The Signal to Move: D. discoideum Go Orienteering Alan R. Kimmel and Carole A.

Parent Science 300, 1525 (2003); DOI: 10.1126/science.1085439

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VIEWPOINT

The Signal to Move: D. discoideum Go Orienteering

Alan R. Kimmel1 and Carole A. Parent2*
Cells migrating directionally toward a chemoattractant source display a highly polarized cytoskeletal organization, with F-actin localized predominantly at the anterior and myosin II at the lateral and posterior regions. Dictyostelium discoideum has proven a useful system for elucidating signaling pathways that regulate this chemotactic response. During development, extracellular adenosine 3, 5 monophosphate (cAMP) functions as a primary signal to activate cell surface cAMP receptors (cARs). These receptors transduce different signals depending on whether or not they are coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) (see the STKE Connections Maps). Multiple G proteinstimulated pathways interact to establish polarity in chemotaxing D. discoideum cells by localizing F-actin at their leading edge and by regulating the phosphorylation state and assembly of myosin II. Many of the molecular interactions described are fundamental to the regulation of chemotaxis in other eukaryotic cells. We live in a world where environmental interactions are essential for survival and cells have developed the capacity to exquisitely respond to their dynamic extracellular milieu. Directional response to chemoattractant gradients is crucial for numerous physiological processes. Most remarkably, cells chemotax directionally in very shallow gradients, indicating that the signaling machinery is able to
1 Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, 2Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

*To whom correspondence should be addressed. Email: parentc@helix.nih.gov

respond functionally to concentrations that differ by only 2% across the cell body (1). The social amoebae Dictyostelium discoideum has proven indispensable to elucidate signaling events that regulate chemotaxis. Furthermore, it is particularly amenable to both genetic (its genome sequence is nearly complete) and biochemical analyses (2). D. discoideum grow as single cells, but upon starvation they enter a developmental program where individual cells chemotax into multicellular aggregation centers; development culminates with the formation of terminally differentiated fruiting bodies comprised of spores atop stalks of vacuolated cells (3). The chemotactic behavior of neutrophils is largely similar to aggregating D. discoideum

cells. In the absence of chemoattractant, cells appear round and apolar. However, when exposed to a gradient of chemoattractant they quickly polarize and migrate directionally at speeds approaching 15 m/min (4 ). The most potent chemoattractant for D. discoideum is adenosine 3,5-monophosphate (cAMP), which is produced and secreted endogenously to direct aggregation upon recognition by a specific family of seven transmembrane receptors, the cAMP receptors (cARs). The binding of cAMP to cARs activates various chemotactic and morphogenetic signaling cascades that regulate cytoskeletal organization, gene expression, and the synthesis of additional cAMP for signal-relay. Two STKE Connection Maps accompany this Viewpoint: One focuses on events that are dependent on heterotrimeric G proteins and the other on pathways that take place independently of G proteins (5, 6 ). Because the details of many connections are still emerging, the Connections Maps are by no means comprehensive but are focused on the most prominent features. Additionally, the maps are dynamic; they will be revised and improved as more data become available. Essential to chemoattractant-mediated signaling is the spatial-temporal control of actin polymerization and myosin II assembly. Actin is predominantly polymerized at the

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evidence demonstrating TAK-1 involvement in TLR signaling is still missing. In addition to JNK and p38 MAPKs, TLRs activate ERK1 and ERK2 MAPKs. The mechanism of ERK activation relies on another member of MAPKKK family known as Tpl2 (3). The MyD88-dependent signaling pathway described above is shared by all members of the TLR family and results in the induction of a core set of responses. However, in addition to these stereotyped inflammatory responses, TLRs can also induce appropriate effector responses to distinct types of microbial infections that they recognize. Indeed, one of the most exciting aspects of TLR signal transduction has been the demonstration that differences exist in the gene expression induced by individual TLRs. In particular, analysis of cells from mice lacking MyD88 has demonstrated that TLR3 and TLR4 are capable of inducing certain signaling pathways independent of this adaptor.

Two additional TIR-containing adaptors have been identified. TIR domain containing adaptor protein (TIRAP, also known as Mal) functions downstream of TLR2 and TLR4 but is not involved in signaling by other TLRs (4, 5). TIR domain containing adaptor-inducing IFN- (TRIF, also known as TICAM-1) appears to function downstream of TLR3, and possibly TLR4 (6, 7 ). TRIF appears to be responsible for the induction of interferon (IFN)- and IFN- (IFN/) genes by these TLRs. The induction of IFN/ expression by TLR3 and TLR4 occurs through a MyD88-independent pathway that leads to the activation of interferon regulatory factor 3 (IRF3)a key transcription factor responsible for the induction of IFN genes. Recently, two noncanonical IKKsIKK and TBK-1 have been shown to function downstream of TRIF and upstream of IRF3 (8, 9). These kinases are likely to be responsible for the MyD88-independent induction of NF-B by TLR3 and TLR4 as well (Fig. 1).

The known TLR signaling components still do not explain all of the known differences in signaling between individual TLRs, indicating that additional gene products and signaling mechanisms have yet to be discovered.
References and Notes

1. K. Takeda, T. Kaisho, S. Akira, Annu. Rev. Immunol. 21, 335 (2003). 2. G. Barton, R. Medzhitov, Toll-like receptor pathway. Sci. STKE (Connections Map, as seen April 2003), http:// stke.sciencemag.org/cgi/cm/stkecm;CMP_8643. 3. M. R. Watereld, M. Zhang, L. P. Norman, S. C. Sun, Mol. Cell 11, 685 (2003). 4. M. Yamamoto et al., Nature 420, 324 (2002). 5. T. Horng, G. M. Barton, R. A. Flavell, R. Medzhitov, Nature 420, 329 (2002). 6. M. Yamamoto et al., J. Immunol. 169, 6668 (2002). 7. H. Oshiumi, M. Matsumoto, K. Funami, T. Akazawa, T. Seya, Nature Immunol. 4, 161 (2003). 8. K. A. Fitzgerald et al., Nature Immunol. 4, 491 (2003). 9. S. Sharma et al., Science 300, 1148 (2003); published online 17 April 2003 (10.1126/ science.1081315).

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sively studied. Although adenylyl cyclase A (ACA) activation per se is not essential for chemotaxis, its enrichment at the back of polarized cells helps them align in a head-totail fashion and stream during aggregation, a trick cells use to relay the chemotactic gradient (9). Fine control of chemotaxis does require intracellular cAMP and protein kinase A (PKA) signaling, because cells lacking RegA (an intracellular cAMP phosphodiesterase) or the regulatory subunit of PKA do not suppress lateral pseudopods efficiently (10, 11). The PI3K-PTEN and GC pathways are the most clearly involved in chemotaxis. PI3K and PTEN appear to control events at the leading edge through antagonistic effects on phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) (Fig. 1). Similarly, their cellular distributions are reciprocal; in quiescent cells, PTEN is localized at the membrane, whereas PI3K is cytosolic. In a chemoattractant gradient, however, PI3Ks are directed to the leading edge membrane, whereas PTEN is excluded from the front and shows lateral and posterior targeting (12, 13). Thus, sustained levels of PI(3,4,5)P3 are produced at the leading edge. This, in turn, recruits the pleckstrin homology (PH) domain containing proteins cytosolic regulator of adenylyl cyclase (CRAC) protein kinase B (PKB), and PhdA (PH domain containing protein A), which spatially localize downstream signaling events. Although, the exact mechanisms that couple PI(3,4,5)P3 at the front with actin polymerization have yet to be delineated in D. discoideum, suppressor of cAR (SCAR) and vasodilator-stimulated phosphoprotein (VASP), as well as small guanosine triphosphatases are certainly involved (14 16 ). Interestingly, ACA and p21-activated kinase (PAKa), downstream effectors of the anteriorly localized CRAC Fig. 1. Signal transduction events leading to the spatial and temporal regulation of chemotaxis. A simplied model depicting and PKB, respectively, are the major effectors (in blue) activated by cAMP receptors. highly enriched at the back of Receptor stimulation ultimately gives rise to actin polymeriza- chemotaxing cells (9, 17 ), tion at the front for propulsion, myosin II assembly at the sides underscoring the complex nato suppress lateral pseudopod formation, and myosin II assembly ture of signaling cascades, also at the rear for retraction. The molecular details leading to where events initiated at the the regulation of PTEN, PI3K, GC, and AC by G are still being elucidated. Ras appears to be directly required for the activation front are integrated with comof PI3K, although its role in PI3K localization is not known. Green ponent function at the back. arrows, enzyme activation; blue arrows, membrane localization; D. discoideum that lack red arrows, product relation; dashed arrow, complex regulations the two GCs (sGC and GCA) that have yet to be fully established. (Image) Myosin heavy chain or the cGMP-binding pro(MHC) and actin lament distribution in polarized cells. Cells teins (Gbp) C and D do not expressing MHC fused with green uorescent protein (GFP) were fully differentiated, xed, and stained with tetramethyl rhoda- efficiently move directionally mine isothiocyanate (TRITC)-phalloidin. The MHC-GFP posterior in chemoattractant gradients. signal appears in green, and the actin laments in red. Bar, 10 m. Conversely, cells lacking the

1. S. H. Zigmond, J. Cell Biol. 75, 606 (1977). 2. L. Kreppel, A. R. Kimmel, Nucleic Acid Res. 30, 84 (2002). 3. L. Aubry, R. A. Firtel, Annu. Rev. Cell Dev. Biol. 15, 469 (1999). 4. C. A. Parent, P. N. Devreotes, Science 284, 765 (1999). 5. A. R. Kimmel, C. A. Parent, Dictyostelium discoideum cAMP receptor, cAMP chemotaxis pathway, Sciences STKE (Connections Map as seen June 2003), http:// stke.sciencemag.org/cgi/cm/stkecm;CMP_7918. 6. A. R. Kimmel, C. A. Parent, Dictyostelium discoideum cAMP receptor, G protein independent pathway, Sciences STKE (Connections Map as seen June 2003), http://stke.sciencemag.org/cgi/cm/stkecm; CMP_11471. 7. A. L. Drayer, J. Van der Kaay, G. W. Mayr, P. J. Van Haastert, EMBO J. 13, 1601 (1994).

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front of cells for anterior pseudopod propulsion through a chemoattractant gradient, and myosin II is assembled at the sides to suppress lateral pseudopod formation and at the rear for retraction. The cascade is initiated by the binding of cAMP to cAR1, one of the four cAMP receptors, and the dissociation of the bound heterotrimeric G protein G2, although the downstream signaling events that regulate chemotaxis appear to be primarily mediated by G (5). The binding of cAMP to cAR1 regulates five main effectors: phospholipase C (PLC), adenylyl cyclase (AC), guanylyl cyclase (GC), phosphoinositide 3-kinase (PI3K), and PTEN phosphatase (Fig. 1). The PLC pathway is not essential for chemotaxis (7, 8) cells lacking either PLC or the putative inositol (1,4,5)-phosphate (IP3) receptor show no marked chemotaxis defects, and, in contrast to mammalian cells, the D. discoideum genome does not appear to encode a definitive protein kinase C (PKC). cAMP acts as both a first and second messenger and the pathways that regulate its synthesis and degradation have been exten-

cGMP phosphodiesterases (GbpA and GbpB), which have elevated cGMP, extend fewer lateral pseudopods and display a higher chemotactic (directional) index (18). The cGMP pathway regulates myosin II (heavy and light chain) phosphorylation as well as lateral tension and retraction (18, 19). Cells with reduced levels of cGMP exhibit decreased myosin II phosphorylation and cytoskeletal association in response to chemoattractants, whereas cells with increased cGMP levels have an opposite phenotype. However, because partial myosin II phosphorylation and association persist in cells that lack cGMP, additional regulatory input must exist, including PAKa (17 ). Moreover, cGMP may regulate other targets. In cells chemotaxing in very shallow attractant gradients, the signal detection machinery comprised of cAR1 and G proteins displays nearly uniform distribution and cAMP sensitivity around the periphery of cells (20). Yet, PI3K, PTEN, F-actin, ACA, PAKa, and myosin II are highly localized in these moving cells (Fig. 1). Thus, cells chemotaxing directionally must transduce and amplify weak extracellular concentration differences into highly orchestrated steep intracellular gradients. Although simple stochastic models can be used to explain activation-based polarity asymmetries, receptor-mediated responses are far more complex, transmitting both excitatory and inhibitory (adaptive) signals (4, 20). The adaptive pathways are poorly understood, yet both inhibitory and excitatory signaling may function cooperatively to spatially and temporally localize signaling events (2124 ). Apart from strains fully deficient in factors essential for signal detection (for example, cARs and G), no cell lines exist that cannot chemotax. Even cells lacking the central signaling components PI3K and PTEN respond chemotactically to cAMP, albeit more slowly and less directionally (12, 13). The complexity of the known pathway components (5, 6 ) attests to the highly robust and plastic nature of the chemoattractant response and predicts redundancies for primary and feedback regulation. Clearly, there is still much to uncover.

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8. D. Traynor, J. L. Milne, R. H. Insall, R. R. Kay, EMBO J. 19, 4846 (2000). 9. P. W. Kriebel, V. A. Barr, C. A. Parent, Cell 112, 549 (2003). 10. D. J. Wessels et al., Mol. Biol. Cell 11, 2803 (2000). 11. H. Zhang et al., Eukaryot. Cell 2, 62 (2003). 12. S. Funamoto, R. Meili, S. Lee, L. Parry, R. A. Firtel, Cell 109, 611 (2002). 13. M. Iijima, P. Devreotes, Cell 109, 599 (2002). 14. Y. H. Han et al., J. Biol. Chem. 277, 49877 (2002). 15. J. E. Bear, J. F. Rawls, C. L. Saxe III, J. Cell. Biol. 142, 1325 (1998). 16. A. Wilkins, R. H. Insall, Trends Genet. 17, 41 (2001).

17. C. Y. Chung, G. Potikyan, R. A. Firtel, Mol. Cell 7, 937 (2001). 18. L. Bosgraaf et al., EMBO J. 21, 4560 (2002). 19. M. A. de la Roche, J. L. Smith, V. Betapudi, T. T. Egelhoff, G. P. Cote, J. Muscle Res. Cell Motil., in press. 20. M. Iijima, Y. E. Huang, P. Devreotes, Dev. Cell 3, 469 (2002). 21. A. Levchenko, P. A. Iglesias, Biophys. J. 82, 50 (2002). 22. M. Postma, P. J. Van Haastert, Biophys. J. 81, 1314 (2001). 23. A. Narang, K. K. Subramanian, D. A. Lauffenburger, Ann. Biomed. Eng. 29, 677 (2001).

24. W. J. Rappel, P. J. Thomas, H. Levine, W. F. Loomis, Biophys. J. 83, 1361 (2002). 25. We are extremely grateful to the members of the Kimmel and Parent laboratories. Special thanks to S. Shu and E. Korn for generating the image in Fig. 1. The pathways described rely on the contributions of the many groups studying chemotaxis, and we are indebted to everyone who has generously provided us access to their unpublished data. An integrated genetic and literature database about D. discoideum can be found at http://dictybase. org.

VIEWPOINT

Interleukin-4 and Interleukin-13 Signaling Connections Maps

Ann E. Kelly-Welch,1 Erica M. Hanson,1 Mark R. Boothby,2 Achsah D. Keegan1*
Cytokines are inammatory mediators important in responding to pathogens and other foreign challenges. Interleukin-4 (IL-4) and IL-13 are two cytokines produced by T helper type 2 cells, mast cells, and basophils. In addition to their physiological roles, these cytokines are also implicated in pathological conditions such as asthma and allergy. IL-4 can stimulate two receptors, type I and type II, whereas IL-13 signaling is mediated only by the type II receptor (see the STKE Connections Maps). These cytokines activate the Janus kinase/signal transducer and activator of transcription ( JAK/STAT ) signaling cascades, which may contribute to allergic responses. In addition, stimulation of the phosphatidylinositol 3-kinase (PI3K) pathway through recruitment of members of the insulin receptor substrate family may contribute to survival and proliferation. The cytokines IL-4 and IL-13 are produced by T helper type 2 (TH2) cells in response to antigen receptor engagement, and by mast cells and basophils upon cross-linkage of the high-affinity receptor for immunoglobulin E (IgE) (1, 2). These cytokines can elicit similar responses, many of which are associated with allergy, asthma, and inhibition of autoimmunity. However, IL-4 is more active in regulating TH2 development, whereas IL-13 is more active in regulating airway hypersensitivity and mucus hypersecretion, and TH2type inflammation of the bowel (24). The signal transduction pathways activated by these cytokines, and the mechanisms by which they regulate cell growth, survival, and gene expression, were characterized with long-term cell lines (15) (Fig. 1). More recently, studies of signaling in primary cell types are revealing new signaling rules (5). Because therapeutic strategies targeting IL-4 and IL-13 or their specific signaling pathways are currently being tested to treat aller1 Department of Immunology, Holland Laboratory, American Red Cross, Rockville, MD 20855, and the Institute for Biomedical Sciences, George Washington Medical Center, Washington, DC 20037, USA. 2Department of Microbiology, Vanderbilt University, Nashville, TN 37232, USA.

*To whom correspondence should be addressed. Email: keegana@usa.redcross.org

gy and asthma (3), understanding these differences is critical. However, the differences in the signaling pathways and target genes of IL-4 and IL-13 are not yet clear. IL-4 receptors are composed of two transmembrane proteins. The IL-4R chain binds IL-4 with high affinity, leading to dimerization with another protein to form either a type I or type II receptor. In cells of hematopoietic lineage, the type I receptor arises by recruitment of a common gamma chain (C) (also a component of receptors for IL-2, -7, -9, -15, and -21) (13). In nonhematopoietic cells, the type II receptor is formed by interaction of IL-4R with IL-13R1 instead of C (24 ). IL-13 also binds two different receptor complexes. IL-13 binds to IL-13R1 with high affinity, inducing heterodimerization with IL4R to form a complex identical to the type II IL-4R [see the IL-4 and IL-13 Pathways in the STKE Connections Maps (6, 7 )]. Because IL-4 and IL-13 both use the type II receptor, the molecular basis of the cytokines differing functional responses is unclear. In contrast to its interaction with the IL-13R1, IL-13 binds IL-13R2 with greater affinity than IL-13R1 but fails to induce a signal, so that IL-13R2 acts as a decoy receptor (8). As with other cytokine receptors, the cytoplasmic tails of IL-4 and IL-13 receptor subunits associate with tyrosine kinases of

the Janus family ( JAK 13, TYK2) (17 ). IL-4R associates with JAK1 and C with JAK3, whereas IL-13R1 interacts with either JAK2 or TYK2, but not JAK3. Dimerization of the receptor subunits enhances JAK activity, leading to phosphorylation of tyrosine residues in the cytoplasmic domain of IL-4R. These phosphotyrosines then act as docking sites for signaling molecules containing protein tyrosine binding domains (PTBs) or Src homology 2 (SH2) domains. One critical signaling pathway activated by the IL-4 receptor leads to tyrosine phosphorylation of STAT6, a latent cytoplasmic transcription factor in the family of signal transducers and activators of transcription (STAT) (9, 10). In cell lines, STAT6 is recruited to IL-4R upon phosphorylation of the second, third, or fourth cytoplasmic tyrosine residues (2). STAT6 becomes tyrosine phosphorylated, disengages from the IL-4R, dimerizes by reciprocal SH2 domain interaction, migrates to the nucleus, and binds to consensus sequences found within promoters of IL-4 and IL-13 regulated genes. STAT6 is central in gene regulation and the IL-4 and IL-13regulated allergic responses, including TH2 differentiation, IgE production, and chemokine and mucus production at sites of allergic inflammation (3). STAT6 also regulates gene expression involved in cell survival, including E4 binding protein 4 (E4BP4) and growth factorinduced gene1 (GFI-1) (2, 11), suggesting that the STAT6 pathway helps regulate lymphocyte growth and survival. A second mechanism of signal transduction activated by IL-4 and IL-13 leads to the insulin receptor substrate (IRS) family (17 ), which consists of four proteins (IRS-1 to IRS-4). IRS1, -2, and -3 can interact with IL-4R, but IRS-2 is the main family member expressed in hematopoietic cells. These large cytoplasmic docking proteins contain a PTB domain and

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