Signalling by Cytokines Brian T. Kawasaki, O. M. Zack Howard, Ana M. Gamero and William L. Farrar National Cancer Institute, Frederick, MD, USA INTRODUCTION Cytokines are small, soluble proteins with a variety of bio- logical effects. Many cell types, not limited to the immune system, secrete these proteins. Cytokines can be grouped into four major families based on structure: haematopoi- etins, IFN, TNF and chemokines. Within the haematopoietin family, groups can be subdivided based on binding to their “common” cognate receptor. For the purposes of brevity, the γ -common (γ c ) chain will be discussed as a model system for the haematopoietins. There are three classes of IFNs (types I, II, and III) and they constitute one of the largest and most divergent subfamily of cytokines. Membrane-bound as well as secreted proteins make up the members of the TNF fam- ily of cytokines, which trimerize upon activation. Lastly, the chemokines form a class of cytokines that bind to a large family of seven transmembrane spanning G-protein-coupled receptors (GPCRs). STRUCTURE AND FUNCTION OF γ C -DEPENDENT CYTOKINES AND RECEPTORS Members of the cytokine haematopoietic superfamily often share a common receptor subunit while retaining their own private receptor subunits. One well-documented example is that six cytokines, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, form one group, which is characterized by using the γ - common (γ c ) chain as a receptor subunit. The common γ c subunit was initially cloned as the γ chain of th IL-2R complex. Soon it was discovered that this γ subunit also participates functionally in the receptors for IL-4, IL-7, IL-9, IL-15, and IL-21 and, therefore, was designated γ c , where c represents “common”. This subunit is constitutively expressed on essentially all cells of haematopoietic origin. It functions to enhance the binding of cytokines to their receptor, presumably by direct interaction with the ligand and to induce intracellular signal transduction events such as Janus-activated kinase – signal transducers and activators of transcription (JAK-STAT) signal pathway (Kovanen and Leonard, 2004). γ c -Dependent Cytokines γ c -Dependent cytokines include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. These peptides or glycoproteins have a molecular mass of 14 – 20 kDa. Crystal structure analysis reveals that IL-2 is an α-helical protein, lacking β -sheet structure, with a four-fork core stabilized by a single intrachain disulphide bond (Wang et al., 2005). IL-4 contains six cysteine residues involved in intramolecular disulphide bridges. The secondary structure of IL-4 reveals a four-helix bundle with a unique up–up–down–down helix topology. IL-7, IL-9, and IL-15 contain a similar α-helical structure (Hage et al., 1999). The most recent addition, IL-21, also contains a 131- residue four-helix bundle cytokine domain with significant sequence homology to IL-2, IL-4, and IL-15 (Parrish-Novak et al., 2000). Each cytokine is secreted by particular cell types in response to a variety of stimuli and produces a characteristic constellation of effects on the growth, motility, differentiation or function of its target cells. These cytokines affect multiple biological functions (Table 1). IL-2, IL-4, IL-9, and IL-21, all produced by activated T cells, are important immune regulatory cytokines, whereas IL-7 and IL-15, which nonlymphoid cells primarily produce, have also been implicated in the regulation of lymphocyte development (Kovanen and Leonard, 2004). γ c -Dependent Cytokine Receptors The γ c subunit belongs to the cytokine receptor superfamily. All members of this superfamily are type I membrane glyco- proteins with a single hydrophobic transmembrane domain containing an evolutionary-related extracellular region or cytokine homology region (CHR) that results in a conserved structural fold for binding to helical cytokines. The CHR contains two major regions of homology. The first region includes four cysteine residues located in the N-terminal half The Cancer Handbook 2nd Edition. Edited by Malcolm R. Alison 2007 John Wiley & Sons, Ltd.
Transcript
Signalling by CytokinesBrian T. Kawasaki, O. M. Zack Howard, Ana M. Gamero and William L. Farrar
National Cancer Institute, Frederick, MD, USA
INTRODUCTION
Cytokines are small, soluble proteins with a variety of bio-logical effects. Many cell types, not limited to the immunesystem, secrete these proteins. Cytokines can be groupedinto four major families based on structure: haematopoi-etins, IFN, TNF and chemokines. Within the haematopoietinfamily, groups can be subdivided based on binding to their“common” cognate receptor. For the purposes of brevity, theγ -common (γc) chain will be discussed as a model system forthe haematopoietins. There are three classes of IFNs (typesI, II, and III) and they constitute one of the largest and mostdivergent subfamily of cytokines. Membrane-bound as wellas secreted proteins make up the members of the TNF fam-ily of cytokines, which trimerize upon activation. Lastly, thechemokines form a class of cytokines that bind to a largefamily of seven transmembrane spanning G-protein-coupledreceptors (GPCRs).
STRUCTURE AND FUNCTION OF γC-DEPENDENTCYTOKINES AND RECEPTORS
Members of the cytokine haematopoietic superfamily oftenshare a common receptor subunit while retaining their ownprivate receptor subunits. One well-documented example isthat six cytokines, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21,form one group, which is characterized by using the γ -common (γc) chain as a receptor subunit. The common γcsubunit was initially cloned as the γ chain of th IL-2Rcomplex. Soon it was discovered that this γ subunit alsoparticipates functionally in the receptors for IL-4, IL-7,IL-9, IL-15, and IL-21 and, therefore, was designated γc,where c represents “common”. This subunit is constitutivelyexpressed on essentially all cells of haematopoietic origin.It functions to enhance the binding of cytokines to theirreceptor, presumably by direct interaction with the ligandand to induce intracellular signal transduction events suchas Janus-activated kinase–signal transducers and activators
of transcription (JAK-STAT) signal pathway (Kovanen andLeonard, 2004).
γc-Dependent Cytokines
γc-Dependent cytokines include IL-2, IL-4, IL-7, IL-9, IL-15,and IL-21. These peptides or glycoproteins have a molecularmass of 14–20 kDa. Crystal structure analysis reveals thatIL-2 is an α-helical protein, lacking β-sheet structure, witha four-fork core stabilized by a single intrachain disulphidebond (Wang et al., 2005). IL-4 contains six cysteine residuesinvolved in intramolecular disulphide bridges. The secondarystructure of IL-4 reveals a four-helix bundle with a uniqueup–up–down–down helix topology. IL-7, IL-9, and IL-15contain a similar α-helical structure (Hage et al., 1999).The most recent addition, IL-21, also contains a 131-residue four-helix bundle cytokine domain with significantsequence homology to IL-2, IL-4, and IL-15 (Parrish-Novaket al., 2000). Each cytokine is secreted by particular celltypes in response to a variety of stimuli and produces acharacteristic constellation of effects on the growth, motility,differentiation or function of its target cells. These cytokinesaffect multiple biological functions (Table 1). IL-2, IL-4,IL-9, and IL-21, all produced by activated T cells, areimportant immune regulatory cytokines, whereas IL-7 andIL-15, which nonlymphoid cells primarily produce, have alsobeen implicated in the regulation of lymphocyte development(Kovanen and Leonard, 2004).
γc-Dependent Cytokine Receptors
The γc subunit belongs to the cytokine receptor superfamily.All members of this superfamily are type I membrane glyco-proteins with a single hydrophobic transmembrane domaincontaining an evolutionary-related extracellular region orcytokine homology region (CHR) that results in a conservedstructural fold for binding to helical cytokines. The CHRcontains two major regions of homology. The first regionincludes four cysteine residues located in the N-terminal half
The Cancer Handbook 2nd Edition. Edited by Malcolm R. Alison 2007 John Wiley & Sons, Ltd.
Program T cells for apoptosisReverse T-cell anergy
IL-4 20 Activated TH2 cells T-cell growthMast cells B-cell growthBasophils IgG1 and IgE class switchMK1 + CD4 + T cells Enhance expression of MHC class II and CD23
IL-7 17 Bone marrow stroma T-cell growthThymic stromal cells Proliferation of pre-B cellsIntestinal epithelial cells Viability of TN thymocytesKeratinocytes Promotes development of CTLc
IL-9 14 Activated T cells Promotes the growth of mast cellsEnhances mast cell secretion of IL-6 and expression of granzyme A and Band FcRε
of the extracellular domain. The second region of homology,the “WS motif” encodes the amino acids, Trp-Ser-X-Trp-Ser (WSXWS). This motif lies close to the transmembraneregion and probably serves as the main ligand-binding site.The C-terminal half of the extracellular domain is com-posed of fibronectin type III domains found in a series ofcell surface molecules with adhesive properties; however,the functional significance of these domains remains to beclarified (Bazan, 1990). The γc-dependent cytokine receptorsare heterodimeric (IL-4R, 7R, 9R, and 21R) or heterotrimeric(IL-2R and 15R). In addition to the γc subunit, each recep-tor contains a subunit that is also a member of the cytokinesuperfamily. The third subunit of the IL-2R and IL-15R (IL-2Rα and IL-15Rα) is not related to the cytokine superfamily,but may also contribute to cytokine binding affinity (Minamiet al., 1993).
JAK–STAT SIGNAL PATHWAY
All known γc-containing receptors signal through the asso-ciated Janus protein-tyrosine kinases, JAK1 and JAK3 pro-teins, although not all γc-dependent cytokines activate thesame STAT molecules (see the following text). Phospho-rylated tyrosines and flanking amino acid residues in theactivated cytokine receptors determine this specificity by pro-viding specific docking sites for the SH2 domains of STATs.Most likely, JAKs mediate tyrosine phosphorylation of thereceptor proteins. The JAK/STAT signal pathway, therefore,connects activation of the receptor complexes directly totranscription of genes. Upon receptor oligomerization, JAKsare activated, presumably by trans-”auto” phosphorylation
on tyrosines. Subsequently, JAKs phosphorylate STAT pro-teins, which form homodimeric or heterodimeric complexesthrough their SH2 domains. These complexes translocate tothe nucleus, where they bind to specific targeting sequencesand influence gene transcription (see Figure 1 and IFN sig-nalling) (Horvath and Darnell, 1997).
Janus Kinases
The JAKs are cytoplasmic tyrosine kinases, which mediatesignalling from a number of cell surface receptors, which lackintrinsic tyrosine kinase activity. Four mammalian membersof the JAK family are known, JAKs 1–3, and TYK2 (Ihle,1995). Whereas JAK1, JAK2, and TYK2 are expressedubiquitously, expression of JAK3 is confined to myeloid andlymphoid cells. Characteristic of the structure of JAKs is thepresence of seven JAK homology (JH) domains, of whichthe C-terminal (JH1) domain has tyrosine kinase activity.The N-terminal JH7 domain associates with proline-richconserved BOX1/BOX2 regions found on the cytoplasmicside of cytokine receptors (Kisseleva et al., 2002). Studies ofknockout mice provided important insights into the functionof JAKS in vivo. The JAK1 and JAK2 knockout mice arenot viable, but the JAK3 knockout survives and is fertile.The JAK1 and JAK2 knockouts show profound defectsin lymphoid development and erythropoiesis, respectively,demonstrating a profound role in cytokine signalling (Rodiget al., 1998; Neubauer et al., 1998). The JAK3 survival canbe associated with its limited expression pattern. However,these mice suffer from severe combined immune deficiency(SCID) (discussed in the following text) that affects Tcells, B cells, and natural killer (NK) cells. Interestingly,
Figure 1 Signalling of γc-dependent receptors through the Janus-activated kinases-signal transducer and activator of transcription (JAK-STAT) signallingpathway. In this illustration, IL-2/IL2R complex is given as a prototypical example of γc-cytokine signalling. Ligand binding to its cognate receptor causestrimerization of the receptor complex followed by activation of JAK. JAK kinases, in turn, phosphorylate cellular substrates, including at least one of thereceptor chains. This allows recruitment of STAT proteins to a phosphorylated SH2 domain of the receptor and subsequent phosphorylation of the STATsby the activated JAKs. The STAT proteins dimerize, translocate to the nucleus, bind DNA and induce transcription. Unphosphorylated STATs can alsotraffick between the nucleus and cytoplasm in the absence of cytokine signalling (Zeng et al., 2002).
the γc-knockout phenotype is virtually indistinguishablefrom JAK3-deficient mice. This similarity strongly suggeststhat the major role of γc is the recruitment of JAK3to each γc-receptor (Nosaka et al., 1995). In many cases,other JAKS, such as JAK1, found in association with theadditional subunits of γc-containing cytokine receptors, donot sufficiently initiate signalling. For example, embryonicfibroblasts from JAK1 knockout mice do not respond toclass II cytokine receptor ligands, Ifnγ and Ifnα (Mulleret al., 1993).
Signal Transducer and Activator of Transcription(STAT)
The STATs constitute a family of signal transduction proteinsthat are activated in the cytoplasm by the binding of extra-cellular polypeptides to transmembrane receptors such ascytokines binding to their cognate receptor. Following theirobligatory tyrosine phosphorylation, induced by a cytokineligand, STATs dimerize, translocate to the nucleus and binddirectly to response elements present in the promoters of tar-get genes in order to trigger induction of transcription. Thusfar, six mammalian STATs (1–6) proteins plus two isoformshave been identified (Darnell, 1997). Encoded by differentgenes, two homologues of STAT5 (STAT5A and STAT5B)
exist. Expression of STAT proteins is ubiquitous, exceptfor STAT4, which is expressed in several tissues includingspleen, heart, brain, peripheral blood cells and testis. MostSTATs can be activated by many different ligands. IL-2, IL-7,IL-9, and IL-15 activate STAT3 and STAT5, in contrast toIL-4, which only activates STAT6. IL-21 activates STAT1,STAT3 and to a lesser extent STAT5A and 5B (Kisselevaet al., 2002). STAT knockout mice show defects in a vari-ety of cytokine-dependent processes that affect both immuneand non-immune processes. STAT1 and STAT2 knockoutmice show defects in IFN-α/β signalling, which promotessusceptibility to viral infections (Durbin et al., 1996; Parket al., 2000). The STAT3 knockout is embryonic lethal, buta conditional knockout of STAT3 in monocytes indicatesSTAT3 mediates the suppressive effects of IL-10 duringan inflammatory response in macrophages and neutrophils(Takeda et al., 1997; Takeda et al., 1999). STAT4-deficiencyresults in unresponsiveness to IL-12 coinciding with a Th1response impairment (Kaplan et al., 1996; Thierfelder et al.,1996). STAT5A and 5B knockouts appear to be related toterminal differentiation and tissue specific gene expression.For example, STAT5A and STAT5B double knockout miceshow loss of function with regard to prolactin and growthhormone receptors causing disturbed ovary and mammarygland development and growth retardation. In addition, these
mice lack NK cells, develop splenomegaly and developT cells with proliferation problems (Moriggl et al., 1999).STAT6-deficient mice have impaired IL-4 and IL-13 sig-nalling that promotes defects in Th2-response development(Takeda et al., 1996).
DISEASES ASSOCIATED WITH PERTURBATIONSIN γC-RECEPTOR/JAK/STAT SIGNALLING
Because γc-dependent cytokines orchestrate a variety ofimmune system responses by activating the γc-receptor/JAK/STAT signalling pathway, it is not surprising that mostcircumstances causing an inappropriate inhibition of thissignalling pathway have genetically immunosuppressive con-sequences. A number of pathological conditions have beenidentified with mutations or deregulation in γc-cytokinereceptors or associated signalling molecules.
SCID
SCID is a hereditary human disease characterized by thecrippling of the adaptive immune response. There are sev-eral forms of SCID, categorised on the basis of the presenceor absence of T, B and NK cells. The most prevalent form, X-linked SCID (X-SCID), which affects more than 50% of allcases, is characterized by a lack of T and NK cells, whereasthe B-cell number is normal (Noguchi et al., 1993; Pucket al., 1993). The most famous case being David Vetter, thebubble boy, who lived for more than 12 years in a germ-free,protective “bubble” environment. The ensuing susceptibil-ity to opportunistic infections is the most prevalent causeof premature mortality in young patients suffering from thisdisease. X-SCID associates with mutations (chromosomallymapping to Xq13) in the γ -chain of the IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors (Leonard, 2001). Strikingly,a form of autosomal SCID exists with clinical symptomsidentical with X-SCID, in which the gene encoding JAK3 isaffected. Another form of SCID with slightly different clin-ical features involves mutations in the IL-7 receptor chain.Here, the patients have defects only in T-cell development,whereas B and NK cell development is normal (Giliani et al.,2005). SCID mice show a different molecular phenotype.Mice deficient in the γc subunit indicate the development ofmultiple lymphoid lineages but not myeloid and erythroidlineages requires signalling through γc. In the mouse, B-celldevelopment is substantially inhibited at the pro-B-cell stage,whereas in human X-SCID, the production of B cells is out-wardly normal. X-SCID mice show hypoplastic thymusesand reduced thymic cellularity (10–25-fold) compared withnormal littermates (Noguchi et al., 1993). The IL-7/IL-7Rknockout appears to follow the γc-dependent cytokine formouse B-cell development; however, their NK cell develop-ment was otherwise normal (He and Malek, 1996). The fail-ure to block B-cell development in human X-SCID suggestsa γc-independent or redundant pathway for the production
of B cells in these patients whereas in mice it is absolutelynecessary. In summary, mouse models can be useful pre-dictors of defects that would be observed in humans butcaution must be employed as differences will exist (Leonard,2001).
Immunosuppressive Diseases and Suppression ofJAK/STAT Signal Transduction
Cytokine receptor–signalling substrates, in particular theJAKs and STATs, contribute to tumourigenesis. JAKsand STATs are known to be constitutively activated inhaematopoietic cells transformed by diverse oncogenic tyro-sine kinases and in a variety of lymphomas and leukaemias.STAT3 association with many different types of canceris well documented, including multiple myeloma, T-cellcutaneous lymphoma and chronic myelogenous leukaemia(CML). STAT3 is implicated in a number of solid tumoursas well, including lung, breast, pancreas and prostate (Hodgeet al., 2005). Together these data indicate a role for the JAK-STAT pathways in tumourigenesis as well as possible targetsof prevention and therapy.
INTERFERON SIGNALLING PATHWAY
IFNs exhibit a wide variety of biological activities includingantiviral and antiproliferative effects and modulation ofinnate and adaptive immunity (Pestka et al., 2004). Thesecytokines have a molecular weight of 20–25 kDa. Studies ofhow IFNs activated gene transcription led to the discovery ofthe classical JAK-STAT pathway and uncovered the existenceof a signalling network that is common to other cytokines(Darnell et al., 1994). IFNs are a large family of cytokinesthat are divided into three functional groups that bind to acommon receptor: types I, II, and III. Type I IFNs representthe largest group with more than 20 members in its class(e.g., IFNα, -β, and -ω), type II IFN consists of a singlemember (IFN-γ ), and the recently identified type III IFNclass consists of three members (IFN-λ1, -λ2, and -λ3 alsoreferred as IL-28A, IL28B, and IL-29) (Kontsek et al., 2003).Signalling with all three types of IFNs begins at the cellsurface when IFNs bind to their specific membrane receptorsresulting in gene activation.
Interferon-γ Signalling Pathway
IFN-γ also known as immune or type II IFN is produced byT cells (both CD4 and CD8), NK, and natural killer T (NKT)cells in response to immune and inflammatory stimuli (Bironand Brossay, 2001; Young and Hardy, 1995). Activationof the IFNγ signalling cascade involves the participationof a pre-assembled complex consisting of five distinctcomponents: two transmembrane receptor chains (IFNGR1and IFNGR2), two Janus kinases (JAK1 and JAK2), andSTAT1 (Schroder et al., 2004).
IFN-γ receptors belong to the class II cytokine receptorfamily and are constitutively expressed on virtually all celltypes (Bach et al., 1997). Both receptor chains lack intrinsickinase and phosphatase activity. The 90-kDa IFNGR1 isconstitutively associated with JAK1 and contains a STAT1binding and tyrosine phosphorylation site that is activatedfollowing phosphorylation by JAK1. In contrast, the 62-kDaIFNGR2 is constitutively associated with JAK2. Expressionof both IFNGR1 and IFNGR2 is required to activate theIFN-γ signalling pathway. While IFNGR1 is the ligand-binding receptor, IFNGR2 is required for proper assemblingof the receptor complex for signal transduction (Sakatsumeet al., 1995).
Genetic and biochemical observations led to the pro-posal of a model that is widely accepted for IFN-γ sig-nalling that is mediated by the JAK-STAT pathway (Figure 2)(Stark et al., 1998). A biologically active IFN-γ homod-imer binds two IFNGR1s on their extracellular domains.As each IFNGR1 chain binds to one IFNGR2, the sta-ble heterodimeric receptor is brought into close proxim-ity, together with their pre-associated, inactive JAK1 andJAK2. The intracellular domains of both receptors openand induce JAK2 auto-phosphorylation and the subse-quent JAK1 trans-phosphorylation by JAK2. Activated JAK1phosphorylates tyrosine residues of each IFNGR1 chain,which serve as docking sites to recruit inactive monomericSTAT1. STAT1 binds to this site through its SH2 domain
and becomes phosphorylated on tyrosine residue 701 byJAK1. This causes STAT1 to dissociate from the receptorand form a reciprocal homodimer through SH2 domain-phosphotyrosyl interactions. STAT1 homodimers translo-cate to the nucleus and bind to a specific DNA elementdesignated as IFN-gamma activated site (GAS) or IFN-gamma responsive element (GRR) found within promot-ers of a subset of IFN-stimulated genes (ISGs) to activategene transcription (Figure 2) (Pearse et al., 1991; Deckeret al., 1991).
The JAK-STAT signalling pathway is not unique to IFNsas other cytokines can activate a similar signal transduc-tion network by inducing the tyrosine phosphorylation of adocking site on a receptor and recruitment of specific mem-bers of the STAT family of transcription factors (Leonardand O’Shea, 1998). Consequently, cytokine-induced activa-tion of the JAK-STAT signalling pathway has become theestablished signal transduction model that demonstrates howcytokine receptors when coupled to specific JAKs and STATsdeliver intracellular signals to the nucleus to mediate geneactivation.
Distinct mechanisms exist to regulate the IFN-γ signallingpathway. Expression of specific members of the family ofSOCS/JAB/SSI proteins, induced by IFN-γ and the protein-tyrosine phosphatase (PTP) SHP2 can bind to and inhibitactivated JAKs (Alexander et al., 1999; You et al., 1999).In the nucleus, the PTP Tc-PTP, tyrosine dephosphorylates
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Figure 2 IFNγ signalling through JAK-STATs (see text).
Figure 3 IFN-(α/β) signalling through JAK-STATs (see text).
STAT1 (ten Hoeve et al., 2002), while PIAS1 interferes withSTAT1 binding to promoters of ISGs (Liu et al., 1998, 2004).These discoveries have broadened our understanding on howactivation of the JAK-STAT pathway in response to specificstimuli is a tightly regulated event.
Interferon-α/β Signalling Pathway
IFN-α/β are produced by most cell types in response to awide array of biological stimuli that include viruses, bacteria,double-stranded RNA, and mitogens. Activation of the IFN-α/β signalling pathway is controlled by seven components:two distinct transmembrane receptor chains (Interferon alphareceptor 1 and 2 IFNAR1 and IFNAR2), two JAK kinases(JAK1 and TYK2), two STATs (STAT1 and STAT2), and thetranscription factor p48/IRF9.
Both chains that comprise the IFNα/β receptor complexdesignated as IFNAR are ubiquitously expressed on all celltypes. All type I IFNs-α, -β, and -ω) recognize and bindto the same receptors (Pestka et al., 2004). The 110-kDaIFNAR1 is a single chain, whereas the IFNAR2 with amolecular weight of 55–100 kDa exists in three differentiallyspliced products of the same gene that differ in the lengthof the cytoplasmic and transmembrane domains: IFNAR2a(short), IFNAR2b (soluble), and IFNAR2c (long) (Domanskiand Colamonici, 1996). IFNAR2c is the high binding affinityligand chain that when dimerized with IFNAR1 forms afunctional receptor complex for signal transduction.
The established model for IFNα/β signalling is illustratedin Figure 3. Binding of an IFNα/β monomer to the IFNARcomplex induces dimerization of the two receptors chains.The IFNAR1 and IFNAR2c chains are constitutively associ-ated with Janus kinases, JAK1 and TYK2, respectively. JAKsbecome activated and tyrosine phosphorylate IFNAR1 andIFNAR2c on specific tyrosine residues. STAT1 and STAT2are recruited to the receptor through their SH2 domain andbecome phosphorylated on tyrosine 701 and tyrosine 690,respectively, by JAKs. Activated STATs dissociate from thephosphorylated IFNAR complex and assemble as STAT1homodimers or STAT1/STAT2 heterodimers and translo-cate to the nucleus (Stark et al., 1998). STAT1/STAT2 het-erodimers associate with the DNA binding subunit p48/IRF9to form the ISGF3 complex that binds to a specific DNAsequence element known as IFN-stimulated response element(ISRE ) found in the promoters of a subset of ISGs to initi-ate gene transcription (Qureshi et al., 1995; Fu et al., 1990).In contrast, STAT1 homodimers assemble independently ofIRF9 and bind to a distinct DNA element designated as GASto drive the expression of a different subset of ISGs (Deckeret al., 1991). IRF9 serves as an adaptor molecule that stabi-lizes the binding of ISGF3 to the ISRE element to activateexpression of selective ISGs (Horvath et al., 1996).
IFN-λ Signalling Pathway
IFN-λs, also referred as interleukin (IL)-28 and IL-29, wererecently discovered as a class of cytokines that exhibit
antiviral and antiproliferative activities (Kotenko et al., 2003;Sheppard et al., 2003; Dumoutier et al., 2004). While IFN-λis produced by peripheral blood mononuclear cells and den-dritic cells in response to viral infection, only a restrictedpanel of cell lines respond to IFN-λ stimulation (Mea-ger et al., 2005). IFN-λ binds to a heterodimeric classII cytokine receptor complex, IFNLR, which consists ofa ligand-binding chain IFNLRI (also known as IL-28Rα)and IL-10 receptor β chain (IL-10Rβ). Limited informa-tion is available regarding the signal transduction pathwayof IFNλ. Thus far, IFNλs appear to signal through activationof the JAK-STAT pathway. The IFNLRI is pre-associatedwith JAK1 and contains three important tyrosine residuesthat are essential for STAT activation (Dumoutier et al.,2004). Binding of IFN-λs to their specific receptor leads totyrosine phosphorylation of IFNLRI and JAK1 activation.STAT1 and STAT2 are recruited to the IFNRL complex,become tyrosine phosphorylated by JAK1, and assembleas STAT1 homodimers or STAT1/STAT2 heterodimers thattranslocate to the nucleus. IFNλ and IFNα/β share somesimilarities. IFN-λ stimulated formation of STAT1 homod-imers bind the GAS sequence, whereas STAT1/STAT2 het-erodimers in association with IRF9 form the ISGF3 complexand bind the ISRE element to activate gene transcription ofISGs that mediate the biological actions of IFNλs (Kotenkoet al., 2003).
TUMOUR NECROSIS FACTOR: RECEPTORSAND SIGNAL TRANSDUCTION PATHWAYS
The TNF superfamily of cytokines comprises a group ofsecreted and membrane-bound proteins that interact withtheir cognate cell surface receptor, which mediates a host ofbiological responses including cellular differentiation, deathand survival. One of the first two members of the TNF super-family identified were TNF (formally known as TNFα) andlymphotoxin-α (LTα) (Carswell et al., 1975; Granger et al.,1969). These two proteins are the prototype members ofa large family of related proteins, which includes CD30,CD40, CD70, Fas ligand, TNF-related apoptosis-inducingligand (TRAIL), OXO-4, and LIGHT. TNF is a major phys-iological mediator of inflammation. It initiates the responseto gram-negative bacteria that produce lipopolysaccharide(LPS). TNF induces fever, activates the coagulation sys-tem, induces hypoglycaemia, depresses cardiac contractility,reduces vascular resistance, induces cachexia, and activatesthe acute phase response in the liver (Odeh, 2001; Traceyet al., 1986).
Interestingly, attempts to use TNF in the clinic actu-ally predate its discovery and characterization. Towards theend of the nineteenth century, a small number of cancerpatients experienced disease regression after suffering sys-temic bacterial infections. Subsequently a mixture of killedStreptococcus pyogenes and Serratia marcescens (“Coley’stoxins”) were administered to patients with advanced cancer,albeit with very limited success. This approach became thetreatment of choice for over three decades until superseded
by advances in radiotherapy, chemotherapy, and surgery.With hindsight, the most likely explanation for the resultsobserved with Coley’s toxins was the production of TNF,largely by macrophages in response to bacterial LPS presentin the cell wall of gram-negative bacteria such as Serratiaspecies (Carswell et al., 1975).
TNF, LTα, and their Receptors
TNF and LTα are closely related homotrimeric proteins(32% identity). Human TNF is synthesized as a 233 aminoacid glycoprotein, containing a long (76 residue) N-terminalleader sequence, which anchors it to the cell membrane asa 25-kDa type II membrane protein. A secreted 17-kDaform of TNF is generated through the enzymatic cleavageof membrane-bound TNF by a metalloproteinase termedTNF-alpha-converting enzyme (TACE). Both soluble andmembrane-bound forms of TNF are biologically active,although they have different affinities for the two TNFreceptors (R1 and R2, see the following text), and probablyas a consequence exhibit different biological properties(Hehlgans and Pfeffer, 2005).
LTα differs from TNF in that it is synthesized as asecreted glycoprotein. Human LTα is synthesized as a 205amino acid glycoprotein, which in native form exists asa 25-kDa homotrimer. LTα can bind both TNF receptorswith affinities comparable to those of TNF, and has similarbiological effects. However, a membrane-bound form ofLT has been identified, which consists of a heterotrimericcomplex containing one LTα subunit noncovalently linkedto two molecules of an LTα-related type II membraneprotein termed LTβ. The LTα1β2 heterotrimer (also knownas mLT) is not cleaved by TACE and is thought to existexclusively as a membrane-bound complex. Membrane-LTdoes not bind either of the two TNF receptors, but ratherexerts its effects on another member of the TNF receptorsuperfamily, the lymphotoxin β receptor (LTβR). TNFand the two LT subunits are encoded by closely linkedsingle copy genes, which are situated in the class IIImajor histocompatibility locus, within a 25-kb region on theshort arm of chromosome 6 in humans, at P21 (Locksleyet al., 2001).
The two receptors for TNF (and LTα) are type I trans-membrane glycoproteins designated tumour necrosis factorreceptor (TNFR) 1 (also termed P60 in humans, P55 inmice) and TNFR2 (also known as P80 in humans, P75 inmice). These receptors are characterized by six cysteine-rich pseudorepeats spanning 40 amino acids in their N-terminal extracellular domains. These cysteine-rich domainsare involved in intrachain disulphide bonds and are thesignature hallmark of the TNFR superfamily. The cyto-plasmic domains of these receptors lack intrinsic enzy-matic activity and are thought to recruit and activate adap-tor proteins to propagate their signal. Signal transductionis therefore achieved by the recruitment and activationof adaptor proteins, which recognize specific sequencesin the cytoplasmic domains of these receptors. Signallingoccurs through two principal classes of adaptor molecules:
death domain (DD) proteins and TNF receptor-associatedfactors (TRAFs) (Dempsey et al., 2003). Recruitment ofadaptor molecules activates a number of characteristic sig-nalling pathways that can lead to a remarkably diverseset of cellular responses including differentiation, activa-tion, release of inflammatory mediators and apoptosis (seealso Apoptosis).
Signal Transduction: DD Proteins and TRAFs
Death Domain Proteins, TRADDs, and FADDs
TNFR-associated death domain (TRADD) and Fas-associated death domain (FADD) are intracellular DD-containing adaptors that can cause activation of thecaspase cascade (see the following text) and subsequentinduction of apoptosis. The principal molecule involved inTNFR1 signal transduction is TRADD, which is recruited to
TNFR1 after activation by TNF. The interaction betweenTNFR1 and TRADD is mediated by the DD, a motiffound in both adaptor molecules and the cytoplasmicdomains of the receptor itself (Figure 4). The binding ofTRADD to TNFR1 leads to recruitment and activationof numerous associated signalling molecules. TNF-inducedapoptosis is generally thought to be achieved by theinteraction of TRADD with FADD, which oligomerizes withTRADD through the DDs contained in both molecules.Recruitment of FADD activates a cascade of events whichultimately lead to apoptosis (Dempsey et al., 2003). Severalmembers of the caspase family bring about this coordinatedactivation.
Caspases are cysteine aspartate proteases, which origi-nally synthesize as zymogens, and typically convert to theiractivated form by proteolytic cleavage, often by a distinctcaspase upstream in the proteolytic cascade. Caspase-8, gen-erally considered the apical caspase in the TNF and Faspathways, recruits to FADD in the activated complex, and
TNFR1
TRAF2
TNFR2
TIM
DD
DD
DDFADD
TRADD
Caspase-8
Caspase-3
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RIP
clAP TRAF1
IKK-a
NF-κB(P65)
1-κB
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Survival
P
Degradation
Ubiquitin
Figure 4 TNFR1 and R2 signalling through DD- and DD-containing adaptor molecules. Receptor association with FADD adaptor molecules throughdead domains (DD) signals cellular apoptosis. Association with TRADD adaptor molecules can activate TRAF (survival responses) or FADD (apoptosis).Assembly of TRAF-mediated survival responses is facilitated by other signalling proteins such as RIP (receptor-interacting protein) and cIAP (cellularinhibitor of apoptosis). Binding of TRAF proteins to TNFR2 is mediated through TIM (TRAF-interacting motifs) domains found on the cytoplasmic tailof the receptor.
Figure 5 Graphic depiction of the chemokine signal cascade. Heterotrimeric G-proteins are composed of α-, β- and γ -subunits; PI-3,4,5-P3 –phosphatidylinositol 3,4,5-triphosphate; PI-4,5-P2 – phosphatidylinositol 4,5-diphosphate; PI-3,4-P2 – phosphatidylinositol 3,4-diphosphate; PTEN –phosphatase and tensin homologue deleted on chromosome ten; PI3K – phosphoinositide 3-kinase; SHIP – SH2-containing inositol 5’phosphatase;GRK – G-protein-coupled receptor kinase; DOCK-2 – downstream of Crk-180 homolog-2 ERK1/2-extracellular signal receptor-activated kinase 1 or2; WASP – Wiskott–Aldrich syndrome protein; Arp2/3 – actin-related protein 2 and 3; SPRED – sprouty-related protein with ENA/vasodilatory-stimulatedphosphoprotein homology-1 domain; RACK1 – Receptor-activated C Kinase-1; ROCK – Rho-associated coiled-coil forming protein kinase; MLCK –myosin light chain kinase; DAG – diacylglycerol; PA – phosphatidic acid; PLC – phospholipase C; RGS – Regulators of G-protein signal; Plic-1 – aubiquitin-related protein; PKC – protein kinase C; and PAK – p21-activated kinases.
activates by self-cleavage induced by an increase in itslocal concentration. Cleaved caspase-8 subsequently acti-vates downstream caspases, notably caspase-3, and therebyinduces apoptosis (Thorburn, 2004).
Interestingly, TNFR1 signals for cell death only inconditions where protein synthesis is blocked. In mostcircumstances, TNFR1 induces transcription and activationof inflammatory genes, suggesting a mechanism to sup-press apoptosis. Despite the well-defined ability of TNFR1signalling to induce cell death, the majority of normal cellsdo not respond to TNF by undergoing apoptosis. This contra-dictory situation can be accounted for by TNFR1 activatingnuclear factor-κB (NF-κB), which induces the expression ofa number of anti-apoptotic proteins. NF-κB transcription fac-tors sequester in the cytoplasm by inhibitor of NF-κB (IκB).Phosphorylation of IκB leads to its degradation through ubiq-uitination by the 26S proteasome. The heterodimeric NF-κB
subunits then translocate to the nucleus where they regulateexpression of a wide variety of genes involved in anti-apoptotic responses (Wajant et al., 2003). The best describedof these are (i) A20, a zinc finger-containing molecule;(ii) A1/BFLI, a BCL-2 homologue; and (iii) c-IAP1/c-IAP2,members of the inhibitor of apoptosis (IAP) family (Karsanet al., 1996; Rath and Aggarwal, 1999). In lieu of theseobservations, TNFR1 associates with TRAF, which protectcells from apoptosis and initiate inflammatory responses.
TRAFs
TRAFs are a group of intracellular intermediates that binddirectly or indirectly to many members of the TNF receptorfamilies. To date, there are six mammalian TRAFs (1–6)identified and are evolutionary conserved in Drosophilamelanogaster and Caenorhabditis elegans (Grech et al.,
2000). Although TRAFs lack intrinsic enzymatic activity,they bind to several serine–threonine kinases, including NF-κB-inducing kinase (NIK), receptor-interacting protein (RIP)and GCK (germinal centre kinase). Through the recruit-ment of these kinases, TRAF2 induces the activation ofseveral transcription factors, particularly NF-κB, as well asdownstream kinases involved in stress responses, notablyc-Jun N-terminal kinase (JNK), which are crucial effectors ofthe TNFR1-mediated pro-inflammatory reaction. TRAF2 isalso a central component of the TNFR2 signalling complex,through a direct interaction with the cytoplasmic domain ofthe receptor, which lacks a DD. TNFR2 signalling inducesproliferation by activating anti-apoptotic signal transductionpathways including NF-κB (Yeh et al., 1997). Paradoxically,cells responding to TNFR2 signalling can also undergo apop-tosis. Mounting evidence suggests TNFR1 is affected by asignal crosstalk mechanism with TNFR1 whereby TNFR2stimulates the expression of membrane-bound TNF, whichsubsequently activates TNFR1. This TNFR2-dependent pro-cess also affects the stability of prosurvival proteins, TRAF2and TRAF1, by inducing proteolysis of these protective pro-teins (Duckett and Thompson, 1997; Grell et al., 1999).
Other Signalling Molecules and Pathways
To account for the diverse range of outcomes followingTNFR1 activation, other signalling proteins have been iden-tified. Notably, silencer of death domains (SODD) was iden-tified on the basis of its ability to bind to the DD of TNFR1.SODD is found in the TNFR1 receptor complex beforereceptor activation, but dissociates from the receptor afterligand binding. As such, SODD-deficient mice alter cytokineproduction when challenged with TNF. Furthermore, treat-ment of cells with TNF dissociates SODD from the DDsof TNFR1, allowing recruitment of proteins such as TRAFand TRADD to activate the receptor-signalling complex. Thissuggests SODD pre-associates with TNFR1 to prevent spon-taneous signalling by DD-containing receptors (Takada et al.,2003; Jiang et al., 1999).
TNF also signals through mitogen-activated protein (MAP)and phosphatidylinositol-3 (PI3) family of kinases. Activa-tion of these kinases by TNF increases osteoclast survival byup to 80%. This increase in survival of mature osteoclastsis mediated by activation of Akt and extracellular signalreceptor-activated kinase (ERK) whereby inhibitors of thesekinases increase osteoclast morbidity (Lee et al., 2001). TNFalso appears to activate neutral sphingomyelinase (N-Smase),which mediates some of the inflammatory and proliferativeresponses to TNF. Factor associated with N-Smase activation(FAN), which binds to distinct sites on the cytoplasmic tailof TNFR1, mediates this activation. The coupling of FAN toN-Smase activity results in the production of ceramide, whichleads to a number of pro-inflammatory responses includingdegradation of IκB (Adam-Klages et al., 1996; Heller andKronke, 1994).
TNF Signalling and Cancer Therapy
The death receptors (DR) of the TNF superfamily representpossible targets for cancer therapy. Treatment includes usingantibodies against the TRAIL receptors that act as agonists.Activation of these DRs would effectively trigger an apop-totic response. This antibody-based therapy already showspromising results. Administration of an agonist anti-DR5antibody exhibits potent antitumour effects without causingsystemic toxicity to the animal. Furthermore, this approachnot only eliminates TRAIL-sensitive tumour cells, but it alsoinduces T-cell memory allowing for long-term preventionagainst a recurrence (Takeda et al., 2004). To date, anti-TRAIL-R agonistic antibodies are in Phase I and II clinicaltrials. In a similar fashion, activating the TRAIL receptorswith truncated versions of TRAIL that contain the extracel-lular domain has been used. Preclinical studies show theserecombinant constructs induce apoptosis in cancer cell lineswhile leaving normal cells unaffected. Furthermore, usingTRAIL in combination with a chemotherapy agent or a his-tone deacetylase inhibitor potentiates the antitumour effectsin cancer cell lines as well as in a mouse xenograft model(Inoue et al., 2004; Naka et al., 2002).
CHEMOKINE RECEPTOR SIGNAL TRANSDUCTION
Chemoattractant cytokines are typically <15-kDa proteins,which are secreted by many tissue and cell types (Zlotnikand Yoshie, 2000). The classical chemokine-induced bio-logical activity is leucocyte migration, but as the field hasmatured other chemokine-mediated physiological functionshave been identified, including regulation of tumour metas-tasis and growth (Howard and Galligan, 2004), lymphocytematuration, stem cell selection and maturation (Kucia et al.,2004; Broxmeyer et al., 2003), regulation of angiogenesisand apoptosis (Guo et al., 2005; Kovanen and Leonard,2004). Although these additional functions have been demon-strated, the components of the distinct signalling cascadesremain unresolved, therefore this section will focus on theaccepted components of the chemokine-induced chemotaxis,cell activation and survival signalling cascades (Figure 5).
Chemokines bind to and activate seven transmembraneGPCRs, which are structurally similar to the rhodopsin (typeA) subfamily, a nomenclature following the systematic onefor chemokines has been proposed (Bacon et al., 2002). TheN-terminus of the receptor, which is also the first extracellu-lar domain, is essential for high-affinity chemokine binding.Disulphide bonds between the extracellular domains main-tain the closely packed positions of the seven transmembranedomains. The disulphide bonding is needed for efficientchemokine-induced signalling but is not necessary for HIV-1
coreceptor activity. The chemokine receptor signal is depen-dent on a ligand-induced dynamic change in the receptor thatresults in an increased affinity for Gi and Gq heterotrimericG-proteins. Fine regulation of the GPCR signal occurs atthe membrane where increased phosphatidylcholine in thelipid bilayer enhances GTPase activity of the G-proteins andis essential for the activity of G-protein-coupled receptorkinase(s) (GRKs).
Heterotrimeric G-Proteins
Chemokine-induced chemotaxis is inhibited by pertussistoxin, indicating that chemokine receptors activate trimericG-proteins in the Go/i subfamily. However, activation ofphospholipase C (PLC), intracellular calcium (Ca2+) mobi-lization and cellular exocytosis can be mediated through per-tussis toxin-insensitive Gq proteins. The type of cell express-ing the GPCR and its activation state regulates which G-protein couples to the receptor, such that chemokine receptorshave been reported to couple to several classes of G-proteins.
Heterotrimeric G-proteins are composed of α-, β-, and γ -subunits. In the resting G-protein, the α-subunit is boundto guanosine diphosphate (GDP). Once activated, GDP isexchanged for GTP and the α-subunit separates from theβ –γ -subunits. The α-subunits are apparently necessary forregulation of the GPCR function. Regulators of G-proteinsignal (RGS) are widely expressed GTPase-activating pro-teins that contain a 130 amino acid domain that binds Gα-GTP subunits accelerating the hydrolysis to Gα-GDP andblocking Gα interaction with PLC. Mice deficient in RGS1,show delayed homologous desensitization and exaggeratedB-cell follicles (Moratz et al., 2004). A multifunctional cyto-plasmic protein, PDZ-RGS3, links the surface anchoredephrin-B to the GPCRs whereby cell-to-cell contact can reg-ulate cell migration (Lu et al., 2001). Additionally, some Gα
subunits are substrates for protein kinase C (PKC) resultingin autoregulation of this G-protein-mediated signal. Gαi sub-units were shown to be nonessential in chemokine-inducedchemotaxis. These data indicated that any Gα linked to β –γ -subunits, which are essential, could transmit a chemotacticsignal. In addition to transmitting the chemotactic signal,β –γ -subunits participate in signal component receptor dock-ing and cell activation.
Heterotrimeric G-proteins interact with several intracellu-lar domains of GPCRs found in the cytoplasmic tail and sec-ond and third intracellular loops and initiate expansive func-tional and regulatory signals. The β –γ -subunits were shownto guide GRK-2 to its phosphorylation site on a GPCR, sug-gesting that both the correct membrane lipid compositionand G-protein components are needed for GRK regulationof GPCR signal (Metaye et al., 2005). Receptor phosphory-lation mediated by GRK’s and various PKC isotypes leadsto β-arrestin binding, which targets the receptor to clathrin-coated pits and internalization (Lefkowitz and Shenoy, 2005).However, the ultimate role of chemokine receptor endocyto-sis remains controversial (Neel et al., 2005).
The G-protein β –γ -subunits directly bind to and activatethe RAS kinase, RAF-1, phosphoinositide 3-kinase gamma
(PI3Kγ ), PLC and other small GTPases. RAF-1 is a serine/threonine kinase that links the mitogen-activated proteinkinase (MAPK) cascade to tyrosine kinase-dependent growthfactor receptors (see Signalling by Ras and Rho GTPases).Mutagenesis studies showed that β –γ -subunits bind to RAF-1 with an affinity similar to that between β –γ -subunits andGRKs, suggesting that there maybe a competition betweenreceptor inactivation by GRKs and the mitogenic signal.
Mutagenesis of G-protein β-subunits resulted in inappro-priate organization of cellular cytoskeleton (Peracino et al.,1998), and additional studies have shown that small GTPasesof the RHO family are essential for chemotaxis and seques-tration of β –γ -subunits leads to rearrangement of the actincytoskeleton, suggesting a link between β-subunits and thesesmall GTPases. Recent work has focussed on identifyingregulators of β –γ -subunits. Two such proteins have beenidentified. PLIC-1, a ubiquitin-related protein, and Receptor-activated C Kinase-1 (RACK-1) directly bind to β –γ -subunits blocking activation (Chen et al., 2004). PLIC-1also regulates receptor internalization, and cell migration(N’Diaye and Brown, 2003).
Phospholipase
Mice lacking PLC-β2 and -β3 were used to show the role ofPLC in chemokine-induced cell signalling (Li et al., 2000).Neutrophils from animals lacking both PLC-β2 and -β3did not produce inositol trisphosphate (IP3), flux calcium orsuperoxide in response to chemokines or chemoattractants.Animals lacking only PLC-β2 clearly showed reduced bothIP3 and Ca2+ flux in response to interleukin-8 (IL-8 alsoknown as Cxcl8) and macrophage inflammatory protein 1beta (MIP1β, also known as Ccl4), but not to the sameextent, suggesting that both PLC isoforms participate insignal transduction in neutrophils. In contrast to the calciumflux signal, the chemotaxic response of the neutrophils fromanimals lacking both PLC-β2 and -β3 was not reduced;rather, there was an enhanced chemotactic response to IL-8.The PLC-β2− and -β3−deficient animals failed to activatePKC in response to chemoattractants, indicating that the PKCpathway is linked to the Gα and not the Gβ –γ signal.Additionally, PLC-deficient animals failed to phosphorylatethe mitogen-activated protein kinase c-jun N-terminal kinase.However, PLC deficiency had no effect on Rac activation,suggesting that cytoskeletal modification is a separate signalin neutrophils.
In addition to activation of PLC, there is evidence thatphospholipase D (PLD) is also activated by chemokines.The position of PLD in the chemokine signal cascade isambiguous because diacylglycerol (DAG) can be intercon-verted to the lipid hydrolysis product of PLD, phosphatidicacid (PA), suggesting that PLC activity may regulate PLDactivity. Additionally, RAS and RHO family members andPKC activate PLD. A function for PA in chemokine-inducedcell activation has not been identified, but PA is stronglyassociated with cell vesicle transport, suggesting that PLDmay act late in the chemokine-induced cascade by regulatingreceptor localization to the membrane.
PI3Ks have been implicated in many cellular responses,including, proliferation, apoptosis, adhesion and chemotaxis.To show the role of PI3Kγ in chemotaxis a number ofgroups generated PI3Kγ deficient mice, all groups observed asevere reduction (≤85%) in chemokine-induced myeloid cellchemotaxis, but lymphocytes were less affected, indicatingthat Gβ –γ may link to other intracellular components andinduce chemotaxis (Ward, 2004). PI3Kγ deficiency had noeffect on chemoattractant-induced actin polymerization orcalcium flux. The role of PI3Kγ in cell migration has beenproposed to be that of providing “steering” for a cell inmotion. PI3Kγ deficiency had a profound inhibitory effect onchemoattractant-induced activation of Akt, ERK1 or ERK2.The activation of ERK1 and ERK2 by PI3Kγ directlylinks the chemokine GPCR signal to both proliferation andactivation signals. An alternative signalling component usedby lymphocytes to respond to chemokines is downstream ofCrk-180 homolog-2 (Dock-2).
Small GTPases Lead to Actin and Kinase Activation
The small GTPases participate in several critical cell migra-tion steps. In myeloid cells, Rac appears to mediate lamel-lipodia formation, while Cdc42 appears to be essential foradhesion to extracellular matrix. Also in myeloid cells,Cdc42 and Rho participate in endocytosis and antigen pre-sentation (Shurin et al., 2005). Tumour cell lines have beenused to show that Rho and Rac are required for tumourcell invasion. In an osteosarcoma cell line, Rho medi-ated actin fibre formation was inhibited by Sprouty-relatedprotein with ENA/vasodilatory-stimulated phosphoproteinhomology-1 domain (SPRED) 1 and 2 resulting in decreasedtumour metastasis (Miyoshi et al., 2004). Thus, the smallGTPases are a target to regulate cell migration.
Subsequent to activation of the small GTPases, p21-activated kinases (PAKs), and Rho-associated coiled-coilforming protein kinase (ROCK), which are serine/threonineprotein kinases, are also required for cell migration. Racactivation leads to LIM kinase 1 activation that in turnphosphorylates cofilin, an actin depolymerizing and severingprotein. Cofilin regulates actin filament polymerization atthe leading edge of lamellipodia of a polarized cell andto degrade polymerized actin at the trailing edge or uropod(Nishita et al., 2005).
Another protein known to regulate actin organization isWiskott–Aldrich syndrome protein (WASP). WASP coordi-nates with several other proteins to regulate cell migrationincluding Cdc42, Rac, actin, actin-related protein 2 and 3(Arp2/3) complex, and Wiskott–Aldrich syndrome protein-interacting protein (WIP) (Gallego et al., 2006). The loss ofWASP results in reduced monocyte and lymphocyte migra-tion in vivo. The WASP and cofilin pathways are independentof each other leading to the observation the WASP is essentialfor cytokine-induced migration but not chemokine-inducedmigration, where it is redundant.
Mitogen-Activated Protein Kinases (MAPKs)
The role of the MAPKs in chemokine-induced migrationis highly controversial because most cells stimulated withchemokines phosphorylate one or all of the classic MAPKs;ERK1 or 2 and p38. Supporting evidence for p38 beingrequired for in vivo cell emigration was shown using p38inhibitors (Cara et al., 2001). Further, p38 defects are oftenfound in leukaemia cells that do not migrate. Most studiessuggest that ERK1/2 are required for cell survival along withPI3K phosphorylation of Akt. However, myosin associationwith actin appears to require myosin light chain kinase, whichis activated by either p38 or ERK1/2 (Adachi et al., 2003).
Phosphatases
As the PI3K pathways have long been associated with cellmigration and survival it is no surprise that phospholipidphosphatases are considered to be equally important. SH2-containing inositol 5’phosphatases (SHIP) 1 and 2 and phos-phatase and tensin homologue deleted on chromosome ten(PTEN) remove phosphate groups from phosphatidylinositol3, 4, 5-triphosphate (PI-3, 4,5-P3) (Sly et al., 2003). Since PI-3, 4,5-P3 is essential for the recruitment and activation of theserine/threonine Akt (also known as protein kinase B), thesekinases balance directional sensing in cell migration and cellsurvival. In addition to the phospholipid phosphatases, sev-eral protein-tyrosine phosphatases have been implicated infine-tuning the chemokine signal (Neel et al., 2003).
Varied Negative Regulatory Pathways
As with all activating signals there must be a mechanism tocounteract the activation. In addition to receptor phospho-rylation and endocytosis, there appear to be several novelmethods to negatively regulate chemokine-induced migra-tion and proliferation. Association of kisspeptin-10 with its’corresponding GPCR, GPR54, blocks cell migration and sur-vival by a poorly understood mechanism. The association ofthe SLIT, a secreted protein, with roundabout receptor (robo)blocks chemokine-induced adhesion by blocking PYK2 tyro-sine phosphorylation through a small GTPase dependentpathway.
CONCLUSIONS
As the field of chemokine-induced signal transduction hasmatured, cell specific signalling cascades have been identi-fied along with additional linkages to other signalling cas-cades. The recognition that different cell types mediate thechemokine signal differently brings hope for selective reg-ulation of pathological conditions. Further, the endogenousregulators of chemokine-induced cell migration and survivalare beginning to be identified, which may lead to novel ther-apeutic targets.
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