Oncogene (2014) 33, 4537–4547 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

REVIEW SRChing for the substrates of Src

AB Reynolds1, SB Kanner2, AH Bouton3, MD Schaller4, SA Weed5, DC Flynn6 and JT Parsons7

By the mid 1980’s, it was clear that the transforming activity of oncogenic Src was linked to the activity of its tyrosine kinase domain and attention turned to identifying substrates, the putative next level of control in the pathway to transformation. Among the first to recognize the potential of phosphotyrosine-specific antibodies, Parsons and colleagues launched a risky shotgun-based approach that led ultimately to the cDNA cloning and functional characterization of many of today’s best-known Src substrates (for example, p85-Cortactin, p110-AFAP1, p130Cas, p125FAK and p120-catenin). Two decades and over 6000 citations later, the original goals of the project may be seen as secondary to the enormous impact of these protein substrates in many areas of biology. At the request of the editors, this review is not restricted to the current status of the substrates, but reflects also on the anatomy of the project itself and some of the challenges and decisions encountered along the way.

Oncogene (2014) 33, 4537–4547; doi:10.1038/onc.2013.416; published online 14 October 2013 Keywords: Src; cortactin; p130Cas; FAK; p120-catenin; AFAP1

INTRODUCTION and/or visualized by western blotting. The technology was rapidly The decade of the 1980’s witnessed an immense leap in the adopted by Parsons and colleagues in the form of a risky endeavor understanding of oncogenic signaling, including that mediated that led ultimately to the cDNA cloning and functional by v-Src, the first identified oncoprotein1 and the first described characterization of several of today’s best-known Src substrates. proteintyrosinekinase(PTK).2 The discovery of Src as a PTK was Contrary to expectations, most shared an ability to regulate the quickly followed by identification of other oncogenic PTKs3,4 actin cytoskeleton, as exemplified by the roles of FAK and p120 in including members of the receptor tyrosine kinase family, and cell–ECM and cell–cell adhesion, respectively. They were, in fact, attention turned to the possibility of downstream effectors. The among the first representatives of a larger network of substrates search for receptor tyrosine kinase substrates was facilitated by that now define one of the better-recognized roles of c-Src. the ligand-induced formation of stable receptor–substrate At the request of the editors, this review is not entirely about complexes that could be immunoprecipitated for analysis. In the substrates, but reflects also on the anatomy of the project contrast, the substrates of Src were slow to emerge owing itself and some of the challenges and decisions encountered primarily to technical constraints. Nonetheless, by mid-decade it along the way. Indeed, despite the enormous long-term impact of was clear that the kinase activity of Src was essential for its the work described below, it was not at all obvious at the time that transforming activity and the possibility of a substrate-depen- the spectrum of apparent Src targets, as indicated by pTyr- dent pathway to transformation was increasingly difficult to antibodies, would turn out to be anything more than a collection ignore. of physiologically irrelevant proteins, phosphorylated unproduc- Lacking the handle provided by today’s phosphotyrosine- tively by an overexpressed and promiscuous kinase. Arguing specific antibodies, identifying putative Src substrates was a slow against that notion was the apparent specificity implied by the and labor-intensive process, requiring high-level 32P-labeling of recognition of just 15–20 well-defined pTyr substrates from a Src-transformed cells, separation of the radiolabelled phospho- proteome of some 30 000 candidates. proteins by 2-D polyacrylamide gel electrophoresis and an in-gel alkaline treatment protocol designed to leave phosphotyrosine residues intact, while cleaving phospho-serine and -threonine. It The search for Src substrates was largely ineffective and by 1988, only a handful of putative In 1987, postdoctoral fellows Kanner and Reynolds, together with substrates had been identified (for example, b1-integrin, talin, Parsons, initiated a high-risk project (never subjected to NIH peer vinculin, phosphatidylinositol-3 kinase, p36 (calpactin) and p42 review) to identify the Src substrates indicated by pTyr-Abs. The (later, identified as MAP kinase)).5 two-pronged strategy was to first use pTyr-antibodies to purify the The technical problems were ultimately resolved by the tyrosine-phosphorylated proteins from Src-transformed cell discovery of phosphotyrosine (pTyr)-specific antibodies,6 which lysates, with the aim to shotgun immunize mice for generation provided a convenient and highly specific molecular handle of monoclonal antibodies (mAbs) to individual substrates. The on pTyr-containing proteins. For the first time, tyrosine- substrates were then to be identified directly by cDNA cloning, phosphorylated proteins could be selectively immunoprecipitated using the mAbs to screen lGT11 expression libraries.

1Department of Cancer Biology, Vanderbilt University, Nashville, TN, USA; 2Arrowhead Research Corporation, Madison, WI, USA; 3Departments of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA, USA; 4Department of Biochemistry, 3124 HSN, Robert C. Byrd Health Sciences Center, West Virginia University School of Medicine, Morgantown, WV, USA; 5Department of Neurobiology and Anatomy, 1833 Mary Babb Randolph Cancer Center, Robert C. Byrd Health Sciences Center, West Virginia University School of Medicine, Morgantown, WV, USA; 6Department of Medical Lab Sciences, College of Health Sciences, University of Delaware, Newark, DE, USA and 7Departments of Microbiology, Immunology and Cancer Biology, University of Virginia Cancer Center, Charlottesville, VA, USA. Correspondence: Dr DC Flynn, College of Health Sciences, University of Delaware, Newark, DE 19716, USA. E-mail: dfl[email protected] Received 31 May 2013; revised 16 August 2013; accepted 17 August 2013; published online 14 October 2013 Substrates of Src AB Reynolds et al 4538 Accordingly, tyrosine-phosphorylated proteins were purified However, a much better measure of the importance of the from cell lysates of Src-transformed chicken embryo fibroblasts, substrates stems from their enormous impact over the past two essentially in one step, using anti-pTyr immunoaffinity chromato- decades in areas of biology that extend well beyond the early graphy.7 Given the small amount of purified protein available, only focus on Src. four mice were immunized, two of which responded robustly to the chicken-derived antigens. As there was almost no information on substrate abundance, direct and indirect ELISAs were P85/CORTACTIN developed, the former for robustness, the latter providing Early work with p85 revealed striking localization to the cortical increased sensitivity.8 Both mice were fused, generating 786 actin cytoskeleton, giving rise to the name ‘cortactin’.9 On western hybridomas, of which 251 were positive in one or both of the blots, cortactin appeared as two to three bands ranging from ELISA assays. After subcloning and biochemical validation, the 80–85 kDa, all of which were phosphorylated on tyrosine by final tally came to 32 individual mAbs recognizing seven distinct Src. Early work in several cell types revealed that cortactin is tyrosine-phosphorylated substrates, and in a few cases, pTyr also tyrosine-phosphorylated by other Src-family members and itself.8 following ligand activation of the FGF receptor.10 To isolate the corresponding cDNAs efficiently, lGT11 libraries were initially screened with an antibody pool comprised of the Structure best mAbs to each substrate. The positive clones were then Cortactin was described well ahead of the other substrates due rescreened with each individual antibody, resulting in partial 11 cDNA clones for most substrates, the lone exception being p120 largely to the rapid isolation of a full-length cDNA clone. (as described elsewhere). Because of the rapid expansion in the Sequence analysis revealed a novel amino-terminal domain scope of the project, it was broken up according to substrate consisting of six-and-one-half 37 amino-acid tandem repeats, (along with corresponding mAbs and partial cDNAs), and later identified as the actin-binding region, and a carboxy-terminal redistributed to lab members to be completed as independent SH3 domain. The human ortholog (EMS1) was cloned shortly thereafter as a component of the 11q13 amplicon in head and projects. 12 Interestingly, sequencing of the open reading frames revealed neck squamous cell carcinoma (HNSCC). that all but tensin encoded entirely novel genes. Moreover, with the possible exception of p185, which was not pursued, all of the Cellular function and effects of phosphorylation proteins were ultimately validated as physiologically relevant Src Consistent with a role in cortical actin regulation, cortactin substrates. Notably, an unexpected finding at the time of original promotes actin polymerization through direct binding to the sequencing was the almost ubiquitous presence of one or more actin related protein (Arp)2/3 complex13–15 and interactions with Src homology domains (for example, SH1, SH2 and SH3). For the Arp2/3 nucleation activator protein, neuronal Wiskott–Aldrich example, FAK itself emerged as an unconventional PTK (that is, Syndrome protein (N-WASp).16,17 Cortactin also stabilizes binding SH1 domain), tensin was the first reported example of an SH2 of Arp2/3 to F-actin networks, consistent with its function in domain in a cytoskeletal protein, and both Cortactin and p130CAS lamellipodia dynamics.18–20 Studies conducted with the N-WASp contained SH3 domains. P120, on the other hand, was similar to activating proteins WIP and Nck1 demonstrate that tyrosine b-catenin, as both proteins contained central Armadillo Repeat phosphorylation of cortactin enhances Arp2/3 activation through Domains. P120 was initially viewed as an outlier based on the lack binding of the adaptor protein Nck1, which in turn activates of PTK-associated domains. However, both p120 and b-catenin are N-WASp directly or through the association of the cortactin–Nck1 prominent Src substrates that together control the dynamic complex with WIP.21 Further work on invadopodia formation interface between classical cadherin complexes and the actin confirmed that Nck1 binding to cortactin is critical for promoting cytoskeleton. Importantly, they bind and regulate all 26 members cytoplasmic N-WASp-mediated Arp2/3 actin nucleation.22,23 These of the human classical cadherin family, thereby accounting for observations indicate a positive role for Src-mediated cortactin substantial control over cell–cell adhesion systems in essentially phosphorylation in regulating Arp2/3-based actin filament all solid tissues. Thus, despite distinctly different roles, all of formation. the substrates share close functional connections to the actin Changes in cellular actin filament polymerization are hallmarks cytoskeleton. of Src-transformation and cortactin is well positioned to regulate The substrates are now well known and firmly established in these changes. Invadosomes (aka, podosomes and invadopo- the literature. The following sections contain brief accounts dia),14 comprise another hallmark of cancer found in most of progress to date for each of the five novel substrates (Table 1). carcinoma cell types, where they direct proteolytic degradation As they were discovered as part of a larger effort to understand of the underlying basement membrane, thereby facilitating local the mechanism(s) underlying transformation by Src, each section tumor cell spread.24,25 Cortactin expression and Src-based at least briefly reflects on the nature of that relationship. phosphorylation are essential for invadosome formation and

Table 1. Tyrosine-phosphorylated substrates

Substrate Cellular function Possible role in transformation identified

p85-Cortactin Dynamic regulation in actin filaments Promotes invadosomes formation, stabilizes lamellipodia p110-AFAP1 Actin filament cross-linking Regulation of Src activity, cell contractility and invadosomes stability p130Cas Protein interactions in focal adhesion complexes and at membrane Regulation of cell adhesion and cell survival protrusions p125FAK Regulation of signaling in focal adhesion complexes Regulation of cell adhesion, survival, and cell–cell contacts p120CTN Regulation of cell–cell adhesion by classical cadherins Regulation of cell–cell contacts The five substrates identified during the course of this study, their cellular function and potential role in Src-directed transformation.

Oncogene (2014) 4537 – 4547 & 2014 Macmillan Publishers Limited Substrates of Src AB Reynolds et al 4539 function,22,26,27 suggesting that tyrosine phosphorylation of dynamic changes in actin filament cross-linking is regulated by cortactin is a critical regulator of the early metastatic cascade. PKCa-mediated phosphorylation, which modulates the con- Indeed, cortactin is now a widely accepted marker for formation and multimer size of AFAP1 in a fashion analogous to invadosomes and may have a role in regulating the dynamic deletion of the Lzip motif.44 Thus, AFAP1 mediates dynamic changes in actin polymerization required for invadopodia function changes in actin filament cross-linking in response to PKCa in transformed cells. phosphorylation. Interestingly, several lines of evidence suggest that AFAP1 can Mouse models and human disease function as an upstream activator of Src. For example, deletion of the AFAP1 Lzip motif enables autonomous activation of c-Src in Interestingly, embryo formation was blocked by constitutive cells.46 In a potentially analogous scenario, PKCa can promote Src cortactin knockout (using gene trap technology) due to mis- activation by an AFAP1-dependent mechanism. Overall, the data localization of the Arp2/3-F-actin network and inability of the 28 suggest that AFAP1 phosphorylation by PKCa induces structural oocyte to undergo proper asymmetric cell division. In adult changes (for example, destabilization of AFAP1 multimers) that mice, however, the effect of global disruption of the gene potentiate both its actin-filament cross-linking activity and its (induced by Cre-lox activation) was relatively mild, the main positive effects on c-Src.47–50 A working model based on these phenotype being increased vascular permeability. A possible data proposes that the Lzip motif functions as an auto inhibitor explanation for the discrepancy is that expression of the amino- of AFAP1 activity by stabilizing the multimeric configuration, terminal end of cortactin by the gene trap vector may have a a condition relieved by upstream activation of PKCa. Notably, dominant-negative affect, as apposed to the null phenotype 29 AFAP1 knockdown has little effect on the Src-transformation associated with the conditional knockout. phenotype. Thus, AFAP1 is apparently an upstream effector of EMS1 amplification occurs in multiple carcinoma types, suggest- 30 c-Src, but its role downstream of activated Src, does not modulate ing a role for cortactin in human malignancy. In HNSCC, increased transformation. copy number correlates with poor patient survival, increased recurrence and lymph node involvement.31,32 In premalignant HNSCC lesions, EMS1 overexpression associates with progression to Mouse models and human cancer malignancy.33 Other effects of ems1 gene amplification in HNSCC To date, AFAP1 has not been extensively investigated with regard include increased invasive disease, Arp2/3 activation, invadopodia- to potential roles in human cancer. Its expression, however, is associated matrix degradation, metalloproteinase secretion and significantly elevated in at least two cancers, namely cancer of the resistance to targeted therapeutics and radiation.34–37 These prostate and neuroblastoma.51,52 Together, these data indicate observations suggest a prominent role for cortactin amplification that AFAP1 may be positioned to promote cancer progression, and/or overexpression in HNSCC. possibly through its function as a signaling protein or as a regulator of cytoskeletal integrity and cell contractility. Its role as a c-Src activating protein and regulator of stress filament cross- P110/AFAP1 linking in tumor formation in mouse models has not been p110/AFAP1 (formerly AFAP-110) was initially identified as a determined. The establishment of mouse models will be 110-kDa tyrosine-phosphorylated protein that bound to activated important to determine a role for AFAP1 in cancer progression. forms of Src in an SH3- and SH2-dependent fashion.7,8,38 Immuno- Early results indicate that AFAP1 knockout mice appear to have fluorescence analysis of normal cells revealed an association with deficits in lactation, indicating AFAP1’s normal function could stress filaments and the cortical actin matrix, giving rise to the have an important role in breast physiology (Cho, Flynn and name ‘AFAP-110’ (actin filament-associated protein 110 kDa).8,38 It Cunnick, manuscript in preparation). This mouse model may also was later re-named ‘AFAP1’ (actin filament-associated protein-1) be a useful tool to determine a role for AFAP1 in tumor formation by the Human Genome Project to distinguish it from newly or tumor progression. discovered family members AFAP1L1 (AFAP1-like 1) and AFAP1L2 (AFAP1-like 2).39,40 P130CAS (CAS) Of the 15–20 Src substrates evident on the original pTyr western Structure blots, p110 (AFAP1) and p130Cas (Cas) were singled out because cDNA library screening initially led to the discovery of the it strongly coimmunoprecipitated with Src, a potential indicator neuronal form of AFAP1, a 120-kDa protein called AFAP-120 of physiological relevance.8,38,53 The p130 interaction turned out expressed exclusively in neurons.41 Further screening turned up to be dependent on the Src SH2 domain, suggesting an SH2- the fibroblast form of AFAP1, which lacks just one codon relative mediated interaction with a phosphotyrosine epitope on p130. to the neuronal form.42 Structure–function analyses revealed an This same p130 protein also associated with the v-Crk actin filament binding motif in the AFAP1 carboxy-terminus,43,44 oncoprotein (p47v-Crk). The p130 cDNA was later cloned by preceded by a series of protein-binding modules, including 2 Hirai et al.,54 who proposed the name p130Cas for ‘Crk-associated pleckstrin homology domains, one of which binds to the protein substrate’. kinase C (PKC) isoforms a, b, g and l. AFAP1 also contained SH2-binding motifs (generated by Src phosphorylation) and an Structure SH3-binding motif that facilitates stable interactions with Src, as well as the Src-family members Fyn and Lyn.41,45 These data Analysis of the amino-acid sequence of p130Cas (Cas) predicted a indicate that AFAP1 may function by bridging interactions unique domain structure consisting of an amino-terminal SH3 between Src-family kinases and the actin cytoskeleton. domain and a centrally located ‘substrate-binding domain’ containing 15 Tyr-X-X-Pro motifs hypothesized to serve as binding sites for the Crk SH2 domains when phosphorylated. The carboxy- Cellular function and effects of phosphorylation terminus contained an additional predicted Src SH2 domain- In addition to direct interactions with actin filaments, AFAP1 can binding site and an adjacent poly-proline motif likely to function multimerize via a carboxy-terminal leucine motif43,44 to function as an SH3 domain-binding site for Src.55 Analysis of the dynamic as an actin filament cross-linking protein. Deletion of this motif interaction between Src and Cas led to a novel paradigm of reduces multimer size, and induces a transition from cortical fibers processive binding between Src and its substrates, whereby the to tighter actin filament bundles. The ability of AFAP1 to affect kinase forms an initial, lower affinity interaction with the substrate

& 2014 Macmillan Publishers Limited Oncogene (2014) 4537 – 4547 Substrates of Src AB Reynolds et al 4540 (in this case via the Cas SH3 domain) before enzymatic response to antiestrogens and cytotoxic agents.84,89,90 The ability modification of the substrate.56,57 Ensuing phosphorylation of of Cas to bind to, and activate, c-Src62,91,92 has an important role in the adjacent tyrosine residue on Cas then provides a binding site this resistance pathway89,90 demonstrating a clear link between for the Src SH2 domain, significantly strengthening the interaction Cas, Src and cell survival. between the two proteins.55 A similar model was predicted for AFAP1, whereby SH3-directed interactions were required for tyrosine phosphorylation of AFAP1 and high-affinity binding FOCAL ADHESION KINASE, P125FAK (FAK) with Src.58,59 Binding of Cas to the c-Src SH3 domain also results Early studies demonstrated that in normal chick embryo in release of c-Src’s autoinhibitory conformation resulting in fibroblast cells, the only major substrate phosphorylated on activation of the kinase.39,56,60–62 Thus, as with AFAP1, p130Cas tyrosine was a single 125 kDa phosphoprotein.93 Ahintregarding can function as an upstream activator of c-Src. Cas is now the function of p125 came from observations that levels of recognized as the prototype of a four-member family of adaptor/ phosphotyrosine increased in integrin-dependent adhesion scaffolding proteins that share common structural and functional structures upon attachment of cells to fibronectin,94 cross- features.63–66 In addition, to Cas (also known as BCAR1 for breast linking cell surface integrins95 or platelet activation and adhesion cancer anti-estrogen resistance-1), this family includes NEDD9 to fibrinogen.96 (also known as HEF1 and Cas-L), EFS (also known as Sin) and CASS4 (also known as HEPL). Structure Identification of the p125 protein elicited excitement during the Cellular function and effects of phosphorylation early phases of this project. At that time, BLAST databases As with several of the other Src substrates discussed in this review, came into use and early identification of coding sequences Cas becomes phosphorylated in response to integrin engagement, revealed that p125 was a tyrosine kinase. Further, studies by localizes to focal adhesions and has a critical role in adhesion- other colleagues in the field demonstrated that integrin dependent cytoskeletal remodeling and cell migration.67–72 These engagement was associated with increased tyrosine phosphor- activities are mediated by the processive phosphorylation of ylation in focal adhesion complexes. With p125 thought to have a tyrosine residues located in the Cas substrate-binding domain and role in regulating integrin engagement and signaling, the lab subsequent association with Crk, leading to activation of Rac1 gave much thought into naming the protein, ultimately deciding and Rap1.73–75 Cas-Crk coupling may also contribute to its function on focal adhesion kinase (FAK), due to its colocalization with as a mechanotransducer, as Cas phosphorylation is signi- focal adhesion complexes and intrinsic kinase activity. The ficantly enhanced under conditions in which the Cas substrate- observation that FAK localized to regions of the cell that closely binding domain is physically stretched in response to integrin adhere to the extracellular matrix indicated that it might function engagement.76 Cas signaling is involved in the regulation of cell in the transduction of biochemical signals both in response to death once apoptosis has been triggered, which is mediated by a and in the control of cell adhesion.97 The domain organization of 31-kDa carboxy-terminal Cas fragment produced by the action of FAK consists of a FERM (Band Four.one/Ezrin/Radixin/Moesin) apoptosis-induced proteases.77,78 Independent expression of the domain, catalytic domain, a domain of unknown structure 31 kDa fragment causes diverse pro-apoptotic cellular activities. containing several proline-rich regions and a C-terminal In addition to adhesion signaling, Cas has been implicated in domain that mediates localization (the focal adhesion targeting the control of cell proliferation and survival. Cas tyrosine domain).98 phosphorylation is induced in response to a number of 79,80 mitogenic stimuli. However, the mechanisms by which Cas Cellular function and effects of phosphorylation phosphorylation regulates cell proliferation are complex and not Tyrosine 397 is a major site of phosphorylation in FAK and has a yet clear. Cas becomes dephosphorylated on tyrosine and critical role in signaling as a docking site for SH2 domain- phosphorylated on serine/threonine residues during mitosis, containing proteins, including Src.97,99 The relationship between which leads to disruption of Cas/FAK/c-Src interactions and FAK and cell adhesion is complex. Integrin-dependent cell dissolution of focal adhesions.81 These changes in adhesion to extracellular matrix proteins stimulates FAK activity phosphorylation may enable Cas to affect dynamic changes in resulting in tyrosine phosphorylation on multiple residues.98 FAK focal adhesions in response to phosphorylation by Src and controls a number of biological responses regulated by cell subsequent dephosphorylation. adhesion, including anoikis, proliferation and cell migration. One role of FAK in the control of cell migration is the regulation of the Mouse models and human disease assembly and disassembly of focal adhesions, adhesive structures Mouse models of breast tumorigenesis further support a role for that tether cells to the underlying extracellular matrix.100–102 FAK Cas in cell proliferation and survival, as mammary gland-specific mutants that cannot be phosphorylated at tyrosine 397 are overexpression of Cas results in tissue hyperplasia coincident defective for disassembly of cell/matrix adhesions. In this regard, with Src and Akt activation.82 Furthermore, when crossed with a Src is likely an important signaling partner for FAK, as Src null cells HER2-neu mammary tumor model, Cas overexpression results in exhibit a similar phenotype.102 shortened tumor latency, while knockdown of Cas in cells isolated FAK regulates cell adhesion through multiple mechanisms. from HER2-neu tumors results in increased apoptosis.82 As an The FAK/Src complex controls phosphorylation and the activity integral component of signaling pathways that control cyto- of PIP5KCI, an isoform of phosphatidylinositol 50 kinase that skeletal dynamics, adhesion, migration, proliferation, survival and discretely localizes to cell/extracellular matrix adhesions, regulates apoptosis, Cas expression positively correlated with poor the generation of PI(4,5)P2 at these sites, and the subsequent prognoses in a number of human cancers, including breast, assembly of these structures.103 FAK can also regulate cell prostate, ovarian and lung.65,83–88 It remains undetermined adhesion through Rho family GTPases.104–108 The focal adhesion whether these pathological states are a direct consequence of targeting domain of FAK mediates binding to two focal adhesion- Cas function and, if so, whether specific Cas signaling nodes can associated proteins, paxillin and talin, and each has been be targeted as a means of disease management. Nonetheless, Cas implicated in directing FAK localization to focal adhesions and in has clearly shown an ability to enhance cell proliferation and the regulation of cell adhesion.109,110 Paxillin had been identified survival. For example, overexpression of Cas in breast cancer cells as a Src substrate by Glenney et al.111 FAK can directly and can result in a failure to undergo cell cycle arrest and apoptosis in indirectly (via Src) promote paxillin phosphorylation on tyrosine.

Oncogene (2014) 4537 – 4547 & 2014 Macmillan Publishers Limited Substrates of Src AB Reynolds et al 4541 Like FAK, paxillin is required for proper turnover of cell/matrix P120-CATENIN, P120CTN, P120 adhesions and mutants of paxillin that are defective for tyrosine Interest in p120 was initially based on the profiling of tyrosine- phosphorylation are defective for regulation of adhesion phosphorylated proteins in cells expressing activated forms of Src 102 turnover. The interaction of FAK and talin is also important. that were either transformation-competent or transformation- FAK is required for the recruitment of talin to nascent focal defective. Remarkably, p120 was unique among the substrates in 112 adhesions but not to mature focal adhesions; Talin has a role in that its phosphorylation correlated with cell transformation, recruiting or stabilizing FAK in mature adhesions. The interaction suggesting a possible role in the mechanism of transformation.53 between FAK and talin113 could offer another mechanism for regulating adhesion. Further, FAK may regulate the cleavage of Structure talin by calpain, which is an important mechanism for regulation of adhesion.112,114 p85-Cortactin also binds via its SH3 domain to Most of the substrates emerged as unique regulators of the actin two proline-rich sequences in the C-terminal domain of FAK. cytoskeleton, each possessing one or more domains typically Mutation of these sites in FAK impairs its ability to regulate cell/ associated with PTK signaling. In contrast, p120 contained tandem 115 armadillo repeats with weak homology to the Armadillo repeats in matrix adhesion turnover. Although these sequences mediate 139,140 interaction with multiple proteins, cortactin may be the critical b-catenin/Armadillo. Further analysis revealed sets of binding partner for controlling adhesion remodeling.115 FAK also tandem repeats in p120 and several other proteins, leading to regulates recruitment of dynamin to adhesive structures, via a the recognition and naming of the Armadillo Repeat Domain, a direct interaction with the FAK FERM domain or an indirect structure that has emerged over the last decade as an important interaction via a phosphorylated tyrosine residue in the protein–protein interaction scaffold. Notably, the similarity to b- C-terminus of FAK (tyrosine 925), and dynamin has been catenin led to testing for an analogous interaction with classical implicated in the disassembly of adhesions.116,117 Thus, FAK may cadherins. It turned out that p120 and b-catenin interact simultaneously with adjacent motifs in the cadherin cytoplasmic regulate cell adhesion through changes in phosphorylation that 141 affect interactions with different binding partners, including Src. tail. Because of these molecular interactions, p120 was originally called ‘Cas’ for cadherin-associated (Src) substrate.142 FAK has an important role in the organization of the 143 extracellular matrix in tissue culture and the basement membrane That name was soon rescinded in favor of p120-catenin (p120) 118,119 to avoid being confused with the previously described Src in vivo. By regulating expression and secretion of matrix 54 metalloproteinases, FAK also controls remodeling of the matrix substrate p130Cas. after its initial organization, which enables FAK to control cell invasion.120,121 FAK functions in regulating the assembly of Cellular function and effects of phosphorylation adhesive structures that mediate matrix degradation. FAK The catenin identifier turned out to be a highly accurate interactions with Cas are required to regulate invadosome descriptor, as p120 and b-catenin are direct stoichiometric binding formation in several cell types, and the subsequent degradation partners for each of the 26 classical cadherins, important of the underlying extracellular matrix.122 FAK also functions in mediators of cell–cell adhesion in solid tissues. b-catenin interacts controlling extracellular matrix degradation at focal adhesions and further with a-catenin, a vinculin-like actin-binding protein at the Cas is an important FAK-binding partner in controlling matrix dynamic functional interface between the cadherin cytoplasmic degradation at these sites as well.123 FAK is implicated in tail and the underlying actin cytoskeleton. The term catenin (that controlling cell proliferation and degradation of the extracellular is, chain or to link), coined well before the discovery of p120, was matrix in response to mechanical signals generated by stiff aimed originally at the a-catenin/b-catenin partnership. However, matrices.124–126 p120 also modulates the actin interface, albeit via direct and/or Although FAK’s role in controlling cell/extracellular matrix indirect regulation of Rho GTPases. For example, cytoplasmic p120 adhesion was not surprising, the finding that FAK has a role in functions as a guanine nucleotide dissociation inhibitor, binding controlling cell–cell adhesions was unexpected. FAK is required for RhoA and blocking its activity by preventing GTP/GDP efficient assembly of N-cadherin- and E-cadherin-mediated cell/ exchange.144,145 Cadherin-bound p120, on the other hand, does cell adhesions and FAK-dependent control of the activity of Rho not bind RhoA directly,144 but instead recruits factors such as family proteins appears responsible for modulating the assembly p190RhoGAP146–148 and ROCK149 that in turn localize RhoA activity of cell/cell junctions.127,128 Impairment of FAK activity also retards to adherens junctions. Hence, p120 and b-catenin are important the disassembly of cell/cell junctions in response to stimuli that regulators of the actin cytoskeleton and participate actively in the promote epithelial to mesenchymal transition.129 Thus, FAK is global integration of cytoskeletal activity. implicated in controlling both the assembly and disassembly of More recently, p120 has emerged as a master regulator of different classes of cell adhesions. classical cadherin stability and cell surface retention. p120 modulates the strength of cell–cell adhesion, in part, by controlling cadherin levels. Nascent cadherin complexes Mouse models and human disease arrive at the cell surface independent of p120. Thereafter, direct FAK is essential for mouse development, as demonstrated by the interaction with p120 is essential as the complex is otherwise embryonic lethal phenotype of FAK-null mice.130 These embryos rapidly internalized and degraded. Thus, loss of p120 in most exhibit defects in mesoderm development, neural tube closure adhesive cell types results in reduced or complete loss of and heart tube formation.131 The analysis of conditional knockout cell–cell adhesion. Mechanistically, p120 prevents cadherin mice has provided additional insight into FAK’s role in endocytosis by physically masking crucial endocytic signals in development. These include essential roles in embryonic the cadherin juxtamembrane domain.150 Recent evidence angiogenesis,132,133 development of the heart/aorta134 and suggests a second role, however, as the cell fate and endocytic differentiation of neural crest cells.135 Further, in mouse models adaptor protein Numb can regulate endocytosis of the entire FAK has also been implicated in the regulation of axonal cadherin complex (p120 included) through direct interaction branching,136 myelination137 and mammary gland development with p120.151 during pregnancy.138 As outlined below, FAK is implicated in a Transformation by Src in many cell types is associated with number of human diseases. Thus, FAK appears to have an disruption of adherens junctions, actin-based cell–cell adhesion important role in regulating cell adhesion, cell–cell interactions complexes organized by classical cadherins. p120 is also tyrosine- and cell survival, all of which are altered in Src-transformed cells phosphorylated by various growth factor receptors, including the and in human cancer. EGF, PDGF and CSF-1 receptors,152,153 which are functionally

& 2014 Macmillan Publishers Limited Oncogene (2014) 4537 – 4547 Substrates of Src AB Reynolds et al 4542 coupled to cadherins by poorly understood mechanisms.154 competitive, including dasatinib, bosutinib and saracatinib, while The role of p120 phosphorylation in these processes is not yet KX-01 is peptidomimetic.169 Additional non-selective kinase known. p120 itself, on the other hand, is clearly essential for inhibitors have been developed, including nilotinib (Tasigna), transformation by Src and Rac1, but not H-Ras, it remains an open that inhibit Src-family kinases amongst their off-target activities.170 question, therefore, as to whether tyrosine phosphorylation of Although anti-Bcr-Abl activity has enabled dasatinib, bosutinib and p120 in fact has an essential role in transformation. nilotinib to be marketed for CML, the activity of the four Src inhibitors in clinical trials of solid tumors has been less robust.165,166 Src regulates tumor cell migration, invasion and Mouse models and human disease adhesion in a variety of model systems, suggesting a role in The in vitro effects of p120 loss are also observed in vivo in mouse tumor cell metastasis.171,172 In addition, Src exhibits downstream models, although the complexity of the phenotypes varies activity in both angiogenesis and bone resorption pathways, depending on the epithelial organ targeted. Global p120 knockout indicating functions in tumor cell maintenance and survival.166.Src B is early embryonic lethal ( day 9.5). Targeted deletion in the also has roles in tumor cell proliferation and cell cycle control in intestinal epithelium causes cell–cell adhesion defects and serious both in vitro and in vivo pre-clinical models of many major solid intestinal bleeding, leading to inflammation and death in the first 155 tumor types, including prostate, breast, colon, lung, pancreatic three weeks of life. Curiously, p120 ablation on a very limited and glioblastoma, among others.173,174 The pre-clinical antitumor B basis (that is, 10% of the intestinal epithelial surface) by itself activities of Src inhibitors provided the rationale, which prompted induces intestinal tumors in about half of the mice, but tumors 156 investigators to evaluate Src inhibitors in multiple solid tumor invariably retain at least one p120 allele. In the squamous clinical trials.166 Early trials show promise, leading to advanced epithelia of skin and esophagus, p120 induces an apparently cell studies evaluating combinations with chemotherapy or other autonomous inflammatory response (that is, it cannot be 157,158 targeted agents. One possibility for the limited success of Src attributed to a barrier defect). These clues suggest an as inhibitors as single agents is their inability to induce apoptosis, yet poorly understood role for p120 in the innate immune system. potentially required for clinical antitumor activity, despite their The esophageal phenotype involves progression to esophageal inhibitory effects on tumor cell proliferation and xenograft cancer that closely resembles the human condition. In contrast, growth.173 The significant effects of Src inhibitors in tumor cell the mammary gland does not form at all in the absence of 159 motility in vitro may impart a critical activity on patient metastases. p120, and the salivary gland displays misshapen ducts that lack Previous and ongoing clinical trials evaluating the effects of acini.160 Remarkably, the epithelium of the prostate is not 159 Src inhibitors in metastatic cancer are critical for shaping our noticeably affected, and is the only system to date lacking an understanding of their antitumor capacity. Evaluating early stages obvious phenotype. Despite significant tissue-specific variation, of malignancy such as in adjuvant settings and studying rational the mouse models in general illustrate multiple connections to combinations of Src inhibitors with chemotherapy or other cancer and will ultimately be essential for understanding the roles targeted agents is warranted. Another limitation for the success of p120 misexpression in human cancer. Indeed, the human of Src inhibitors in solid tumors may be a lack of standard pathology literature reveals frequent mislocalization and/or 161 biomarkers. Such data could identify responsive patients, and downregulation of p120 in most epithelial cancers. The clinical trials could focus on those patients that demonstrate a mechanism(s) of p120 dysregulation in human cancer are not molecular response to treatment. The potential for establishing yet clear but the involvement of receptor- and Src-family tyrosine novel Src biomarkers may exist within the rich panel of Src kinases remain a strong possibility. substrates.8 The discovery of Src substrates provided an important cornerstone in the development of agents targeting downstream PHARMACEUTICAL TARGETING OF SRC AND ITS SUBSTRATES effectors of Src function.5 One key target is FAK, implicated in Among the substrates identified in this study, FAK, as well as Src, multiple cancer types.175–178 Similar to its upstream activator Src, appear to have the greatest promise as druggable targets for the FAK promotes tumor cell motility, survival and angiogenesis, treatment of cancer. The approach to identify new signaling thereby validating its potential utility in the clinical setting.175–180 pathway components remains an important element in the Five small-molecule FAK inhibitors have been described, several discovery of targets for pharmaceutical development. However, having advanced into clinical trials.181–183 Although early in the lengthy path from target identification to drug registration development, these agents show promise for use in the shifted the effort toward later stages of preclinical development. armamentarium against different tumor types. Similarly as Companies save time and money if their starting point involves a observed for Src inhibitors, perhaps the pre-clinical validation of preclinically, well-validated target before initiating a drug hunting FAK in survival and cell motility points to the potential activity of campaign.162–164 For these reasons, drug targeting of Src and its inhibitors as potent anti-metastatic agents rather than impacting substrates proves a critical opportunity for the discovery and primary tumors.179–183 Combination therapies that address tumor development of new therapies for cancer and other diseases. de-bulking (for example, chemotherapy) with an anti-metastatic There have been significant strides in developing novel therapies drug (for example, potentially FAK inhibitors) may have the best inhibiting Src.165,166 However, the pharmaceutical industry should patient response rate and progression-free survival. Future and explore new avenues of target discovery to ensure future ongoing clinical evaluation of existing and new inhibitors should development of highly effective treatments. Targeting Src help to answer these fundamental questions. substrates represents a rational next step toward the deliberate Other substrates of Src have been studied exten- intervention of this key signaling cascade in multiple disease sively.5,46,65,68,184–187 The substrate proteins p130Cas, p120Ctn, states. p110-AFAP1, p85-cortactin and p210 tensin exhibit functional To date, four small-molecule inhibitors of Src have been interactions with important partner proteins.5,46,65,68,184–187 Such developed for treatment of various malignancies, including chronic interactions are crucial for their functions in tumor cell biology. myelogenous leukemia (CML).165,166 Among these, dasatinib Pharmaceutical intervention of protein–protein interactions (Sprycel) and bosutinib (Bosulif) exhibit low nanomolar activity emerged as a potential area for further exploration.188,189 against Src-family kinases.167,168 However, they both equally inhibit Although many biological therapies are marketed based on Bcr-Abl kinase, the pivotal target in CML, and other tyrosine- disruption or stabilization of protein–protein interactions, the and serine/threonine-specific kinases, thereby promoting their approach remains challenging for small-molecule development anti-CML activity.167–169 Three of the inhibitors are ATP- targeting intracellular proteins. The large surface area between

Oncogene (2014) 4537 – 4547 & 2014 Macmillan Publishers Limited Substrates of Src AB Reynolds et al 4543

Figure 1. The five substrates identified during the course of this study largely regulated cytoskeletal processes that are affected by Src- transformation. P120CTN regulates E-cadherin interactions between cells, p85-cortactin regulates invadopodia formation and actin polymerization, p110-AFAP1 regulates actin filament cross-linking, p125FAK and p130Cas regulate cell adhesion complexes.

protein domains and few binding pockets amenable to small- application of appropriate combinations of new medicines molecule docking limits this approach. Peptide scaffolds provided customized for each patient. the basis for generating some protein–protein interface inhibi- tors.188,189 Recently, protein–complex stabilizers that enhance the retention time of key complexes have been generated and may SUMMARY 188,189 have therapeutic utility. Allosteric site modulators offer an Twenty years of study has provided much information about the alternative approach when targeting outside of enzymatic structure and function of Src substrates. The work highlighted domains, such as those that inhibit myristate binding in the herein was among the first to describe an important group of Src 190 autoinhibitory pocket of the Bcr-Abl kinase. A variety of substrates involved in the regulation of cell adhesion and actin potential therapeutic agents have been generated for protein dynamics (Figure 1). Notably, the main roadblock from outset had interfaces that possess high affinity, but are larger than typical little to do with the technical challenges. Indeed, by 1989, 188,189 pharmaceuticals. Overall, the approach has significant merit, just 2 years into the project, both mAbs and partial cDNAs for and the industry is poised to harvest novel medicines from the most of the substrates were already in hand, but it was not a large breadth of protein-complexes involved in tumor cell biology. project for the risk averse. Although the jury is perhaps still out on The Src substrates and their cognate interactions may serve p120, the substrates identified did not turn out to be essential for as targets for pharmaceutical development, possibly by RNA transformation. They were, however, among the first bona fide Src interference, and as clinical biomarkers for drugs that interfere substrates identified, and taught us a great deal about Src and its with Src-associated pathways. substrates. Two decades later, the original goals of the project Targeting Src, FAK or other substrates may provide critical new seem rather unimportant compared to the enormous impact of drugs for cancer treatment, but may best be served in many of the substrates in areas that could not have been combinations with other drugs in parallel or convergent path- anticipated a priori. ways. The development of resistance is common among Driven by a compelling question, application of novel targeted agents, particularly with kinase inhibitors. Some muta- technologies, and the willingness to invest time and energy in a tions in kinases remain sensitive to drugs, while others are ‘risky’ venture, we began the search for the Src substrates. In resistant, such as the T315I mutation of Bcr-Abl not inhibited today’s difficult funding environment, we must consider whether 169 by either dasatinib or bosutinib. Drug combinations that this type of scientific effort will garner support. The substantial include multiple Src pathway inhibitors may provide a rational effort to identify and characterize Src substrates involved not only approach for reducing relapse for malignancies where Src exhibits the authors of this review, but a number of post-doctoral fellows, a direct role. graduate students and technicians. Importantly, the rewards have The discovery of the Src substrates exemplifies a ‘shotgun’ been notable. A survey of PubMed citations lists over 6000 papers 8 approach to identify multiple proteins involved in Src biology. on these substrates discussed above. Understanding the function Today, microarrays, protein arrays, yeast two-hybrid systems, of these proteins far exceeded the original goal of understanding synthetic lethality screens and other technologies enable how Src functions to transform cells and has in addition provided researchers to identify putative targets in multiple settings, novel insights as to how these proteins contribute to the complex including signal transduction pathways, protein networks and regulation of cellular dynamics. patient responses in clinical trials to experimental agents. Broad approaches to the discovery of critical connections between cellular proteins require exploration to enhance our ACKNOWLEDGEMENTS understanding of the cell system beyond an individual Many thanks to Steven Bernhardt for critical review and suggestions for organization 191 molecular pathway. Defining the system biology and of this review. Also, we are grateful for the help of Jenny Michelle Reed in the understanding tumor microenvironments may hasten the clinical preparation of this review.

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