeting these molecules to the plasma membrane. Abbreviations: DAG, diacylglycerol; IP3, inositol (1,4,5)-trisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol (4,5)-bisphosphate.

SH2-domain-containing protein of the correct molecular mass for p36 was characterized22. However, Lnk lacked any relevant phosphotyrosine-binding sites for Grb2 and PLC␥1 and was dropped as contender for this crucial T-cell adaptor. The problem was the low level of expression of p36: antiphosphotyrosine western blots of p36 using chemiluminescence technology could detect the protein in cell extracts prepared from 1–2 ⫻ 106 activated T cells – but this was misleading; Zhang et al. now report that purification of sufficient quantities of p36 for amino acid sequencing required 1011 activated T cells9. From partial amino acid sequence information, it was possible to clone the cDNA encoding p36 and establish the complete amino acid sequence of this molecule. The protein isolated by the Samelson group has been called LAT (for: linker for activation of T cells) because it has all the characteristics of a protein that would couple the TCR to downstream signalling pathways. As LAT is an integral membrane protein, it is in the correct intracellular location to act as a linker for TCR-activated PTKs. LAT appears to have a short extracellular ‘domain’ and a long cytosolic tail with nine tyrosine residues conserved between mouse and human. These tyrosine residues include sites that are good consensus docking sites for Grb2 and PLC␥1, indicating that these molecules probably bind directly to p36. LAT complexes with PI 3-K and SLP76 have also been noted, although these associations might not be direct. Expression of a LAT protein mutated in two tyrosines that act as Grb2-binding sites blocks TCR activation of the transcription factors AP-1 and NFAT. Although it remains to be proved formally that LAT is a direct substrate for Syk and ZAP70, clearly LAT is tyrosine phosphorylated in response to activation of these kinases in heterologous cells. trends in CELL BIOLOGY (Vol. 8) May 1998

The future for LAT? LAT will undoubtedly be the focus of attention for investigators seeking to understand antigen receptor dysfunction in T-cell anergy/tolerance. It will also be of interest to see how LAT interprets the different signalling thresholds achieved by antigen receptor occupancy by ligands with different avidity. In addition, the future for LAT will also include analysis of its function in natural killer (NK) cells and mast cells. The name ‘LAT’ infers a T-cell function for this molecule, but it should be remembered that LAT is expressed in mast cells and NK cells9. Indeed, a functionally analogous protein to the T-cell ‘p36’ has been described in mast cells and NK cells: a 33-kDa protein substrate for F⑀R1-activated PTKs couples antigen receptors to Grb2–Sos and Grb2–SLP76 complexes in mast cells23. Similarly, in NK cells, p36–Grb2 complexes have been described. The data presented by Samelson’s group justify naming p36 as a linker molecule. LAT looks like the crucial link between TCRactivated PTKs and the signalling pathways that regulate induction of cytokine gene expression. Further work may well reveal that ‘LAT’ is in fact a ‘LAL’ – linker for activation of lymphocytes. References

Acknowledgements

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I thank Karin Reif, Helen Turner and Narin Osman for their carefully conceived and meticulous experiments characterizing signal-transduction pathways in T cells and mast cells.

Wange, R. L. and Samelson, L. E. (1996) Immunity 5, 197–205 Genot, E. et al. (1996) EMBO J. 15, 3923–3933 Cantrell, D. (1996) Annu. Rev. Immunol. 14, 259–274 June, C. H. et al. (1990) J. Immunol. 144, 1591–1599 Buday, L. et al. (1994) J. Biol. Chem. 269, 9019–9023 Reif, K. et al. (1994) J. Biol. Chem. 269, 14081–14089 Sieh, M. et al. (1994) Mol. Cell. Biol. 14, 4435–4442 Gilliland, L. K. et al. (1992) J. Biol. Chem. 267, 13610–13616 Zhang, W. et al. (1998) Cell 92, 83–92 McCormick, F. (1994) Curr. Biol. 4, 71–76

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11 Osman, N. et al. (1996) Eur. J. Immunol. 26, 1063–1068 12 Ravichandran, K. S. and Burakoff, S. J. (1994) J. Biol. Chem. 269, 1599–1602 13 Osman, N. et al. (1995) J. Biol. Chem. 270, 13981–13986 14 Smit, L. et al. (1994) J. Biol. Chem. 269, 20209–20212 15 Ravichandran, K. S. et al. (1993) Science 262, 902–905 16 Nunes, J. A. et al. (1996) J. Biol. Chem. 251, 1591–1598 17 Motto, D. G. et al. (1994) J. Biol. Chem. 269, 21608–21613

HDAC, a key to pRb tumour suppression? BREHM, A., MISKA, E. A., McCANCE, D. J., REID, J. L., BANNISTER, A. J. and KOUZARIDES, T. (1998) Retinoblastoma protein recruits histone deacetylase to repress transcription Nature 391, 597–601 MAGNAGHI-JAULIN, L. et al. (1998) Retinoblastoma protein represses transcription by recruiting a histone deacetylase Nature 391, 601–605 LUO, R. X., POSTIGO, A. A. and DEAN, D. C. (1998) Rb interacts with histone deacetylase to repress transcription Cell 92, 463–473

This month’s headlines were contributed by Laura Attardi, Jim Brugarolas, Arianne Heinrichs, Tom Misteli, Fiona Townsley and Gary Whittaker.

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The retinoblastoma tumour-suppressor protein (pRb) inhibits progression through the G1 phase of the mammalian cell cycle. pRb acts by inhibiting the expression of cell-cycle regulatory genes upon recruitment to the promoter through interaction with the E2F transcription factor. While originally envisioned to act simply by masking the E2F activation domain, pRb is now recognized to actively repress transcription. pRb represses sometimes through binding and inhibiting neighbouring transcription factors at the promoter and, as shown here, sometimes through interaction with histone deacetylase (HDAC). A variety of repressor proteins have been shown to act by recruiting HDAC to promoters, resulting in histone deacetylation and, consequently, chromatin reorganization and gene silencing. Here, the authors investigated the hypothesis that HDAC mediates pRb repression. They showed that trichostatin A (TSA), an inhibitor of histone deacetylase, relieved pRb repression of a variety of reporter constructs in which pRb was recruited to the promoter, although not in cases in which pRb was known to act by direct binding and inhibition of nearby transcription factors. Various protein– protein interaction assays showed that pRb interacted with HDAC1, but not necessarily directly. Mutations in the pRb pocket domain, such as those found in tumours, abolished the interaction with HDAC1 and blocked repression. The pRb family member p107, by contrast, did not interact with HDAC1, nor was repression by p107 affected by TSA. These data suggest that pRb, but not p107, once bound to E2F, recruits HDAC1, leading to repression. Thus, pRb repression seems to be mediated in two distinct ways: by blocking adjacent transcription factors and by binding

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Motto, D. G. et al. (1996) J. Exp. Med. 183, 1937–1943 Jackman, J. K. et al. (1995) J. Biol. Chem. 270, 7029–7032 Wu, J. et al. (1996) Immunity 4, 593–602 Tuosto, L., Michel, F. and Acuto, O. (1996) J. Exp. Med. 184, 1161–1166 22 Huang, X. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11618–11622 23 Turner, H. et al. (1995) J. Biol. Chem. 270, 9500–9506

HDAC1. Strong in vivo support for this model comes from experiments that showed that some E2F-responsive genes were induced by TSA treatment, whereas others were not. A number of questions still need to be addressed. For example, is the interaction between pRb and HDAC1 direct? Does the ability to recruit HDAC1 also explain the ability of pRB to repress pol I and pol III promoters? What is the significance of the fact that pRb acts through HDAC and p107 does not? Could this difference between pRb, a known tumour suppressor, and p107, which is not a tumour suppressor, reflect the fact that pRb-mediated repression through histone deacetylation is crucial for tumour suppression? Answering these questions will be essential for understanding why pRb loss can lead to cancer.

Lipid kinases help deliver the goods LI, E., STUPAK, D., KLEMKE, R., CHERESH, D. A. and NEMEROW, G. R. (1998) Adenovirus endocytosis via ␣v integrins requires phosphoinositide-3-OH kinase J. Virol. 72, 2055–2061 Adenoviruses are increasingly being used for delivery of heterologous genes into cells and tissues. Because of their promising gene-therapy application, the entry mechanisms of adenoviruses are among the best understood of all viruses. The virus first binds to the cell surface through two functionally linked receptors, CAR (coxsackie-adenovirus receptor) and ␣v integrins. There then follows an integrindependent rupture of the endosome, and a programmed series of uncoating steps leading to the ultimate delivery of viral DNA into the nucleus. Nemerow and colleagues investigate adenovirus endocytosis and the requirement in this process for two molecules involved in a wide variety of cell-signalling events – focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI 3-K). They show that both FAK and PI 3-K become phosphorylated upon adenovirus entry and that PI 3-K becomes activated – a process mediated apparently by the virus penton base protein. Treatment of cells with kinase inhibitors such as wortmannin, and expression of the iSH2 domain of PI 3-K (p85), reduces adenovirus internalization and gene delivery by about three fold. However, the mechanism of entry is different from that of transferrin (Tfn) as iSH2 expression has no effect on Tfn internalization. trends in CELL BIOLOGY (Vol. 8) May 1998