Nck/Dock: an Adapter Between Cell Surface Receptors and the Actin Cytoskeleton

Nck/Dock: an adapter between cell surface receptors and the actin cytoskeleton

Wei Li*,1,2, Jianhua Fan1 and David T Woodley1,2

1The Department of Medicine, Division of Dermatology and the Norris Cancer Center, the University of Southern California Keck School of Medicine, 1303 North Mission Road, Los Angeles, California CA 90033, USA; 2The Greater Los Angeles Veteran Administration Healthcare System, Los Angeles, California, USA

In response to extracellular signals, cell surface receptors cellular targets. SH2 domains enable their host engage in connections with multiple intracellular signal- proteins to form complexes with phosphotyrosine ing pathways, leading to the cellular responses such as (pY) proteins by binding to pY residues within these survival, migration, proliferation and dierentiation. The proteins. The stability and speci®city of the binding `pY?SH2/SH3?eector' connection is a frequently also depend upon the additional three to six amino used scheme by many cell surface receptors, in which acid residues immediately carboxyl to the pY residue, SH2/SH3-containing adapters connect protein tyrosine pY-(X)3±6. Therefore, SH2 domains from dierent phosphorylation to a variety of downstream eector host molecules bind dierent pY-containing peptides. pathways. Following the initial landmark ®nding that SH3 domains bind proline-rich segments of target Grb2 adapter links the receptors to the Ras pathway molecules with a minimum consensus of P-x-x-P leading to DNA synthesis, recent studies have revealed (Mayer, 2001). Each proline is often preceded by an that the biological function of the SH2/SH3 adapter aliphatic amino acid residue, and the aliphatic-proline Nck/Dock is to link cell surface receptors to the actin pair binds to a hydrophobic pocket on SH3 domains. cytoskeleton. For example, in the evolutionarily-con- A major dierence between the SH2-pY binding and served signaling network, GEF-Rac-Nck-Pak, Nck `®xes SH3-PxxP interactions is that the former is always up' the interaction of Pak with its upstream activator, transiently induced by extracellular signals, whereas Rac. The activated Pak then regulates the cytoskeletal the latter is often, but not always, constitutive. For dynamics. The fact that the majority of the more than 20 instance, the SH2 domain of the adapter protein Grb2 Nck-SH3-associated eectors are regulators of the actin only binds to tyrosine phosphorylated/activated cytoskeleton suggests that Nck/Dock regulates, via EGFR, whereas its SH3 domain-associated GEF, binding to distinct eectors, various cell type-speci®c Sos, does not change in the absence or presence of motogenic responses. This review focuses on our current EGF. SH2 and SH3 domains can either individually understanding of Nck/Dock function. Due to the number reside in dierent host molecules or co-exist in the and complexity of the terminologies used in this review, a same host molecule. When SH2 and SH3 domains `Glossary of Terms' is provided to help reduce confu- work together, they connect pY signaling at the cell sions. Oncogene (2001) 20, 6403 ± 6417. surface to a variety of signaling pathways via the P-x- x-P/SH3 interactions. Keywords: Src-homology domains; adapters; tyrosine kinases; signal transduction; cytoskeleton Nck/Dock belongs to a group of SH2/SH3 adapter molecules The SH2/SH3 adapters A group of SH2- and SH3-containing proteins is The src-homology 2 (SH2) and src-homlogy 3 (SH3) composed exclusively of the SH domains and does not domains are protein ± protein interaction modules, have any other enzymatic or known functional motifs found in a variety of structurally and functionally (see review by Birge et al., 1996; Pawson, 1995). These distinct signaling molecules, including protein tyrosine proteins are therefore called SH domain-containing kinases/phosphatases, lipid kinase/phosphatase, gua- adapters. This group of proteins includes Crk (Crk-II nine nucleotide exchange factors/GTPase activation and Crk-L) (see the review by Feller, 2001 in this proteins, docking proteins/adapters, cytoskeletal bind- issue), Grb2 (Sem-5Ash/Dor) (Schlessinger, 1994), ing proteins and even transcription factors (Pawson, Gads/Grap2/Grf40/GrpL/Mona/GRID (see review by 1995; Mayer, 1999). Except for the terminology, SH2 McGlade, 2001 in this issue), Nck (Ncka/Nck/Nck1 and SH3 domains share no other common features and Nckb/Grb4/Nck-2) (also see previous reviews by regarding their structures, binding speci®city, and McCarty, 1998; Li and She, 2000), Grb10 (Morrione, 2000), SLAP (Pandey et al., 1995) and Grap (Feng et al., 1996). The mechanism of action by these adapters *Correspondence: W Li; E-mail: [email protected] is to link cell surface receptors, via their SH2 domains, Nck signaling to the actin cytoskeleton WLiet al 6404 to downstream eectors, through their SH3 domains. expected, Ncka and Nckb in both humans and mice Dierent adapter proteins appear to connect either shared only 68% amino acid identity. In contrast, a common or distinct surface receptors to dierent comparison of mouse Ncka with human Ncka and a downstream pathways. comparison of mouse Nckb with human Nckb showed In the past, genetic studies in invertebrates have over 95% amino acid identity (Chen et al., 1998). Only greatly helped to understand the biological functions of a single Nck-like gene has been identi®ed in several these adapter proteins. For example, based largely invertebrate species, including Xenopus's Nck (Wong upon studies in the C. elegans' CED-24CED- and Mayer, GenBank accession number U85781, 9, 54CED-10 pathway, Crk has been shown to play an 10), Drosophila's Dock (Garrity et al., 1996) and C. important role in cell engulfment/phagocytosis via the elegans (by genome sequence search, see ref. by Chen et Crk/CRKL4DOCk1804Rac pathway (Feller, 2001, al., 1998). The relationships of these invertebrate Nck this issue). Moreover, the search for the function of genes with mammalian Nck genes was analysed as Grb2 in mammalian cells also fortuitously received a described by Chen et al. (1998), who showed that they critical hint from a genetic study in C. elegans. Horwirz shared similar sequence homology with both Ncka and and colleagues reported that the Grb2 homologue in C. Nckb. elegans, Sem-5, acted between the let-23/EGFR and the Let-60/Ras during C. elegans' vulval development (Clark et al., 1992). It was subsequently shown by Nck binds diverse downstream eectors Schelssinger's group that Grb2 acted similarly in mammalian cells in response to epidermal growth Following the initial identi®cation of Nck/Ncka/Nck1 factor (EGF) (Lowenstein et al., 1992). For more than gene in 1990 (Lehmann et al., 1990), four papers 6 years after its ®rst cloning in 1990, the initial search appeared back to back in the September issue of for the biological function of Nck in receptor tyrosine Molecular and Cellular Biology in 1992 (Chou et al., kinase signaling lacked directions. Then, a genetic 1992; Li et al., 1992; Meisenhelder and Hunter, 1992; study in Drosophila reported by Zipursky and his Park and Rhee, 1992). These studies showed that Nck collaborators revealed that Nck/Dock links growth underwent induced tyrosine and serine/threonine cone receptor signaling to the actin cytoskeleton during phosphorylation in cells in response to growth factor photoreceptor axon guidance and targeting (Garrity et treatment or Src transformation. Antibodies against al., 1996). This sentinel ®nding suggested that Nck may Nck co-immunoprecipitated activated receptor tyrosine ®ll in the long-sought-after mechanism by which cell kinases (RTKs) such as EGFR and PDGFR-b. In vitro surface receptors communicate with the actin cytoske- binding experiments using GST-Nck fusion proteins leton (vide infra). indicated that the SH2 domain was fully responsible A cDNA for Nck was ®rst isolated by screening a for the interactions with RTKs. More interestingly, cDNA expression library from human melanoma cell overexpression of Nck caused ®broblast cell transfor- line, apparently due to cross reactivity of the mation in the absence of increased cellular protein monoclonal antibody probe against the melanoma- tyrosine phosphorylation and injected Nck-overexpres- associated membrane antigen MUC18 (Lehmann et al., sing cells caused tumor formation in nude mice (Li et 1990). Its sequence revealed that Nck has, from its N- al., 1992; Chou et al., 1992). terminus, three consecutive SH3 domains, followed by To further understand the cellular function of Nck, a SH2 domain at the C-terminus of the gene. A partial four major technical approaches have been utilized to sequence of a mouse Nck homologue, called Grb4, was identify the downstream targets of Nck, i.e. Nck-SH3- subsequently isolated from an embryonic cDNA interacting molecules. These approaches include: (i) the expression library using a radioactively-labeled C- CORT (cloning of receptor targets) by screening lgt11 terminus of the human EGF receptor as the probe cDNA expression libraries (Margolis et al., 1992); (ii) (Margolis et al., 1992). The human Nck and the mouse by yeast two-hybrid system; (iii) by protein puri®cation Grb4 shared less than 70% identity at both the using anity chromatography; and (iv) by antibody nucleotide and deduced amino acid levels. This co-immunoprecipitations. Some 20 Nck-SH3-binding suggests that human Nck and mouse Grb4 genes were proteins have so far been reported (see reviews by likely homologue, but not orthologue, genes between McCarty, 1998; Li and She, 2000; Zhao et al., 2000b). these two species. Therefore, additional Nck-related Interestingly, with a couple of exceptions, the majority genes may be present. Chen et al. (1998) carried out of the target proteins bind to the middle SH3 domain high stringency screening of mouse cDNA libraries of Nck (Most of the in vitro binding experiments, using human Nck as the probe and human cDNA however, were using Ncka gene product, except the libraries with mouse Grb4 as the probe. This approach studies by Chen et al. (1998), Tu et al. (1998) and led to the discovery of the mouse Nck/Ncka gene and Braverman and Quilliam (1999). An elegant study from the human Grb4/Nckb gene. Three other groups Lim's group showed that the middle SH3 domain of independently reported isolation of the Grb4/Nckb/ Ncka bound almost all the target proteins in three Nck2 gene (Park, 1997, Tu et al., 1998; Braverman and dierent tissues of rat. In contrast, the N'-SH3 and C'- Quilliam, 1999). (Throughout the rest of this article, we SH3 domains of Ncka contributed neither to the will refer the two Nck genes as Ncka and Nckb, for the capacity nor the stability of the SH3 binding (Zhao et sake of simplicity but certainly not of insouciance.) As al., 2000b). Further, they showed that all of the middle

Oncogene Nck signaling to the actin cytoskeleton WLiet al 6405 SH3-binding proteins shared the consensus motif tion, chemotaxis and endocytosis. We will review the PxxPxRxxS, which was critical for binding to Pak1 evidence for this conclusion by going through the and NIK (Zhao et al., 2000b). What about those studies from several well characterized biological previously reported N'-SH3- and C'-SH3-binding systems. For other previously reported functions of proteins? It is possible that those proteins were present Nck in mammalian cells, such as regulation of gene at very low levels in these tissues or their binding expression, cell proliferation and transformation, we anities to Ncka were too weak to be detected by the refer readers to three comprehensive reviews (Birge et novel technique used (Zhao et al., 2000b). It is also al., 1996; McCarty, 1998; Li and She, 2000). possible that the rest of the targets prefer to bind Nckb (such as PINCH for the C'-SH3 of Nck2/Nckb,Tuet In growth cone receptor signaling in Drosophila's visual al., 1998), which was not included in the studies by and embryonic nervous systems Zhao et al. (2000b). Figure 1 summarizes the current list of Ncka- and Nckb-binding proteins and their Morphogenesis of the nervous system requires the either known or possible cellular functions. It is directed migration of post-mitotic neurons to desig- interesting to note that: (i) most (56%) of the Nck- nated locations in the nervous system and the guidance SH3 targets are either proven or potential regulators of of axon growth cones to their synaptic targets. The the actin cytoskeleton; and (ii) binding to many directional information is provided to migrating dierent eector molecules may enable Nck to regulate neurons and growth cones in the form of extracellular various aspects of actin assembly in dierent types of cues, whose changes are interpreted by the guidance cells. cue receptors and transduced into eects on the cytoskeletal machinery and then motility (Song and Poo, 1999). In Drosophila's compound eye, each of the Nck links cell surface receptors to the actin cytoskeleton eight photoreceptor cells (R1 ± R8) represents a distinct neuron and acts as a photoreceptor during the Studies over the last few years in both invertebrates development of the lamina (R1 ± R6) and medulla and in mammals have indicated that the major cellular (R7 and R8). The neurons' growth cone, a sensor- function of Nck is to link cell surface receptors to the imotor structure that resides at the leading edge of the actin cytoskeleton, which is the prerequisite for various extending axon, detects speci®c signals from the biological responses such as axon path®nding, migra- outside of the cell and translates them, via intracellular

Figure 1 The current list of the Ncka/b-binding proteins. The majority of the Nck-SH2-receptor/mediator interactions are transiently induced by the extracellular signals, whereas most of the Nck-SH3-eector interactions appear to be constitutive, with an exception of the Pak1. The eector proteins with an asterisk only bind to Nckb/Nck2. PINCH's binding to Nck-SH3 is independent of the PxxP motif. Several proteins such as Descam and IRS-1 bind Nck through both SH2 and SH3 domains. Certain interactions appear to occur in many types of cells, whereas the rest are cell type speci®c. The percentages re¯ect those Nck-SH3-binding proteins that are potentially involved in the various cellular responses. In most of the cases, only the mammalian terminology of the genes was given, and their invertebrate counterparts, if known, may be found in `Glossary of Terms'

Oncogene Nck signaling to the actin cytoskeleton WLiet al 6406 signal transduction pathways, into directed movement the N'- SH3 and the C'-SH3 domains compensated of the axon body. Once in the target region, the growth each other in working in concert with the middle SH3 cone-led movement stops and synapses are formed. domain. Therefore, the middle SH3 domain can either Zipursky's and Pawson's groups, which also included pair in cis with the SH2 domain or with the remaining Garrity, Rao, Salecker, and McGlade found that the N'- and C'-SH3 domains, depending upon the types of Drosophila homologue gene of Nck, dreadlocks neurons, to control the growth cone motility (Rao and (Dock), was concentrated in the R cell growth cone Zipursky, 1998). These mechanisms of action by Dock and plays an important role in axon guidance in are in marked contrast to those by the closely related response to extracellular cues during Drosophila eye SH2/SH3-containing adapter Grb2's homologues in development (Garrity et al., 1996). Mutations in the Drosophila, Drk, and in C. elegans, Sem-5, in which the Dock gene disrupted signaling from the surface of the SH2 domains play an essential role in EGFR signaling growth cone to the intracellular actin cytoskeleton, (Clark et al., 1992; Olivier et al., 1993). One possible resulting in defects in R cell projection and targeting explanation is that Dock may be involved in connect- (Garrity et al., 1996). Further genetic mosaic analysis ing both tyrosine kinase receptors, via its SH2/middle showed that the dockp1/dockp1 mutant R cells in the eye SH3 pair, and non-tyrosine kinase receptors, via its N'- failed to achieve a normal connectivity, creating gaps and C'-SH3s/middle SH3 pair, to the actin cytoskele- in the array, hyperinnervation and crossing of ®bers ton. Indeed, as described, the growth cone receptor from adjacent columns in the medulla termini (Garrity Descam binds to Nck through both SH2 and SH3 et al., 1996). In addition to its role in the adult ¯y domains (Schmucker et al., 2000). visual system, Dock also plays an important role in The question then was what upstream receptors and synapse formation in embryonic nervous system (Desai downstream eectors of Dock were involved in the et al., 1999; Schmucker et al., 2000). Given the control of photoreceptor axon guidance and targeting. extensive sequence conservation between Dock and In mammals, two yeast Ste-20-related mammalian mammalian Nck, Garrity and colleagues proposed that kinases, the p21rac/cdc42-activated serine/threonine kinases Nck plays a similar role in forming precise patterns of (Paks) and NIK (Nck-interacting kinase), as well as the neuronal connections in vertebrates. Further, they PRK2, a novel serine/threonine protein kinase related hypothesized that Dock mediates growth cone gui- to the Rho eector PKN, and NAP4 (Nck-associated dance by transmitting upstream tyrosine phosphoryla- protein 4) all bind to the middle SH3 domain of Nck. tion signals through its SH2 domain to changes in the These proteins were implicated in the control of the actin-based cytoskeleton via its SH3 domains (Garrity actin cytoskeleton (Li and She, 2000). Furthermore, et al., 1996). This groundbreaking discovery has since Nck mediates growth factor-stimulated membrane guided the direction of the studies on Nck genes. relocation and activation of Pak1 and NIK. (Galisteo To test the above hypothesis, Rao in Zipursky's et al., 1996; Becker et al., 2000). These studies group studied various mutant forms of Dock for suggested that these Ste-20-related protein kinases are rescuing the dock phenotype (Rao and Zipursky, 1998). important downstream targets for Nck. In collabora- They found that the wild-type human Ncka was able to tion with Louis Lim's group, Zipursky and colleagues rescue the R cell projection defects in dock mutants, reported that, Pak, one of the three Drosophila Pak- supporting their hypothesis that Dock and Nck are related molecules (Pak, Mbt and Dpak2), is required functionally conserved during evolution. They assessed for the Dock pathway regulating R cell axon guidance each of four domains of Dock for its rescue ability of and targeting (Hing et al., 1999). Mbt and Dpak2 did the dock phenotype. They introduced point mutations not bind Dock and had no eect on the R cell axon into the key amino acids of SH2 and SH3 domains, guidance. Their studies showed that the loss-of- which abolish the binding to pY or PxxP peptides, and function phenotypes of Pak and Dock were indis- expressed these mutant transgenes in the third instar tinguishable, i.e. there was disruption of R cell axon larvae by germ-line transformation. In wild-type third guidance and targeting but not R cell dierentiation instar larvae, the dierent photoreceptor cells stop their (Hing et al., 1999). Drosophila Pak and Dock co- axonal growth in two distinct layers of the optic lobe, localize to axons and growth cones and, similar to the the lamina and the medulla. In contrast, dock mutant mammalian Nck and Pak, physically interact with each photoreceptor cells fail to establish this speci®c other, via the middle SH3 of Dock and the N-terminal targeting, leading to disruption of the lamina neuropile PxxP site of Pak. Furthermore, rescue of the dock organization. These results indicated that: (i) for the phenotype by Pak requires its protein kinase activity, restoration of R cell connectivity, the SH2 domain of the Dock-binding and the Cdc42/Rac-binding sites Dock was not required, and of the three SH3 domains, (Hing et al., 1999). Also, consistent with the notion only the middle SH3 was essential. However, neither that recruitment of Pak to the membrane by Nck is an the SH2 mutant nor the domain deletion transgenes essential regulatory step for Pak function (Manser et could rescue lethality, suggesting that the SH2 domain al., 1997; Lu et al., 1997), a membrane-attached Pak, of Dock may also play a role in other types of neurons; but not the kinase-dead (GMR-Pakmyr-k459A) or the non- (ii) both the middle SH3 and the SH2 domain were membrane-bound but constitutively active (GMR- essential for the formation of ®ber patterns in the inner PakL115F) Pak, was able to rescue the dock phenotype optic ganglia; (iii) the middle SH3 domain itself was (Hing et al., 1999). These results clearly placed Pak as insucient for the rescue, instead the SH2 domain and a main downstream player of Dock signaling. It should

Oncogene Nck signaling to the actin cytoskeleton WLiet al 6407 be noticed, however, that this ®nding did not support its mammalian homologue, HEM-2/NAP1, which the previous observation that Pak (Pak3) could also act binds the N-terminal SH3 of Nck, Kette and Dock upstream of Rac independently of its kinase activity genetically interact. It remains to be studied whether and its Rac-binding domains in PC12 cells (Obermeier these Dock-interacting molecules act independently or et al., 1998), by possibly bringing to a GEF for Rac coordinately in the Dock-mediated function. (Zhao et al., 2000a). In mammalian cells, Pak acts as a downstream In mediating growth cone receptor signaling, the role eector of the Rho family GTPases, Rac and Cdc42, of the Dock-Pak pathway did not appear to be as but Pak must be membrane-bound in order to bind simple as one would expect it to be. Earlier, Treisman and to be activated by Rac/Cdc42 (Bagrodia and et al. (1997) identi®ed a novel protein serine/threonine Cerion, 1999). As previously mentioned, the role of kinase encoded by the misshapen (Msn) gene, which is Nck is to target Pak to the plasma membrane, where required for the normal shape and orientation of Pak interacts with the GTP-bound Rac/Cdc42 (Lu et Drosophila photoreceptor cells. It was proposed that al., 1997; Manser et al., 1997). The question was Msn is involved in the signal transduction pathway whether the Dock-Pak pathway acts downstream of the leading to cytoskeletal re-arrangements (Treisman et Rho family GTPases in photoreceptor axon guidance al., 1997). Msn belongs to the germinal center kinase in Drosophila? Dickson's group conducted a saturation (GCK) subfamily of the yeast Ste20-related mamma- mutagenesis of the Drosophila genome, and found that, lian serine/threonine kinases, which also include GCK, in addition to Pak and dock alleles, a third NIK, GLK and HPK1. These kinases dier from the complementation group was mapped to a gene which Pak subfamily by lacking the Rho family GTPase- was most closely related to the human Trio and the C. binding domain (reviewed by Sells and Cherno, 1997; elegans UNC-73A. Trio and UNC-73A are multiple- Kyriakis, 1999). Su et al. (1997) showed that Msn plays domain proteins, including a GEF domain for Rho an important role in the embryonic dorsal closure and GTPases (Debant et al., 1996; Steven et al., 1998). acts as an upstream activator of the JNK-MAPK UNC-73A activates Rac and plays an important role in pathway. Two groups led by Rao, and by Treisman cell growth and migration (Steven et al., 1998). Among and Skolnik, respectively, showed that Msn, like Pak, other structural features, the Drosophila Trio contains binds Dock and also played a role in photoreceptor a motif of DH-PH ± DH-PH tandem domains, the axon path®nding. However, the results of the studies characteristic feature of GEFs speci®c for the Rho by the two groups dier in whether or not Msn acts, family GTPases. It was shown that Trio localizes at the like Pak, directly downstream of Dock. The study by growth cone membrane and that the trio mutant Rao's group showed that overexpression of Msn in disrupted the photoreceptor axon projection patterns dock mutant ¯ies largely suppressed the R1 ± R6 (Newsome et al., 2000), defects similar to those seen in nonstop phenotype, and concluded that Msn acts both Pak and dock mutants (Garrity et al., 1996; Hing downstream of Dock. Under these conditions, they et al., 1999). This function of Trio in axon guidance also observed premature termination of many R cell depended upon its GEF activity, since mutation in one growth cones within the optic stalk, suggesting that of two GEF domains of Trio completely abolished the Dock also negatively regulated the Msn action (Ruan Trio function. Newsome et al. (2000) went on to et al., 1999). The studies by the Treisman and Skolnik identify the downstream signaling events activated by groups showed that Msn was mainly involved in dorsal the Drosophila Trio, and showed that Trio activated closure of the embryo, and an activated form of Msn Rac and Mtl (Mig-two-like), but not Cdc42 and Rho, was not sucient to rescue the dock mutant phenotype which in turn bound Pak and induced membrane (Su et al., 2000). They suggested that, although Msn ruing and lamellipodium formation. These results played some role in R cell axon path®nding, it indicated that the Trio-Rac-Dock-Pak pathway func- probably was not the main downstream mediator of tions in the control of growth cone guidance and Dock signaling in the control of photoreceptor axon motility. guidance and targeting. As it will be discussed in the The search for the growth cone receptor, which following sections, recent studies showed that Decam mediates the extracellular cues to the intracellular Trio- turns out to be the cell surface receptor for the Nck- Rac-Dock-Pak pathway in photoreceptor axons, con- Pak pathway in axon path®nding, whereas Frizzled tinued. Since Dock is an SH2/SH3 domain-containing receptor activates Msn kinase in the control of planar adapter, it was initially predicted that tyrosine kinase polarity signaling. receptors or tyrosine phosphorylation of non-kinase Clemens et al. (1996) reported that dPTP61F, a receptors at the growth cone interacted with Dock protein tyrosine phosphatase in Drosophila, bound and (Garrity et al., 1996). After examination of a collection co-localized with Dock in Drosophila's embryonic of mutations in genes that encode proteins in nervous system. The transmembrane receptor-like phosphotyrosine signaling and that have previously protein KETTE, the Drosophila HEM-2/NAP1 homo- been implicated in growth cone function, Garrity et al. logue, has also been reported to control VUM (ventral (1999) reported that mutations in PTP69D, one of unpaired median) axonal path®nding in the CNS three Drosophila receptor protein tyrosine phosphatases (central nervous system) midline and the cytoskeletal (RPTPs) that regulate axon guidance, disrupted R1 ± organization and cell morphology of mesodermal and R6 axon targeting to the lamina. Since the tyrosine epidermal derivatives (Hummel et al., 2000). Similar to phosphatase domain is required, it was postulated that

Oncogene Nck signaling to the actin cytoskeleton WLiet al 6408 PTP69D generates a stop signal to the growth cone macrophage chemotaxis to the wound bed, as well as motility machinery by protein tyrosine dephosphoryla- new tissue formation and contraction of the wound tion (Garrity et al., 1999). However, whether or not (reviewed by Duel et al., 1991). PDGF-bb is so far the PTP69D binds to Dock and acts along with the Trio- only Food and Drug Administration (FDA)-approved Rac-Nck-Pak pathway remains unknown. To search growth factor for the clinical treatment of human skin for Dock-SH2-interacting proteins in Drosophila, wounds. PDGF-bb also plays an important role in the Schmucker and colleagues generated a cell line, S2, accumulation of SMC (smooth muscle cells), a key that constitutively expressed a his-tagged SH2 domain event in the formation and progression of lesions of of Dock (Schmucker et al., 2000). Lysates of this cell atherosclerosis and in re-stenosis after angioplasty and line were subjected to Ni-agarose anity chromato- bypass surgery (Ross, 1993). In both of these biological graphy, and the eluates were subjected to an anti- processes, PDGF-bb-stimulated actin polymerization phosphotyrosine anity chromatography. The bound and cell motility are essential. The signaling mechan- proteins were separated by SDS ± PAGE. One of the isms, by which PDGF induces cell proliferation, have ®ve bands, p270, was excised from the gel, digested been well studied (Jones and Kazlauskas, 2000). with Trypsin and microsequenced. The gene that However, our understanding of how PDGF stimulates encodes the p270 protein was most highly related to actin cytoskeletal assembly/re-assembly, the prerequi- human DSCAM, the gene that has been mapped to the site for cell migration and chemotaxis, still remains Down syndrome region and implicated in human metal fragmentary. Hall's group ®rst showed that the PDGF- retardation (Yamakawa et al., 1998). The cytoplasmic bb-stimulated cell membrane ruing and lamellepo- region of the Drosophila Dscam showed multiple dium formation in Swiss 3T3 ®broblasts were mediated potential phosphotyrosine sites including a Nck/Dock by PI-3K4Rac signaling (Ridley and Hall, 1992; SH2 binding motif, and several putative SH3-binding Nobes et al., 1995). The involvement of PI-3K in PxxP sites (Schmucker et al., 2000). Interestingly, PDGF-bb-stimulated chemotaxis has also been re- Dscam indeed bound both the SH2 and SH3 domains ported in CHO cells (Kundra et al., 1994), in human of Dock and acted as an upstream activator of the arterial SMC (Bornfeldt et al., 1994, 1995), and in Dock-Pak guidance pathway. Since the Dscam gene osteoblasts MC3T3-E1 (Godwin and Solto, 1997). could potentially have as many as 38 016 alternatively Similar results were also reported for PDGF-aa- spliced forms, these authors proposed that such stimulated NIH3T3 cells (Rosenkranz et al., 1999). diversity, which is comparable to that of individual The action of PI-3K under these conditions is to immunoglobulin molecules through gene rearrange- connect the PDGFR to the Rac signaling pathway ments, may contribute to axon connection speci®city possibly by catalyzing the production of phosphatidy- (Schmucker et al., 2000). Thus, the Decam-Trio-Rac- linositol 3,4,5 triphosphate, which binds and activates Dock-Pak pathway regulates photoreceptor axon the GEF for Rac. Activation of Rac is essential for guidance and targeting in Drosophila. Figure 2 PDGF-induced cell motility. In Rat1 ®broblasts, schematically depicts the current working model for Symons' group showed that PDGF-bb-induced migra- the growth cone receptor mediated signal transduction tion required the Ras?(PI3-K)??Rac pathway in photoreceptor axon guidance in Drosophila. (Anand-Apte et al., 1997). One of the main downstream targets for activated Rac is Pak1. Pak1 has the similar eect on actin In PDGF receptor-b signaling to the actin cytoskeleton cytoskeletal assembly as Rac, when overexpressed in and cell motility cells (Sells and Cherno, 1997; Manser et al., 1997; PDGF acts both as a strong mitogen and as a Zhao et al., 1998). It also has similar eect as Rac on chemoattractant in mesenchymally derived cells. Dur- cell motility (Sells et al., 1999). PDGF-bb stimulates ing wound repair, PDGF-bb is one of the prominent the activation of Pak1 kinase and its localization in the growth factors in the control of human dermal focal adhesion complexes in NIH3T3 cells (Dharma- ®broblast proliferation and migration, neutrophil and wardhane et al., 1997; Sells et al., 2000). Activated

Figure 2 Summary of the Dock/Nck signaling in Drosophila axon guidance and targeting. Activation of Dscam receptor recruits Nck-Pak complex to the membrane, and also activates the GEF, Trio, for Rac/Cdc42. The membrane-associated Pak binds to the GTP-bound Rac, which in turn activates the Pak, leading to actin polymerization and gene expression. Whether or not Msn is a parallel (to Pak) downstream target for Dock in photoreceptor guidance and targeting is not entirely clear. The speci®c target(s) for PTP69D and the function of dPTP61F in axon path®nding remain to be further investigated. Msn has other functions such as dorsal closure, and Rac/Cdc42 may have Pak independent pathways to regulate actin polymerization

Oncogene Nck signaling to the actin cytoskeleton WLiet al 6409 PDGFR recruits Pak1 to the cell membrane (Galisteo however, is unknown. Chen et al. (2000) studied et al., 1996). Among the two dozens of Nck-SH3- whether or not Nck mediates PDGF-stimulated, Rac- interacting proteins (as shown in Figure 1), Pak1 is by dependent membrane ruing and lamellepodium far the only reported Nck-bound protein kinase that is formation in NIH3T3 ®broblasts. Using both mutants activated in cells by PDGF-bb stimulation. Galisteo et of Ncka and Nckb and microinjection of antibodies al., and Bokoch et al. showed that Nck interacts, via its speci®c against Ncka or Nckb, they showed that Nckb middle SH3 domain, with Pak1/aPak through the was speci®cally involved in the PDGF-bb-induced QDKPPAPPMRN sequence at its N'-terminus in Cos- cytoskeletal changes, in which the SH2 and the middle 7, Swiss 3T3 and L6 cells (Bokoch et al., 1996; Galisteo SH3 domain of Nckb played an essential role (Chen et al., 1996). Pak1 is recruited to the membrane by the et al., 2000). Nck's binding to the PDGFR. Its kinase activity is Whether or not Nck also plays a role in the then further enhanced (Galisteo et al., 1996), pre- PDGF-stimulated Rac activation appears to depend sumably by PDGFR-activated Rac or by another Rac/ upon the types of cells tested. In the contexts of Cdc42-independent GTPase (Lu et al., 1997, Lu and 293T human embryonic kidney cells and transfected Mayer, 1999; Yoshii et al., 1999). To demonstrate that genes, Yoshii et al. (1999) from Sugimura's group the Nck-mediated relocation of Pak1 is crucial for its showed that PDGF stimulation caused association of activation, Lu and Mayer (1999) showed that over- the aPIX, a GEF for Rac, with the p85 subunit of expression of a myristoylated Nck-SH3-II domain, PI-3K and Nck. This association results in activation which directly bound to Pak1, in 293T cells caused of the aPIX, as assayed by the Pak1 kinase activity activation of Pak1 even in the absence of GTP-loaded and GTP-loading of Cdc42 and Rac (Yoshii et al., Rac and Cdc42 (Lu et al., 1997). Expression of an 1999). In this case, Nck appears to participate (likely unmyristoylated Nck-SH3-II or myristoylated but together with the product of PI-3K) in the relocation Pak1-binding defective Nck-SH3-II mutant in the same of the Pak1-bound PIX to the membrane, where it cells did not activate Pak1. More importantly, several activates Rac. Therefore, Nck acts upstream of Rac groups showed that the Pak-induced actin cytoskeletal in these cells. In the context of NIH3T3 cells, PDGF- assembly, cell motility and directed cell migration bb stimulation activates the Rac pathway via depended upon its N-terminal proline-rich sequence, translocation and phosphorylation of another Rac where Nck-SH3-II binds (The N'-PxxP motif may also GEF, Tiam1, in a calcium/calmodulin kinase II bind other SH3-containing proteins) (Sells and Chern- (CaMKII)- and PKC-dependent fashion (Buchanan o, 1997; Kiosses et al., 1999). et al., 2000). Treatment of these cells with wortman- Ncka/Nck/Nck1 binds at the tyr-751 and Nckb/ nin, an inhibitor of PI-3K, showed no inhibitory Nck2/Grb4 binds at the tyr-1009 in activated human eect on the PDGF-induced Tiam1 activation. This PDGFR-b (Nishimura et al., 1993; Chen et al., 2000). suggests that activation of PIX and Tiam1 by PDGF Following PDGF stimulation, there is an increased was mediated by distinct signaling pathways. Taken phosphorylation of Nck on serines and tyrosines (Li together, the GEF4Rac4Nck4Pak pathway, which et al., 1992; Meisenhelder and Hunter, 1992). plays an essential role in Drosophila's axon guidance Meisenhelder in Hunter's group has mapped the and targeting, also mediates PDGF-stimulated actin tyrosine and the majority of serine sites of phosphor- cytoskeletal assembly, morphological alternation and ylation in the intervening sequence between the ®rst cell migration in mammals. Figure 3 summarizes and second SH3 domains for both Ncka and Nckb. Nck's role in PDGFR-b signaling to the actin These sites are conserved in both isoforms but are cytoskeleton. It should be emphasized that migration phosphorylated to varying degrees in the two Nck is a complex decision for a cell to make and no molecules. They found that PKA and PKC appeared single pathway is sucient to drive cell migration. to be responsible for the serine phosphorylations Therefore, it is anticipated that other parallel (Meisenhelder and Hunter, personal communication). signaling pathways will likely be identi®ed for the The functional relevance of these phosphorylations, control of cell motility.

Figure 3 PDGFR-b signaling to chemotaxis. Activation of PDGFR-b recruits and activates in parallel a number of signaling pathways, including Ras, PI-3K, PLCg and Nck. The Ras-PI-3K pathway activates the GEFs (PIX abd Tiam1) for Rac, which may also be activated by Pak1-associated PIX. Nckb binding to PDGFR relocates Nckb-bound Pak1 to the membrane, where Pak1 binds to GTP-Rac and becomes activated. Pak1 in turn phosphorylates MLCK to regulate the cytoskeleton dynamics. Collaboration with other parallel pathways, such as PLCg and other Rac-regulated eectors leads to chemotaxis

Oncogene Nck signaling to the actin cytoskeleton WLiet al 6410 Pak1, and the GTP-bound Cdc42 possibly activates In T-cell receptor signaling to the actin cytoskeleton and the WASp / Arp2/3 complex (Bubeck Wardenburg et gene expression al., 1998). Overexpression of dominant negative forms Speci®c T-cell recognition of antigen triggers a of SLP-76 (Y3F3), Nck (SH2 deletion) or vav (Vav- cascade of diverse intracellular signaling events that SH2 mutant) in Jurkat T cells reduced TCR-induced together lead to T-cell activation as de®ned by F-actin polymerization (Bubeck Wardenburg et al., proliferation, dierentiation or activation of eector 1998). Overexpression of Grb2 had no eect. These functions, such as antigen recognition. What is the investigators concluded that SLP-76 forms the tri- role of the GEF-Rac/Cdc42-Nck-Pak pathway in molecular complex of Nck/SLP-76/Vav in response to TCR signaling? Weiss's group ®rst showed that TCR activation and mediates TCR-induced Pak TCR stimulation robustly activated Pak1 in a Lck- activation and actin polymerization. Ku et al. (2001) dependent fashion in Jurkat cells (Yablonski et al., from Weiss's group argued that overexpression of an 1998). Overexpression of a kinase domain-deletion exogenous gene in cells can cause non-speci®c eects mutant of Pak1 blocked TCR-induced the activation and, therefore, compromises the physiologic signi®- of the endogenous Pak1 and NFAT-mediated gene cance of the ®nding. They set out to re-examine the expression, in which the dominant-negative eect of mechanisms by which TCR activates Pak1 in Jurkat the mutant depended upon the CRIB domain of mutant cell lines, which do not express endogenous Pak1 (Yablonski et al., 1998), suggesting the ZAP-70 (P116), LAT (J.Cam2), or SLP-76 (J14). involvement of Rho GTPases. Following TCR They found that Pak1 activation required ZAP-70, activation, Nck was tyrosine phosphorylated and but not LAT, SLP-76. To study the role of Nck in induced to form a complex with Pak1 (Yablonski et Pak1 activation, they tested whether the Pak1-P13A al., 1998). Overexpression of a Nck-SH3-II mutant mutant, which fails to bind Nck, could still be showed a similar dominant negative eect as that of activated following TCR activation. The results of Pak1 C-terminal deletion mutant. These results the experiments showed that Pak1-P13A was still suggested that Nck-Pak1 complex acted downstream activated to a level comparable to that of wild-type of vav and Cdc42 but upstream of Ras (Yablonski et Pak1 (Ku et al., 2001). These data indicated that al., 1998). Therefore, the evolutionarily conserved binding to Nck was not necessary for Pak1 activation GEF4Rac/Cdc424Nck4Pak pathway appeared to by TCR in Jurkat T cells. While neither SLP-76 nor play an important role in T cell activation. LAT was required for Pak1 activation, LAT appeared The question was how TCR is linked to the Pak to mediate TCR-induced Rac activation, possibly pathway? Studies of this question focused on two through vav (Ku et al., 2001). These results indicated unique adapter proteins in T cells, SLP-76 (SH2- that TCR activates Pak and Rac via distinct signaling domain containing leukocyte protein of p76) and pathways. How does TCR activate Pak1 then? LAT (linker for activation of T cells). SLP-76 is an Manser and Lim's groups puri®ed and cloned a new adapter protein that is comprised of three motifs class of Rho-p21 guanine nucleotide exchange factors, allowing for protein ± protein interactions: an amino- PIX (PAK-interacting exchange factor, aPIX and terminal acidic region containing tyrosine phosphor- bPIX) (Manser et al., 1998). By yeast two-hybrid ylation sites, a middle proline-rich motif that binds to screening, Cerione's group identi®ed p85Cool-1 and the SH3 domain of Grb2 family members, and a p85Cool-2, which appear to be identical to aPIX and carboxyl-terminal SH2 domain that associates with bPIX (Bagrodia et al., 1998). These proteins share SLP-76-associated phosphoprotein of 130 kDa tandem SH3, Dbl homology, and pleckstrin homology (SLAP-130 or FYB, for Fyn-binding protein) and domains, and bind with high anity through its N- another unidenti®ed 62 -kDa phosphoprotein (Jack- terminal SH3 domain to a conserved proline-rich man et al., 1995). LAT is a 36 kDa transmembrane sequence in Pak. It was shown that PIX/p85Cool-1 protein containing no previously known protein ± induces membrane ruing possibly by activating Rac, protein interacting domains such as SH2, SH3, PTB which in turn activates Pak1 (Manser et al., 1998). Ku or PH, just to mention a few. The palmitoylation of et al. (2001) tested whether the PIX/Pak1 complex LAT targets it to cholesterol-rich lipid rafts and is exists in T cells and, further, plays an important role in required for its function in TCR signaling (Zhang et TCR-induced Pak1 activation. They found that PIX al., 1998b; Lin et al., 1999). LAT is tyrosine and Pak1 are constitutively associated and TCR failed phosphorylated following TCR activation and the to activate Pak1 with mutations at P192 and P193, the phosphotyrosine residues recruit the SH2 domains of binding sites for PIX. Moreover, overexpression of Grb2 and PLCg1 (Zhang et al., 1998a). LAT-de®cient either the Dbl homology domain mutant or the SH3 Jutkat T cells failed to activate either Ras or domain mutant of PIX blocked TCR-induced Pak1 phosphatidylinositol signaling pathways (Finco et al., activation. Interestingly, in replacing the role of Nck in 1998; Zhang et al., 1999). Chan and his collaborators relocating the PIX/Pak1 complex to the membrane, the showed that tyrosine phosphorylated SLP-76 provides p95PKL (paxillin-kinase linker) appeared to recruit a scaold to recruit the SH2 domains of vav, a GEF PIX-Pak1 to upstream activators of Pak1 (Ku et al., for Rho GTPases in T cells, and Nck to form a tri- 2001). p95PKL is a multi-domain protein containing molecular complex. Vav activates Rac and Cdc42. an N'-terminal ARF-GAP domain, three ankyrin-like The GTP-bound Rac in turn binds and activates repeats, a potential calcium-binding EF hand, calmo-

Oncogene Nck signaling to the actin cytoskeleton WLiet al 6411 dulin-binding IQ motifs, a myosin homology domain, cell membrane-bound ligands, called ephrins. Increas- and two paxillin-binding subdomains (PBS). p95PKL ing evidence suggests that the Eph family receptors mediates the interaction of the PIX/Nck/Pak complex play important roles in control of cell migration in the with paxillin, by binding directly to paxillin, LD4 and peripheral nervous system and axon guidance at the PIX (Turner et al., 1999). The discrepancy over Nck- growth cones in the CNS (Flanagan and Vanderhae- dependent, i.e. SLIP-76/Vav/Rac/Nck/Pak1 signaling ghen, 1998; O'Leary and Wilkinson, 1999). In contrast, (Bubeck Wardenburg et al., 1998), or Nck-indepen- the mitogenic eect of Eph receptors was reported as dent, i.e. PKL/PIX/Rac/Pak signaling (Ku et al., 2001), relatively weak (Holland et al., 1998). Therefore, Eph activation of Pak1 kinase in Jurkat T cells remains to receptors mainly activate the signaling pathways which be further assessed. One approach would be to study regulate cytoskeletal architecture and cell adhesions. TCR-mediated Pak1 activation in Nck-de®cient T The primary candidates which act downstream of the lymphocytes. Ncka-, Nckb single or Ncka/b double Eph receptors include PI-3K and the Rho family knockout mice have recently been generated (T GTPases (Rho, Rac and Cdc42). The Eck receptor Pawson, personal communication). Availability of the binds and stimulates PI-3K activity (Pandey et al., cells derived from these animals/embryos will provide 1994). The process of ephrin-A5-induced collapse of invaluable tools for de®nitively assessing the function growth cones is mediated by activation of the small of Nck in TCR, as well as in other receptor signaling. GTPase Rho and its downstream eector Rho kinase (Wahl et al., 2000). Ncka binds indirectly, via p62dok, to EphB2 and In VEGFR signaling in the control of directly to EphB1 receptors (Holland et al., 1997; Stein endothelial cell migration et al., 1998). Stein et al. (1998) mapped the Ncka Vascular endothelial growth factor (VEGF) represents binding site to the tyr-594 in the juxtamembrane region a family of growth and chemotactic factors speci®c for of EphB1 following ephrin B1 treatment. EphB1 cells of endothelial origins. Their receptors belong to receptor with a mutation at the tyr-594 failed to bind the PDGF/CSF-1 receptor tyrosine kinase subfamily, Ncka, activate JNK pathway or transmit attachment including VEGFR-1/FLT-1 (fms-like tyrosine kinase) signals. These signaling events appeared to be mediated and VEGFR-2/KDR (kinase-insert domain containing by NIK, a mammalian homologue of the yeast Ste20 receptor). VEGF is believed to play a direct role in kinase and the Drosophila's Msn. Becker et al. (2000) regulation of capillary cell proliferation, migration and recently reported that the wild-type EphB1, but not tube formation during angiogenesis. Stimulation of EphB1-Y594F mutant, and EphB2 activate NIK in bovine aortic endothelial cells (BAEC) with VEGF mouse teratocarcinoma cell line P19, mouse neuro- increased tyrosine phosphorylation of Nck and its blastoma cells and rat glioma hybrid cells NG108. NIK association with PI-3K and a complex containing activation by EphB1 requires EphB1's tyrosine kinase GAP, p190RhoGAP and p62dok (Guo et al., 1995). activity and its binding to Ncka. Similar to Stein's Nck binds directly to the activated VEGFR-1 at both observation, Becker et al. (2000) showed that both a Y-1213 and Y-1333 (Igarashi et al., 1998) and also to kinase-defective NIK (NIK-D152N) and the Ncka- activated VEGFR-2/KDR (Kroll and Waltenberger, binding-defective NIK (NIK-P611, 614A) blocked 1997). Stoletov and colleagues have recently shown ephrinB1-and ephronB2-induced JNK activation and that VEGF stimulation recruited the Nck-Pak complex P19 cell attachment to a ®brinogen matrix. Ncka to focal adhesions, and the Nck-Pak complexed with appeared to bind selectively to certain, such as EphB1 FAK in human umbilical vein endothelial cells and EphB2, but not others, such as HEK2/EphA3 and (HUVE). Introducing antisense oligonucleotides EphA4, Eph receptors (Ellis et al., 1996; Hock et al., against Ncka into these cells inhibited VEGF-stimu- 1998), although the binding motif for Ncka pY594(I/ lated focal adhesion formation and migration (Stoletov V)DP is conserved in all the Eph family receptors (Ellis et al., 2001). This ®nding suggests that Ncka has a et al., 1996). It will be of interest to test if any of the speci®c role in VEGF signaling, since the 19-mer Eph receptors also binds to Nckb, and to compare the antisense oligonucleotide used by Stoletov et al. (2001) biological eects between Ncka-bound and Nckb- unlikely cross reacted with Nckb mRNA and previous unbound Eph receptors. studies showed that both Ncka and Nckb are co- present in almost all cell types (Chen et al., 1998, 2000), Nonetheless, it is important to test if down Nck has multiple connections to the actin cytoskelton regulation of Nckb in these cells has any inhibitory eects. Activation of the cell surface receptors (tyrosine kinase or non-tyrosine kinase receptors) provides binding sites for SH2 and/or SH3 domains of Nck, which is either In Eph receptor signaling in nerve cell migration constitutively associated or induced to associate, via its With 14 dierent members, the Eph receptor family three SH3 domains, with PxxP-containing proteins represents so far the largest subfamily of receptor such as PAKs, PRK2, NAP-1, NAP4, WASPs, WIP, tyrosine kinases (RTK) (Drescher, 1997; Flanagan and NIK, and PINCH (see Figure 1). The copy number of Vanderhaeghen, 1998; Holland et al., 1998; O'Leary the two Ncks might be high enough so that divided and Wilkinson, 1999). These receptors are activated by pools of Ncks bind each of the target proteins,

Oncogene Nck signaling to the actin cytoskeleton WLiet al 6412 especially to those that share the same SH3 domain 1998). Activated Pak1 phosphorylates itself at serine- (such as the middle SH3 domain) of Ncka. For 20, serine-198 and serine-203 (Manser et al., 1997). instance, the conserved PxxPxRxxS motif in both Phosphorylation of serine-20 dissociates Pak1 from Pak1 and NIK bind to the middle SH3 domain of binding to Nck, and phosphorylation of serines198 Ncka (Zhao et al., 2000a). Likewise, the copy number and 203 disconnects Pak1 from PIX (Zhao et al., of the receptors might be high enough so that dierent 2000b). Activated and dissociated Pak1 acts on its pools of the receptors bind to dierent downstream downstream eectors. Pak1 has been shown to Nck-eector complexes. As previously shown in Figure regulate cell morphology via either a kinase- 1, the majority of the Nck-SH3-associated proteins are dependent or kinase-independent mechanism (Manser potentially involved in the control of the actin et al., 1997; Sells and Cherno, 1997). Nck and PIX cytoskeleton. These Nck-eector complexes: Nck- appear to participate in the Pak1 signaling that is Pak1-PIX-GITI (Zhao et al., 2000b), Nck-Pak3 not related to its kinase activity (Sells and Cherno, (Bagrodia and Cerion, 1997), Nck-Prk2 (Quilliam et 1997; Daniels et al., 1998; Obermeier et al., 1998). al., 1996), Nck-NIK (Su et al., 1997), Nck-WASp One of Pak1's downstream targets is myosin light (Rivero-Lezcano et al., 1995; Quilliam et al., 1996), chain kinase (MLCK) (reviewed by Bagrodia and Nck-WIP (Anton et al., 1998), Nck-NAP-1 (Kitamura Cerion, 1999). Phosphorylation of MLCs by MLCK et al., 1997), Nck-DOCK180 (Tu et al., 2001) and promotes their dimerization and their interaction Nck-PINCH (Tu et al., 1998), all have the potential to with actin to drive contraction. Pak1 phosphorylates connect their upstream activators to the actin and inhibits MLCK activity, and, therefore, regulates cytoskeleton. Some of these complexes are widely contraction. The Nck-Pak-PIX complex can also distributed and the others are present in dierent cell promote cell motility by regulating focal complex types. For example, the Nck-WASp complex is dynamics. To do so, PIX binds the G-protein- predominantly found in hematopoietic cells and the coupled receptor kinase-interacting protein, GIT1, Nck-NIK complex is preferentially formed in neuronal which binds to and works in concert with FAK cells. Each of these complexes may either have distinct (Zhao et al., 2000b). functions or share common functions. Thus, to gain further insights into the major unanswered question of Nck-WASp complex in Cdc42 signaling to actin how Nck integrate signals from a variety of cell nucleation surface receptors to control when and where actin polymerizes, elucidation of the speci®city and redun- Independent from the Nck-Pak-PIX-GIT1 complex, dancy of the various Nck-eector complexes in actions the Nck-WASp (including ®ve WASp/Scar/WAVE should be a focus of future studies. proteins) complex can also link cell surface receptors to actin nucleation, especially for the receptors that control actin assembly by the activation of Cdc42. Nck-Pak complex in Rac signaling to the actin Nck-bound WASp in the cytosol is inactive because cytoskelton of its self folding autoinhibition due to its N- Among the Nck-SH3-eector complexes, the Nck- terminal and C-terminal interaction (Zigmond, 2000). Pak1-PIX/Cool-GITI complex is so far the best Following Nck's binding to the cell surface receptor, studied. Nck binding to the receptor relocates Pak1- WASp gets unfolded and activated by at least two PIX/Cool to the membrane proximity. The Pak1- activators, PIP2 and GTP-Cdc42, which are pro- associated PIX/Cool can then act upstream of Rac duced and activated by independent pathways from by converting GDP-bound Rac to GTP-bound Rac the receptor (Higgs and Pollard, 1999). The (Obermeier et al., 1998). Depending upon the cell unfolded WASp exposes its VCA/WH2 (verprolin types and cell surface receptors, Rac may also be homology, co®lin homology, acidic/WASp homology activated by other Rac GEFs, such as Tiam1 in 2) region that then binds to the Arp2/3 complex, PDGF-stimulated ®broblasts (Buchanan et al., 2000). leading to actin nucleation (Higgs and Pollard, The GTP-bound Rac `circles' back to bind to the 1999). The best example for the important role of PBD domain of Pak1 and release the intramolecular the Nck-WASp-Arp2/3 signaling pathway in regula- autoinhibition of the PIX-associated Pak1. Pak1 can tion of the actin cytoskeleton came from studies of even be activated, in the absence of direct binding a 50-kDa phosphotyrosine protein, A36R, of the to Rac/Cdc42, through interaction with PIX (Manser vaccinia virus, which uses actin-based motility to et al., 1998) or by a novel and unidenti®ed spread between cells. Way's group showed that the mechanism(s) (Lu and Mayer, 1999; Bokoch et al., viral integral membrane protein A36R acts as a 1998). The latter results may explain the con¯icting receptor that activates Src tyrosine kinase. Src ®nding by others that Pak is dispensable for Rac phosphorylates A36R at tyrosine-112, which in turn and Cdc42 signaling. It was shown that the Rac and recruits the Nck-WASp-Arp2/3 signaling complex, Cdc42 mutants that failed to bind all the three Paks via the SH2 domain of Nck, to the site of actin still induced F-actin assembly (Joneson et al., 1996; assembly. The Nck-WASp-Arp2/3 complex plays an Lamarche et al., 1996; Westwick et al., 1997) and a essential role in the actin-based motility of vaccinia membrane-associated and Rac- and Cdc42-binding virus (Frischknecht et al., 1999; Moreau et al., defective Pak1 was still activated (Bokoch et al., 2000).

Oncogene Nck signaling to the actin cytoskeleton WLiet al 6413 of Nck2/Nckb, via interestingly a PxxP motif-indepen- Nck-NIK complex works without direct binding to dent mechanism (Tu et al., 1998). A similar novel RhoGTPases? mechanism was also reported for the Pak-PIX/Cool As previously mentioned, EphB1 and EphB2 activates interaction, in which the SH3 domain of PIX/Cool NIK kinase activity in living cells (Becker et al., 2000). binds with a high anity to a unique sequence (Manser An interesting question, in comparison to the activation et al., 1998). It is a notion that Nck2 mediates the of Pak1 by membrane relocation, by Rho GTPases or interaction of the PINCH-ILK signaling complex with by both, is what the activator(s) for NIK at the both integrin and growth factor receptors. This would membrane site is. NIK does not have PBD domain then lead to focal adhesion formation and other and, therefore, lacks direct binding to Rac/Cdc42. It biological responses such as gene expression and cell was proposed that the increased local concentration of proliferation. NIK by the Nck's membrane-recruiting eort enables to NIK to transphophorylate and activate each other, similar to the mechanism of EGFR activation. In More Nck-eector complexes in the regulation response to Eph receptor activation, p62dok and of the actin cytoskeleton p120rasGAP are added to the Nck-NIK complex (Holland et al., 1997; Becker et al., 2000). While p62dok and The potential role of the Nck-PRK2 complex in p120rasGAP are membrane-bound proteins following the control of the actin assembly came from the studies receptor activation, whether or not they play a role in that PRK2 is a downstream target for Rho (Quilliam NIK activation remains unknown. Finally, it can not be et al., 1996). Analogous to the Nck-Pak and Nck- entirely excluded that Rho GTPases still activate NIK WASp complexes, which participate in Rac and Cdc42 through indirect mechanisms. signaling, respectively, Nck-PRK2 complex may act in receptors/Rho signaling pathway. Nck directly binds, via its middle SH3 domain, to endogenous WIP Nck-NAP1 (HEM-2 or KETTE) complex in axon (WASP-interacting protein), which associates with the pathfinding and mesodermal and epidermal development actin polymerization regulatory protein pro®ling. This The 125 kDa NAP1 (Nck-associated protein 1) is a suggests that Nck may also couple extracellular signals receptor-like protein with six transmembrane domains to the cytoskeleton via its interaction with WIP and that belongs to the evolutionarily-conserved Hem gene pro®ling (Anton et al., 1998). Nckb (Nck2) binds, via family (Kitamura et al., 1996). NAP1 indirectly its middle and C-terminal SH3 domains, to DOCK180 associates, through a 140 kDa protein, with the GTP- (Tu et al., 2001), which was reported to participate in bound Rac1 (Kitamura et al., 1997), and binds directly integrin4Crk4p130cas4DOCK1804Rac signaling to the N-terminal SH3 domain of Nck (Kitamura et by activating Rac to induce membrane ruing and al., 1996). While the function of Hem proteins in cell migration (Kiyokawa et al., 1998a, b; Cheresh et mammals remains unclear, its Drosophila homologue al., 1999). Nck in Nck-Abl complex appears to activate Kette/Hem-2 appears to act either as a cell surface the Abl kinase activity, which plays a critical role in receptor or a membrane docking protein to control gene expression and development (Smith et al., 1999; VUM axon path®nding and cytoskeletal organization Adler et al., 2000). The Nck-dynamin and Nck- (Hummel et al., 2000). Consistently, KETTE requires synaptojanin complexes may couple cell surface DRac1 for signaling. While the kette and dock mutants receptors to endocytosis and vesicle tracking (Zhao genetically interact (Hummel et al., 2000), whether or et al., 2000b). not Dock mediates KETTE's signaling or vice versa remains unclear. Conclusions Nck2-PINCH-ILK complex as a liaison between integrins Studies of Nck have ®lled a critical gap between cell and growth factor receptors surface receptors and an important cellular biological Integrin-linked kinase (ILK) is a focal adhesion serine/ response. Nck proteins are designed to help assembly threonine protein kinase that is under regulation by protein complexes that transmit signals from the cell both integrin- and growth factor-signaling pathways in surface receptors to the actin cytoskeleton. While the a PI-3K-dependent manner (Wu, 1999). ILK binds to same two Nck proteins are expressed in dierent types PINCH, an evolutionarily conserved LIM only protein, of mammalian cells, they are capable of utilizing and the binding is crucial for the focal adhesion multiple distinct eector molecules to mediate dierent localization of ILK. In C. elegans, partial and complete actin cytoskeletal changes, leading to cell type speci®c loss-of-function studies demonstrated that UNC-97/ responses. Figure 4 emphasizes two important extra- PINCH aects the structural integrity of the integrin- cellular signal-induced biological responses, DNA containing muscle adherens junctions and contributes synthesis and the actin cytoskeletal rearrangements. to the mechanosensory functions of touch neurons. In The signal of growth factor-induced DNA synthesis is Drosophila, the PINCH homologue plays a critical role mediated by Grb2 adapter and the signal of the actin in adherens junction assembly and stability (reviewed dynamics is propagated by Nck adapters. Crosstalk by Wu, 1999). PINCH binds to the third SH3 domain between these two pathways and engagement with

Oncogene Nck signaling to the actin cytoskeleton WLiet al 6414 (2) Cbl: the protooncogene of the murine CasNS-1 retrovirus transformaing protein; mediates ubiquitin- dependent protein degradation and growth inhibi- tion. (3) CKIg2: casein kinase Ig2; function unknown (4) Dock: drealocks in Drosophila; the homologue of mammalian Nck (see Nck) (5) DOCK180: 180-kDa protein downstream of CRK; a signaling protein implicated in integrin- and Rac- regulated membrane ruing and migration (6) Dscam: from the human protein `Down syndrome cell adhesion molecule'; cell surface proteins involved in axon guidance. (7) Dynamin: large molecular weight GTPase; involved in clathrin-coated vesicle formation. Figure 4 Grb2 for mitogenesis and Nck for motogenesis. Grb2 (8) FLT-1: fms-like tyrosine kinase. a VEGF receptor links receptors to Ras pathways to induce to DNA synthesis, and Nck works with the Rho GTPases to connect the same receptors family member. to the actin cytoskeleton. Grb2 acts between the receptor and (9) GEF: guanine nucleotide exchange factor; activa- Ras, and Nck acts both upstream and downstream of Rac. The tors of ras superfamily GTPases. much larger number of the Nck-SH3 targets suggests that Nck (10) Grb2: growth factor receptor-binding protein 2;a deals with dierent aspects of actin reorganizations. Not all Nck- SH3 eectors interact with RhoGTPases. The activated receptor SH2/SH3 adapter, via Sos, to ras pathway tyrosine receptor should be a dimmer, and it can be replaced by (11) Grb4: growth factor receptor-binding protein 4; non-kinase receptors. The membrane locations of the proteins and mouse Nckb/Nck2. phosphorylation sites are not accurately drawn for simplicity (12) Grb7: growth factor receptor-binding protein 10;a reasons member of SH2-containing adapter family (also Grb10 and Grb14) involved focal adhesion formation and cell migration. other parallel pathways would determine the fate of a (13) Hem-2: hemorrhage (Hem-1 and Hem-2); essential cell, such as division, migration and dierentiation. in oocyte development (see NAP1 and KETTE) Further elucidation of Nck signaling networks may (14) IRS-1: insulin receptor substrate-1; a phosphotyr- shed light on the mechanisms, which regulate endocy- osine linker between receptors and downstream path- tosis, cell migration, chemotaxis, dierentiation and ways. tumor invasion. (15) LAT: linker for activation of T cells, a phosphotyrosine adapter known in TCR signaling. (16) KDR: kinase-insert domain containing receptor; a Future studies VEGFR family member. (17) KETTE: a Drosophila homologue of Hem family A daunting future challenge is to continue investigating gene; controls axon path®nding in VUM midline neu- the speci®c function of each of the increasing number rons (see Hem-2 and NAP1). of Nck-SH3-bound signaling molecules (so far the (18) MLCK: myosin light chain kinase; a serine/threo- number has already surpassed 20). It is important to nine kinase that phosphorylates and understand which of the 20 some Nck-SH3-bound (19) Msn: misshapen; a Drosophila homologue of yeast proteins is Ncka-, or Nckb-speci®c or shared by both Ste-20 and mammalian NIK serine/threonine kinases, Ncks, since Ncka and Nckb are co-present in most cell involved in dorsal closure and axon path®nding. types (Chen et al., 1998, 2000). Mice de®cient in Ncka (20) NAP1: Nck-associated protein 1; a Hem-2 family or Nckb alone are normal, whereas a double de®ciency transmembrane protein, see Hem-2 and KETTE. causes embryonic lethality (Pawson, personal commu- (21) NAP4: Nck-associated protein 4; a SH2- and nu- nication). This suggests that Ncka and Nckb are clear localization motif-containing protein with un- functionally redundant during development. On the known function. other hand, individual Nck can play speci®c roles in (22) Nck: a two-gene family of SH2/SH3-containing various cell types (Chen et al., 1998, 2000; Tu et al., adapters including Ncka/Nck/Nck1 and Nckb/nck2/ 1998; Stoletov et al., 2001). Therefore, cell lines derived Grb4 (see this paper). from various tissues of the Nck-knockout mice could (23) NIK: Nck-interacting kinase; a mammalian homo- further help our understanding of Nck function. logue of yeast Ste-20 and Drosophila misshapen (Msn) serine/threonine kinases (see Msn). (24) Pak: p21Rac/Cdc42-activated kinases; eectors of Rac Glossary of terms and Cdc42 signaling. (25) PKL: paxillin kinase linker, mediates binding of (1) Arp2/3: actin-related protein 2 and 3 complex, in- PIX (i.e. PIX-Nck-Pak complex) with paxillin. itially puri®ed from Acanthamoeba castellani; a key (26) PINCH: a LIM domain only protein in integrin- regulator of acin nucleation in motile cells. linked kinase signaling (also see UNC-97).

Oncogene Nck signaling to the actin cytoskeleton WLiet al 6415 (27) PIX: Pak-interacting exchange factor/Cool (cloned (39) Tiam1: mouse T-lymphoma invasion and metasta- out of library); a group of guanine nucleotide exchange sis-1 gene; a GEF for Rho family, especially for Rac1. factors involved in Pak signaling. (40) Trio: name based on the fact it contains three en- (28) PRK2: protein kinase C-related kinase-2; related to zymatic domains, two GEF domains and a PSK do- PKN as a potential RhoGTPase eector. main; GEF for Rho family GTPases. (29) p60Dok: downstream of tyrosine kinases; the (41) UNC-97: C. elegans' homologue (also PIN-2) of p120GAP-binding protein and an inhibitor of mitogen- the human PINCH and the Drosophila d-pinch; in- esis. volved in focal adhesions formation in muscles and (30) p130Cas: p130 Crk-associated substrate; a FAK possible functions in the nucleus (see PINCH). substrate and an adapter in integrin signaling. (42) UNC-73A: C. elegans' gene of a GEF for Rac. (31) dPTP61F: Drosophila protein tyrosine phopha- (43) Vav: product of oncogene vav and a guanine nu- tase; involved in axon guidance. cleotide exchange factor in hematopoietic cells; known (32) PTP69D: Drosophila receptor tyrosine phospha- for its role in TCR signaling. tase; involved in axon guidance. (44) WASp: Wiskott-Aldrich Syndrome proteins (33) HnRNP-k: heterogeneous nuclear ribonucleopro- (WASp and N-WASp); involved in Cdc42 signaling teins k; involved in translation regulation. via Arp2/3 to the actin cytoskeleton. (34) Sam68: Src-associated in mitosis, 68 kDa; a RNA- (45) WAVE: WASp family Verprolin-homologous pro- binding protein and a substrate of Src kinase. tein; involved in Rac signaling (see WASp and Scar). (35) Scar: suppressor of cAR (cAR are G protein- (46) WIP: WASp-interacting protein; together with N- coupled receptors); the Dictyostelium discoideum homo- WASp to regulate ®lopodium formation. logue of the mammalian WASp (see WASp and (47) ZAP-70: zeta chain-associated 70-kD protein; a WAVE). key tyrosine kinase in TCR signaling. (36) SLP-76: SH2-containing leukocyte protein of 76 kDa, a phosphotyrosine adapter in TCR signaling. (37) Sos: Drosophila son of sevenless gene product: Drk/Grb2-associated GEF for ras. Ste-20 (38) Synaptojanin: a group of polyphosphoinositide Acknowledgments phosphatases; possible Rac eectors in control of en- This study was supported by NIH Grants CA65567 (to W docytosis and growth. Lei) and AR46538 (to DT Woodley).

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Oncogene