(2004) 23, 7906–7909 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc

Src family , key regulators of signal

Sarah J Parsons*,1 and J Thomas Parsons*,1

1Department of Microbiology and Center, University of Virginia Health System, Charlottesville, VA 22908-0734, USA

The Src family of kinases (SFKs) plays cellular gene (the proto-oncogene, c-Src); Srcactivity key roles in regulating by a diverse set was regulated by intramolecular interactions controlled of surface receptors in the context of a variety of by tyrosine ; and domains of Src(SH2 cellular environments. SFKs have evolved many ingenious and SH3) mediated protein–protein interactions with molecular strategies to couple receptors with the cyto- sequences containing phosphotyrosine and proline-rich plasmic signaling machinery. The contributions to this motifs. Concurrent with the studies on Src came the issue of ONCOGENE describe how this machinery recognition that Src was a member of a large family of regulates fundamental cellular processes, including cell structurally related kinases (Table 1) (Thomas and growth, differentiation, cell shape, migration and survival, Brugge, 1997), many of which were expressed predomi- and specialized cell signals. The pleotropic functions of nantly in highly differentiated cell types. As described in Src and Src family members underscore the importance of the chapters of this issue, Src and family members play these kinases and explain why many of the members of an important role in regulating signals from cell surface this family have been identified as cellular . In receptors. SFKs have evolved many ingenious molecular this volume, we have attempted to provide the reader with strategies to couple receptors with the cytoplasmic an overview of the current understanding of the function of signaling machinery. As described herein, this machin- Src family kinases in the regulation of selected cellular ery regulates many fundamental cellular processes, signaling pathways. including , differentiation, cell shape, migra- Oncogene (2004) 23, 7906–7909. doi:10.1038/sj.onc.1208160 tion and survival, and specialized cell signals. The pleotropicfunctions of Srcand Srcfamily members Keywords: tyrosine ; transformation; cancer; cell underscore the importance of these kinases and explain proliferation why many of the members of this family have been identified as cellular oncogenes (Blume-Jensen and Hunter, 2001). In this volume, we have attempted to provide the reader with an overview of the current Introduction understanding of the function of Src family kinases in the regulation of selected cellular signaling pathways. This issue of ONCOGENE is devoted to the Srcfamily While not comprehensive, we have attempted to of protein tyrosine kinases (SFKs), a family of kinases assemble well-studied examples of how Srcfamily that plays key roles in regulating signal transduction by kinases contribute to the regulation of cellular events. a diverse set of cell surface receptors in the context of Unfortunately, we have not been able to include multiple cellular environments. The origins of the family discussions of all of the interesting and undoubtedly (Martin, 2001) can be traced back almost a century to important roles of Srcin cellularregulation (e.g., the pioneering studies of Peyton Rous, who recognized (Clark et al., 1994; Obergfell et al., 2002), that injection of cell-free extracts from chicken tumors osteoclasts (Rodan and Rodan, 1997; Duong et al., could cause tumors in naive animals, giving rise to the 2000)), or discussions of important Src substrates (e.g., scandalous hypothesis that cancer could be caused by a p190Rho-GAP, Cortactin (Parsons and Parsons, 1997)). transmissible agent (e.g., a virus). Indeed, it took almost 50 years to establish this concept in the scientific literature, but with the acceptance of these ideas came renewed studies on the virus that Rous had originally Historical overview described, Rous sarcoma virus (RSV; Martin, this issue). Over the next 20 years, studies on RSV yielded While the origins of the Srcfamily of kinases are nearly an incredibly rich medley of new and startling informa- a century old, the contemporary understanding of the tion; cellular transformation was caused by a single gene activity of various family members, both as mediators of (Src)(Figure 1); Srcwas a ; Srccatalysed biological signals and as kinases, is recent. Steven the phosphorylation of tyrosine; Srcwas derived from a Martin provides a historical overview of the studies on avian retroviruses and RSV, in particular. He describes *Correspondence: SJ Parsons; E-mail: [email protected] the discoveries that led to the concept of cell transfor- JT Parsons; E-mail: [email protected] mation and the identification of the RSV transforming Src family kinases SJ Parsons and JT Parsons 7907 gene (v-Src), as well as its protein product, and how this SFKs and tyrosine kinases (RTKs) information in turn led to the discovery of cellular Src. The ability of oncogenic forms of SFKs to induce cell transformation pointed early onto a role for Srcand its family members in regulating cell growth. Bromann et al. Structure and regulation of SFKs: structural dissection of provide an overview of how SFKs interact with, and a prototypical modular signaling protein participate in signaling from RTKs. These authors provide a summary of how SFKs are activated by Members of the family exhibit a conserved domain RTKs, and how SFKs in turn activate RTKs. In organization, which includes a myristoylated N-terminal addition, SFKs promote signaling from segment, followed by SH3, SH2, linker and tyrosine receptors in multiple ways, including the direct partici- kinase domains, and a short C-terminal tail (Figure 1). pation in pathways required for DNA synthesis as well As discussed by Boggon and Eck, the structural as controlling the turnover of cell surface receptors, dissection of Src kinases has elucidated canonical modulating rearrangements and mechanisms of phosphotyrosine recognition by the promoting cell motility and survival. SH2 domain and proline-motif recognition by the SH3 domain. Crystallographicanalysis of nearly intactSrc kinases in the autoinhibited state has revealed that these protein interaction motifs turn inward and lock the G-protein-coupled receptors (GPCRs) and SFKs kinase in an inactive conformation via intramolecular interactions. The autoinhibited Src kinase structures GPCRs serve as -activated guanine nucleotide reveal a mode of domain assembly used by other exchange factors (GEFs) for a class of heterotrimeric tyrosine kinases outside the Srcfamily, including Abl guanine nucleotide-binding (G) . and Tecfamily kinases. In addition, the underlying binding catalyses the conformation-dependent exchange regulatory principles uncovered by the analysis of Src of GTP for GDP on the Ga subunit of the hetero- family kinases have proven to be surprisingly general trimericG protein. This, in turn, leads to the dissocia- among diverse modular signaling proteins. Although tion of the GTP-bound Ga subunit from the Gbg considerable structural information is available for the subunit heterodimer, rendering it free to regulate the autoinhibited conformation of Src kinases, how these activity of enzymatic effectors. Ligand activation of kinases assemble into active signaling complexes with many growth factor RTKs leads directly to activation of substrates and regulators is an unresolved question. SFKs, an event essential for propagation of downstream signals. Luttrell and Luttrell discuss the unexpected role for SFKs in GPCR signaling. The strictly dichotomous model of transmembrane signaling by GPCRs has been challenged by the discovery that GPCRs also exert control over cellular growth, proliferation and differ- entiation, and do so by stimulating tyrosine phosphor- ylation cascades. Several mechanisms, from the direct association of SFKs with GPCRs or receptor-associated proteins to the of RTKs and complexes by G-protein-mediated signals, Figure 1 Domain structure of Src family kinases permit GPCRs to activate SFKs. Conversely, Src

Table 1 Characteristics of Src family kinases Src family Pattern of expression Isoforms Oncogenic formsb membera

Blk B cells Fgr Myeloid cells, B cells Oncogenic fusion with gag sequences in feline sarcoma virus; overexpressed in some leukemias and lymphomas Fyn Ubiquitous T-cell-specific isoform (Fyn T) Hck Myeloid cells Two different translational starts Lck T cells, NK cells, brain Overexpressed in T-cell acute lymphocytic leukemias Lyn Brain, B cells, myeloid cells Two alternatively spliced forms Src Ubiquitous -specific isoforms Mutated and truncated in retroviruses; truncated in colon cancer; overexpressed in mammary, pancreatic and other Yes Ubiquitous Oncogenic fusion with gag sequences in avian sarcoma viruses; highly expressed in colon, malignant melanoma and other cancers Yrk Ubiquitous aThomas and Brugge (1997). bBlume-Jensen and Hunter (2001)

Oncogene Src family kinases SJ Parsons and JT Parsons 7908 activity plays a central role in controlling GPCR Emerging roles for p120-catenin in and trafficking and effects on and cytoske- cancer letal rearrangement. As Luttrell and Luttrell point out, GPCRs and Srcfamily kinases do not belong to A large body of evidence points to a role for Src in the separate, exclusive clubs; rather, these strange bed- regulation of cell–cell adhesion. Among the molecules fellows are intimately involved in multilayered forms of that have been implicated in this regulatory pathway is cross-talk that influence a host of cellular processes. p120-catenin, a protein originally identified as an Src substrate in v-Src transformed cells. As described by Reynolds and Roczniak-Ferguson, p120-catenin (p120) is now known to regulate cell–cell adhesion through its SFKs and the regulation of cell adhesion and migration interaction with the cytoplasmic tail of classical and type II . New evidence indicates that p120 regulates The oncogenic potential of several of the SFKs under- turnover at the cell surface, thereby controlling scores the role of these kinases in regulating cellular the amount of cadherin available for cell–cell adhesion. events distinct from those controlling cell proliferation. This function is necessary but not sufficient to promote Indeed the pronounced effects of oncogenic v-Src on cell strong adhesion, which is further controlled by signals morphology, cell adhesion and motility clearly indicated acting on the amino-terminal p120 regulatory domain. a role for Srcin the regulation of cytoskeletal-linked The activities of RhoA, Rac and Cdc42 are also events. Indeed, a growing number of studies directly modulated by p120, suggesting that, along with other points to the role of SFKs in human cancers. Playford Srcsubstrates, p120 contributes to the regulation of and Schaller discuss the connections between SFKs and cytoskeletal dynamics. Reynolds and Roczniak-Fergu- the family of cell adhesion molecules. These son suggest that p120 is a master regulator of cadherin authors review the role of Srcand integrin signaling in abundance and activity and likely participates in normal cells and how integrin signaling is altered in regulating the balance between adhesive and motile human cancer. SFKs target a number of molecules that cellular phenotypes. contribute to the regulation of integrin-mediated signal- ing pathways, including focal adhesion kinase (FAK), and p130CAS. How Srcsignaling affectsthe Role of STATs as downstream signal transducers formation of focal adhesions and interplay with the in SFK-mediated tumorigenesis and how changes in the signals transduced by this pathway contribute to cancer cell The signal transducers and activators of progression are also described. (STATs) were originally identified as components of signaling pathways activated by non-- containing receptors. As a result, the role of Cross-talk between receptors and the c-Src-RTK STATs in regulation of cellular processes is best receptor pathways understood in the context of hematopoietic cell signal- ing. For cytokine receptors, activation of STAT tyrosine In recent years, evidence has emerged showing that both phosphorylation occurs via ligand-induced recruitment steroid and growth factors stimulate prolif- and activation of the intracellular JAK kinases. How- eration of steroid-dependent tumor cells. Regulation of ever, as discussed by Silva, STATs can also be activated steroid receptors is well understood. These receptors are by growth factor receptors, particularly the epidermal ligand-activated transcription factors, which, upon growth factor receptor (EGFR), as well as by SFKs, ligand binding, dimerize, recruit co-regulatory molecules particularly Src. In many cases, a differential activation and activate target gene transcription. As described by of the STATs is mediated by these tyrosine kinases, Shupnick, response to steroid ligands can be function- compared to activation of the /JAK ally linked to activation of Src or RTKs. Ligand- pathway. This difference provides potential for unique independent stimulation of steroid receptor-mediated actions of STATs in response to growth factor receptor transcription by growth factors is now believed to occur and SFK activation. Many cancers, including breast and through activated protein kinases that phosphorylate colon, exhibit elevated expression of SFKs and Src in the receptors and co-regulatory proteins. Recently, particular. In addition, many of these cancers also steroid hormones themselves have been shown to overexpress EGFR and related growth factor receptors. rapidly activate intracellular signaling cascades by virtue As Silva points out, in these cancers, STATs may play of their binding to cognate cytoplasmic or membrane- an important role in the tumorigenesis process. associated receptors. In some contexts, steroid receptors interact directly with Src and other cytoplasmic signal- ing molecules, such as Shc, PI3K and p130Cas.As Src in synaptic transmission and plasticity Shupnick discusses, cross-talk between growth factors and in both the and nucleus may The role of Src in regulating specialized cellular have a profound impact on complex biological pro- functions has been intensely studied in the context of cesses, such as cell growth, and play a significant role in the (CNS). In the CNS, Src and the treatment of steroid-dependent cancers. other SFKs are widely expressed and are abundant in

Oncogene Src family kinases SJ Parsons and JT Parsons 7909 . Srchas been implicated in proliferation and SFKs in B-cell development and signaling differentiation during the development of the CNS. However, Srcis also highly expressed in fully differ- The SFKs play key roles in initiating signal transduc- entiated neurons in the developed CNS, implying tion by the B-cell antigen receptor (BCR). In addition, additional functions. As discussed by Kalia et al., a as discussed by Gauld and Cambier, numerous studies large body of evidence has accumulated showing that a have shown that this family of kinases also contributes primary function of Src is to upregulate the activity of to signaling by BCR surrogates during B-lymphocyte N-methyl-D-aspartate (NMDA) receptors and other ion lineage development and maturation. Paradoxically, channels. NMDA receptors (NMDARs) are a principal loss of function of SFKs not only results in the subtype of glutamate receptors that mediate fast expected defects in B-cell development but also impacts excitatory transmission at most central . Kalia the onset of autoimmunity. Thus, SFKs, most notably et al. describe how Src serves as a regulator of Lyn, play both activating and inhibitory roles in B-cell NMDARs and the role of Srcin NMDAR-dependent function. As described by Gauld and Cambier, an synaptic plasticity. Recent studies provide insights into important, but confounding, issue relating to SFK the regulation of Srcitself at glutamatergicsynapses. By function in B cells is the varied co-expression of family upregulating the function of NMDARs, Src gates the members that mediate redundant as well as unique production of NMDAR-dependent synaptic potentia- functions. tion and plasticity. Thus, Src is critical for processes underlying physiological plasticity, including learning and memory, and pathological plasticity, such as pain and epilepsy. Molecular interdiction of SFK signaling in hematopoietic cells

Function of the SFKs, Lck and Fyn, in T-cell development The ability of SFKs to mediate signaling from cell and activation surface receptors in hematopoietic cells is a function of their catalytic activity, location and binding partners. Studies in cell lines and animal models point to a critical SFK activity is regulated in the cell by regulatory role for Lck and Fyn in proximal T-cell antigen receptor kinases and that alter the state of (TCR) signal transduction. As discussed by Palacios and phosphorylation of key tyrosine residues, and by Weiss, these kinases are recruited to the TCR, where protein-binding partners that stabilize the kinase in they phosphorylate TCR at sequence motifs referred to active or inactive conformations or localize the as ITAMs (immunoreceptor tyrosine-based activation to specific subcellular or submembrane domains. As motifs) on the CD3 and z chains. Phosphorylation of Geahlen et al. discuss, kinase activity and function can these sites, in turn, serves to create sites for Syk be modulated experimentally through the use of small- family kinases. The recruited Syk family kinases are then molecule inhibitors designed to directly target catalytic activated by SFK-dependent phosphorylation. Within or binding domains or regulate the location of the the , Lck and Fyn are spatially segregated protein by altering its state of acylation. The authors by localization to different lipid rafts and are thought to discuss the significant potential for using such small undergo sequential activation. SFKs are linked to the molecules as therapeutic agents. TCR via association with the CD4 and CD8 co- receptors; however, additional adaptor proteins, for example, Unc119, may also serve to link SFKs with the TCR. CD45 and Csk serve as positive and negative Epilogue regulators of SFK functions. Several stages of T-cell development require TCR-based signaling, including As readers will glean from the following chapters, pre-TCR signaling, positive selection, peripheral main- studies on SFKs have provided a rich experimental tenance of naive T cells and lymphopenia-induced milieu for understanding cell regulation. We wish to proliferation. SFKs, particularly Lck, appear necessary thank our colleagues who have devoted their valuable for each of these TCR-based signals. time in helping compile this volume on the SFKs.

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