Selected Glimpses Into the Activation and Function of Src Kinase

Jerey D Bjorge1, Andrew Jakymiw1 and Donald J Fujita*,1

1Cancer Biology Research Group, Department of Biochemistry and Molecular Biology, University of Calgary Medical Center, 3330 Hospital Dr. N.W., Calgary, Alberta T2N 4N1, Canada

Since the discovery of the v-src and c-src genes and their ment of the focus assay by Temin and Rubin (Temin products, much progress has been made in the elucidation and Rubin, 1958), and advances made in the under- of the structure, regulation, localization, and function of standing of biological properties and host range the Src protein. Src is a non-receptor protein tyrosine speci®city of avian tumor virus in cell culture by the kinase that transduces signals that are involved in the laboratories of P Vogt, H Hanafusa and others control of a variety of cellular processes such as (Hanafusa, 1965; Vogt and Ishizaki, 1966) established proliferation, dierentiation, motility, and adhesion. Src RSV as a highly favorable model tumor virus system. is normally maintained in an inactive state, but can be A series of elegant pioneering genetic and biochemical activated transiently during cellular events such as studies in the late 1960's and 1970's by several mitosis, or constitutively by abnormal events such as laboratories ushered in a period of rapid and exciting mutation (i.e. v-Src and some human cancers). Activation progress in RSV and Src research. These studies led to of Src occurs as a result of disruption of the negative the elucidation of RSV gene functions and genome regulatory processes that normally suppress Src activity, structure (Robinson et al., 1967; Wang et al., 1975a,b; and understanding the various mechanisms behind Src Joho et al., 1975), identi®cation of v-src as the only activation has been a target of intense study. Src RSV gene that was required for triggering cell associates with cellular membranes, in particular the transformation (Martin et al., 1971; Vogt, 1971; Lai plasma membrane, and endosomal membranes. Studies et al., 1973; Wang et al., 1975a) and the discovery indicate that the dierent subcellular localizations of Src that the v-src gene product pp60v-src, or v-Src, was a could be important for the regulation of speci®c cellular phosphoprotein with an apparent molecular mass of processes such as mitogenesis, cytoskeletal organization, 60 kDa (Brugge and Erikson, 1977; Purchio et al., and/or membrane tracking. This review will discuss the 1978). Shortly thereafter, it was found ®rst that the v- history behind the discovery and initial characterization Src protein possessed an intrinsic protein kinase of Src and the regulatory mechanisms of Src activation, activity (Collett and Erikson, 1978; Levinson et al., in particular, regulation by modi®cation of the carboxy- 1978), followed by the discovery that Src was a terminal regulatory tyrosine by phosphatases and protein kinase that phosphorylated tyrosine residues kinases. Its focus will then turn to the dierent (Hunter and Sefton, 1980; Collett et al., 1980). In subcellular localizations of Src and the possible roles of contrast, most other protein kinases at that time were nuclear and perinuclear targets of Src. Finally, a brief known to phosphorylate only serine or threonine section will review some of our present knowledge residues. In addition, research on RSV, as well as regarding Src involvement in human cancers. Oncogene independent studies on the murine leukemia retrovirus (2000) 19, 5620 ± 5635. MLV, led to the co-discovery of reverse transcriptase (RNA-dependent DNA polymerase) (Temin and Keywords: Src; tyrosine kinase; protein tyrosine phos- Mizutani, 1970; Baltimore, 1970). phatases; oncogene; cancer; cellular localization The discovery of reverse transcriptase not only greatly facilitated the development of the modern era of molecular cloning, but also was instrumental in Introduction the synthesis and puri®cation of a v-src speci®c cDNA, which was an essential reagent in the The cytoplasmic tyrosine kinase Src has been the discovery of c-src, the cellular homologue and subject of widespread interest for several years, either progenitor of v-src, in normal cellular DNA in the form of v-Src, an activated form encoded by (Stehelin et al., 1976a,b). The discovery of c-src, the v-src transforming gene of Rous Sarcoma virus which was the ®rst cellular homologue or proto- (RSV), or in the form of c-Src, encoded by the c-src oncogene form of a viral oncogene to be uncovered, gene of multicellular organisms. Important break- stimulated the search for, and discovery of, numer- throughs in this ®eld arose from studies on Rous ous other cellular proto-oncogenes, which has sarcoma virus, a chicken tumor virus discovered in ultimately led to important knowledge regarding 1910 by Peyton Rous (Rous, 1910), which became the these proto-oncogenes and the signaling pathways subject of intensive investigation in the 1960's and that they are involved in. 1970's. Although RSV possessed a small genome The discovery of the cellular form of Src kinase, encoding only a few gene products, it was capable pp60c-src, or c-Src, in uninfected cells (Collett et al., of causing tumors in chickens and rapidly transform- 1979; Oppermann et al., 1979), followed by molecular ing cells in culture with high eciency. The develop- cloning and sequencing of the chicken c-src gene (Parker et al., 1981; Shalloway et al., 1981; Takeya et al., 1982) and the RSV v-src gene (Czernilofsky et al., *Correspondence: DJ Fujita 1980, 1983) led to the ®nding that the cellular c-Src Src localization and activation by PTPs JD Bjorge et al 5621 and RSV v-Src proteins were closely related but that Src regulation they diered substantially at their carboxy-termini (Takeya and Hanafusa, 1983). In particular, although With the discovery that v-Src is a constitutively c-Src and v-Src exhibited several single amino acid activated form of c-Src, and that v-Src is capable of dierences, the most striking dierence was the eliciting many dramatic biological responses within replacement of the carboxy-terminal 19 amino acids cells, including cellular transformation, investigators of chicken c-Src by 12 completely dierent amino acids proceeded to search for situations where c-Src becomes in RSV v-Src. Therefore v-Src, which contained 526 activated, and then to characterize the molecular amino acids was a truncated and altered form of mechanisms behind Src activation. The results of these chicken c-Src which contained 533 amino acids (see studies revealed that there are many dierent processes Figure 1) (Takeya and Hanafusa, 1983). This basic that can alter c-Src activity, and ultimately, the signals structural dierence between v-Src and c-Src was also generated downstream of Src. In many cases, these or con®rmed by the sequencing of cloned forms of the similar mechanisms of regulation are applicable to the human c-src gene (Anderson et al., 1985; Tanaka et al., multiple members of the Src family of tyrosine kinases, 1987). c-Src also diered from v-Src in two important which include Src, Fyn, Yes, Lck, Hck, Blk, Fgr, Lyn, respects: (1) The protein tyrosine kinase activity of c- and Yrk. For the purposes of this section of the review, Src in immunoprecipitates was generally lower than we will brie¯y discuss some of these activation/ that of v-Src (Iba et al., 1984; Coussens et al., 1985; inactivation processes, with a focus upon the kinases Tanaka and Fujita, 1986) and (2) c-Src, unlike v-Src, and phosphatases which directly alter the phosphoryla- did not cause cell transformation as measured by focus tion status of a critical carboxy-terminal negative formation in chicken embryo ®broblast cells under regulatory tyrosine residue of c-Src, Tyr(527/530), conditions of moderate overexpression (Iba et al., 1984; and how these proteins are regulated. Shalloway et al., 1984; Tanaka and Fujita, 1986). Experiments using chimeric forms of c-Src and v-Src Regulation of c-Src by phosphorylation indicated that replacement of the c-Src carboxy- terminus with that of v-Src could confer high kinase An important mechanism for regulation of c-Src activity and transforming activity upon the resulting tyrosine kinase activity is through control of its Src chimera (Iba et al., 1984; Tanaka and Fujita, 1986). phosphorylation status. There are two major phos- These experiments, and others, suggested that the phorylation sites present on human c-Src (see Figure carboxy-terminal regions of c-Src and v-Src played 1), Tyr419 (Tyr416 in chicken) and Tyr530 (Tyr527 in important roles in regulating Src activity, as discussed chicken). To avoid confusion when referring to the further in the next section. phosphorylation sites in the context of these two dierent species, we will refer to the positive regulatory autophosphorylation site as Tyr(416/419) and the negative regulatory carboxy-terminal phosphorylation site as Tyr(527/530) throughout this review. A major phosphorylation site that appears critical for regulation of c-Src tyrosine kinase activity is the carboxy-terminal tyrosine Tyr(527/530) (Cooper et al., 1986; Kmiecik and Shalloway, 1987; Piwnica-Worms et al., 1987), that is not present in v-Src. Biochemical data has been obtained supporting the hypothesis that the carboxy-terminal tyrosine residue, when phosphorylated, interacts with the SH2 domain of Src (Roussel et al., 1991; Liu et al., 1993; Bibbins et al., 1993). This interaction results in the suppression of the tyrosine kinase activity of c-Src. More recently, structural data from X-ray crystallography of Src (Xu et al., 1997) and other Src-family members (Yamaguchi and Hendrick- son, 1996; Sicheri et al., 1997) has supported the presence of an intramolecular interaction between the Figure 1 Src is the founding member of the Src family of SH2 domain and the phosphorylated carboxy-terminal structurally-related protein tyrosine kinases. Src and Src-family tyrosine. In addition, other intramolecular interactions, members possess (starting on the left, at the amino-terminus) a in particular between the SH3 domain and the SH2- domain that undergoes fatty acid modi®cation (myristoylation in the case of Src; Myr), a unique region, an SH3 domain, an SH2 kinase linker, also appear to play a role in suppressing domain, the SH2-kinase linker, a kinase domain, and a carboxy- c-Src tyrosine kinase activity (see Figure 2). Dephos- terminal regulatory domain. Chicken Src contains 533 amino phorylation of Tyr(527/530) relieves the intramolecular acids, but human Src contains a total of three additional amino inhibition of c-Src kinase activity, and c-Src becomes acids inserted at two sites within the unique domain resulting in a active. Several tyrosine kinases and PTPs (protein protein that is 536 amino acids. Human Src possesses a positive- regulatory autophosphorylation site at Tyr419 (Tyr416 in chicken tyrosine phosphatases) have been implicated in the Src) and a negative regulatory phosphorylation site at its carboxy- control of the phosphorylation status of this key terminal at Tyr530 (Tyr527 in chicken Src). v-Src, the virally- residue, and will be discussed in detail in subsequent transduced and mutated form of chicken Src, lacks several amino- sections of this review. acids at its carboxy-terminus, and therefore is only 526 amino acids in length. v-Src also possesses several additional point A second major phosphorylation site, Tyr(416/419), mutations (not shown here) which contribute to its transforming is present within the `activation loop', a sequence ability which is conserved amongst many tyrosine kinases and

Oncogene Src localization and activation by PTPs JD Bjorge et al 5622

Figure 2 Src is normally maintained in an inactive conformation (center) with the SH2 domain engaged with Tyr530, the SH3 domain engaged with the SH2-kinase linker, and Tyr419 dephosphorylated. Src activity is regulated by a variety of mechanisms, some of which are illustrated in detail here. Dephosphorylation of Tyr530 as a result of protein tyrosine phosphatase (PTP) action (right) disrupts the intramolecular interaction between the SH2 domain and Tyr530, Tyr419 can then become autophosphorylated, resulting in Src activation. Phosphorylation of Tyr530 by CSK allows this interaction to reform, resulting in Src inactivation. Binding of Src to tyrosine phosphorylated growth factor receptors via its SH2 domain (left) results in displacement of Tyr530, allowing Tyr419 phosphorylation and Src activation. Dissociation of Src from the growth factor receptor allows the intramolecular interactions to reform, resulting in Src inactivation. In addition to growth factor binding and Tyr530 dephosphorylation, a variety of SH2 and SH3 competitors may serve to alter Src structure, leading to Src activation. Some of these competitors may also serve to displace Tyr530 from SH2, allowing exposure to PTP, which would then activate Src by dephosphorylating Tyr530. Mutations within Src can also lead to disruption of the intramolecular interactions that stabilize Src in an inactive conformation, resulting in Src activation

kinases in general (Johnson et al., 1996). When this site ner et al., 1996; Sun et al., 1998), suggesting ®rst, that becomes phosphorylated, it is displaced from the active Src phosphorylated at Tyr(416/419) cannot be substrate binding pocket, allowing the kinase access fully inactivated by phosphorylation at Tyr(527/530) to substrates (Xu et al., 1997, 1999). Thus, when alone, but also requires dephosphorylation of Tyr(416/ Tyr(416/419) is phosphorylated, it is a positive 419). Secondly, this supports recent ®ndings that regulator of Src activity, and when dephosphorylated, inactive Src family members may be the target of it is a negative regulator of Src activity. Tyr(416/419) is other kinases that can phosphorylate Tyr(416/419) and a target for intermolecular autophosphorylation (Smart cause their activation (Chiang and Sefton, 2000). et al., 1981) and may also be a target of other tyrosine Other phosphorylation sites have been identi®ed kinases, resulting in Src activation (Chiang and Sefton, within c-Src and are also thought to modulate the 2000). The importance of Tyr(416/419) autophosphor- activity of the enzyme. During mitosis, phosphoryla- ylation in normal c-Src regulation has been addressed tions within the unique domain of c-Src at Thr34, by examining cells expressing c-Src protein possessing a Thr46, and Ser72 (Shenoy et al., 1989) appear to Phe substitution at Tyr(416/419) (Kmiecik and Shallo- destabilize the SH2/Tyr(527/530) interactions, thereby way, 1987). This Src mutant displays drastically sensitizing c-Src to the action of a PTP capable of reduced biological activity as compared to wild-type dephosphorylating Tyr(527/530) (Bagrodia et al., 1991, Src. Interestingly, mutation of Tyr(416/419) in the 1994; Shenoy et al., 1992; Stover et al., 1994), which context of some transforming mutants of Src (i.e. v- leads to c-Src activation. c-Src is also a target of the Src) has more modest eects (Snyder and Bishop, 1984; activated PDGF receptor and becomes phosphorylated Snyder et al., 1983), suggesting that other alterations within its SH3 domain at Y138 (Broome and Hunter, can perturb the normal regulatory processes imparted 1997) and within its SH2 domain at Y213 (Stover et by the activation loop. Phosphorylation of Tyr(416/ al., 1996). Phosphorylation of these sites reduces the 419) can also activate Src activity in the context of ability of these domains to bind ligands in vitro, simultaneous phosphorylation at Tyr(527/530) (Boer- suggesting that these phosphorylations can disrupt the

Oncogene Src localization and activation by PTPs JD Bjorge et al 5623 intramolecular interactions that stabilize the inactive The interaction of Src with binding proteins can also conformation of Src. Although phosphorylation of serve to either bring Src into direct contact with Y138 has the potential to activate Src kinase activity potential substrates and/or relocalize Src within the cell and appears necessary for PDGF-induced DNA to bring it within close proximity to potential synthesis (Broome and Hunter, 1996), this phosphor- substrates. An example of regulation by localization ylation does not appear necessary for the observed is FAK autophosphorylation, which creates a binding elevations in c-Src activity following PDGF stimulation site for the Src SH2 domain (Schaller et al., 1994; Eide of cells (Broome and Hunter, 1997). Y213, a site et al., 1995). Src can then associate with FAK in focal phosphorylated both in vitro by PDGF receptor and in adhesions where it phosphorylates FAK, leading to vivo upon PDGF stimulation of Balb/c 3T3 cells further activation of FAK tyrosine kinase activity (Stover et al., 1996), also appears capable of activating (Calalb et al., 1995) and downstream activation of the Src and may be responsible for PDGF-mediated Src Ras, MEK, ERK pathway (Schlaepfer et al., 1998). Src activation. Tyrosine sites within the unique domain of localization to focal adhesions also results in the some Src-family members, in particular Hck, also phosphorylation of the cytoskeletal protein paxillin appear to be the target of phosphorylation, with (Richardson et al., 1997). regulatory consequences. Hck can undergo autophosphorylation at Tyr29 within its unique domain in Factors that regulate the phosphorylation status of addition to phosphorylation on Tyr388 within its Tyr(527/530) activation loop (Johnson et al., 2000). Both of these phosphorylation events can lead to Hck activation. Src The phosphorylation status of Tyr(527/530), and does not possess any tyrosine residues within its unique therefore Src activity, is dependent upon a balance domain, and thus is unlikely to be regulated in this between the rate at which phosphate is removed by manner. PTPs and the rate at which it is replaced by kinases. Under most normal circumstances, the Tyr(527/530) site is phosphorylated and Src is inactive in cells, Regulation of Src activity through its interaction with suggesting that the net Tyr(527/530) phosphorylation binding proteins exceeds Tyr(527/530) dephosphorylation. Factors that The binding of Src to various proteins is thought to alter the balance between phosphorylation/dephos- play an important role in Src regulation. This direct phorylation can cause changes in Src activity. In the association can have at least two important regulatory next section, we will discuss the kinases and phospha- consequences, including activation of the intrinsic tases responsible for maintaining this balance, and in tyrosine kinase activity of Src and relocalization of particular, discuss what is known about how these Src to sites of action. proteins are regulated. Several structural features of Src and Src-family members facilitate interactions with proteins that Phosphatases that dephosphorylate regulate Src-family kinase activity. Biochemical and Tyr(527/530) ± PTP-a X-ray crystallographic analyses have revealed various internal interactions within the Src molecule, which Several PTPs have been characterized as being capable maintain it in an inactive state. These include of dephosphorylating Tyr(527/530) and are implicated interactions between the SH2 domain and the in the regulation of c-Src tyrosine kinase activity. One carboxy-terminal tyrosine, Tyr(527/530), as well as PTP that can dephosphorylate the carboxy-terminal interactions between the SH3 domain and the SH2- regulatory tyrosine of c-Src is PTP-a (Zheng et al., kinase linker (see Figure 2). A variety of Src-binding 1992). PTP-a is a receptor-like PTP that is ubiquitously proteins have been detected which can compete for expressed, and is particularly abundant in brain tissue binding to these components and disturb the (Krueger et al., 1990; Kaplan et al., 1990a). It was intramolecular interactions, allowing for Src kinase initially noted that when PTP-a was overexpressed in activation. The platelet-derived growth factor receptor Fisher rat embryo ®broblasts, the stably-transfected (PDGFR) (Kypta et al., 1990; Alonso et al., 1995) cells exhibited a transformed phenotype (Zheng et al., and focal adhesion kinase (FAK) (Cobb et al., 1994; 1992). This phenotype included a rounded and Schaller et al., 1994; Eide et al., 1995) are examples refractile cellular morphology, lack of contact-inhibi- of proteins that bind to the Src SH2 domain and tion, growth in multilayers, the ability to form foci, activate Src. Nef (Moare® et al., 1997) and Sin growth in low serum concentrations, anchorage- (Alexandropoulos and Baltimore, 1996) are examples independent growth in soft agar, and tumor formation of proteins that can bind to SH3 domains and in nude mice. Examination of the phosphorylation activate the Src-family members Hck and Src, status of c-Src in these cells revealed a pronounced respectively. Recently, p130Cas, a protein that is reduction in the Tyr(527/530) phosphorylation level thought to function as a docking protein because of which was accompanied by an increase in c-Src its large number of binding motifs, has been tyrosine kinase activity. PTP-a also appeared to be demonstrated to bind to Src SH2 and SH3 domains, able to dephosphorylate Tyr(416/419) as evidenced by resulting in Src activation (Burnham et al., 2000). No the lack of Tyr(416/419) phosphorylation in PTP-a proteins have currently been identi®ed that can overexpressing cells (Zheng et al., 1992) and by the interrupt the intramolecular interactions and activate ability of chicken PTP-a to dephosphorylate v-Src in Src by binding to either the carboxy-terminal tyrosine vitro (Fang et al., 1994a). or to the SH2-kinase linker. Such proteins, if they do PTP-a has also been demonstrated to dephosphor- exist, would represent two new classes of Src ylate Tyr(527/530) and activate c-Src in P19 EC cells, regulatory molecules. where it has also been shown to alter the dierentiation

Oncogene Src localization and activation by PTPs JD Bjorge et al 5624 fate of these cells in favor of neuronal dierentiation exposing the phosphorylated Src carboxy-terminal (den Hertog et al., 1993). PTP-a overexpression can tyrosine to the action of the phosphatase (Zheng et also dephosphorylate and activate c-Src in the human al., 2000). epidermoid carcinoma cell line A431, where it can also Another mechanism by which PTP-a is proposed to enhance cell-substratum adhesion, without altering be regulated is through dimerization (Bilwes et al., growth characteristics of the cells (Harder et al., 1996; Majeti et al., 1998; Jiang et al., 1999, 2000). 1998). Although most studies have addressed the Evidence derived from X-ray crystallography (Bilwes eects of PTP-a on c-Src, it appears that other Src- et al., 1996) and through the generation of stable family kinases may also be targets for dephosphoryla- disulphide-linked homodimers of PTP-a (Jiang et al., tion and activation by this particular PTP. c-Yes 1999) suggests that dimerization inhibits PTP-a (Harder et al., 1998) as well as c-Fyn (Bhandari et al., activity. PTP-a dimers have recently been detected 1998) have also been found to be both dephos- on the cell surface (Jiang et al., 2000), suggesting that phorylated and activated by PTP-a. This implies that the process of dimerization may be important in PTP-a's eects on cells might not be limited to its cellular regulation of PTP-a phosphatase activity. The eects on c-Src alone. process of dimerization appears to require only the Thus, overexpression of PTP-a in a variety of cell extracellular and transmembrane domains and the types appears to increase c-Src and Src-family member dimer is stabilized by multiple weak dimerization kinase activities. Conversely, what happens when PTP- interfaces by a mechanism referred to as the `zipper a activity is reduced in cells? Antisense constructs have model' (Jiang et al., 2000). There is currently no been employed to address this question and the results physiological mechanism through which the forma- appear to corroborate the idea that PTP-a is a positive tion/breakage of these dimers can occur. It is regulator of c-Src activity (Arnott et al., 1999). interesting to speculate that, like growth factor Expression of antisense PTP-a in 3T3-L1 adipocytes receptors, PTP-a may have a ligand that facilitates reduces PTP-a levels in these cells and simultaneously the stabilization of either monomeric or dimeric forms reduces c-Src kinase activity. In addition to the of PTP-a, which ultimately aects the protein's antisense strategy of reducing PTP-a levels in cells, phosphatase activity. This postulated ligand might PTP-a 7/7 mice have also been generated and also be expected to play a role in the control of Src characterized (Ponniah et al., 1999; Su et al., 1999). activity. Although the mice appeared phenotypically normal, ®broblasts derived from the mice and mouse brain Protein tyrosine phosphatase l tissue were found to have signi®cantly reduced levels of both c-Src and c-Fyn tyrosine kinase activities. This Screening a chicken brain cDNA library with a probe reduction in kinase activity was accompanied by an corresponding to the intracellular domain of CD45 (a enhanced phosphorylation of Tyr(527/530) in c-Src. transmembrane PTP capable of dephosphorylating the Cellular characteristics and pathways downstream of Src-family member Lck) resulted in the identi®cation of Src were also aected. These included a reduction in a related transmembrane PTP that was named PTPl spreading on ®bronectin, decreased phosphorylation of (Fang et al., 1994b). PTPl transcripts are found FAK and p130Cas, and a reduction in activation of abundantly expressed in spleen, intestine, and ®bro- MAP kinases (Su et al., 1999). blasts transformed by oncogenic ras or erbA/B. PTPl Although there is evidence supporting a role for appears to be capable of dephosphorylating both PTP-a in helping to maintain the overall phosphoryla- Tyr(416/419) and Tyr(527/530) as evidenced by its tion status and activity of Src, it is still currently ability to dephosphorylate both v-Src and c-Src. In unknown whether PTP-a is regulated during any studies comparing the activities of PTPl with chicken normal cellular signaling process such that it causes PTP-a and human leukocyte common antigen-related direct changes in c-Src activity. Investigations into molecule (LAR) (Fang et al., 1994a), all three PTPs PTP-a regulation are currently proceeding, and some were capable of dephosphorylating v-Src. However, data is now forthcoming that addresses this important only PTPl was capable of dephosphorylating and question. PTP-a has been identi®ed as a target of activating c-Src in both in vitro and in vivo experi- phosphorylation by protein kinase C on two residues ments, thus inferring some degree of speci®city. The near its transmembrane domain (Tracy et al., 1995) reason for the inability of PTP-a to dephosphorylate and these phosphorylations result in stimulation of the and activate c-Src in this particular model system is protein's phosphatase activity (den Hertog et al., 1995). unknown. Tyr789 in the carboxy-terminus of PTP-a is also a Recently, PTPl has been implicated in the target for phosphorylation, perhaps by c-Src itself (den modulation of c-Src kinase activity in osteoclast Hertog et al., 1994; Su et al., 1994). This phosphoryla- precursor cells (Chappel et al., 1997). The authors tion site appears to be a binding site for Grb2 (den found that when osteoclast precursor cells were Hertog et al., 1994; Su et al., 1994); as well it mediates treated with 1,25-dihydroxyvitamin D3 to induce the association of PTP-a with focal adhesion plaques dierentiation, there was a dramatic increase in c- (Lammers et al., 2000), a known site of c-Src Src speci®c activity in the absence of changes in c-Src localization. Interestingly, experiments utilizing a protein levels. This change in activity was accom- Tyr789Phe mutant of PTP-a (Zheng et al., 2000) panied by a reduction in the phosphotyrosine content identi®ed this residue as being essential for PTP-a's of c-Src, presumably on Tyr(527/530). Interestingly, ability to bind to, dephosphorylate, and activate c-Src. both PTPl mRNA and protein levels were both It also appeared necessary for the transforming ability upregulated, suggesting the possibility that PTPl of PTP-a. Experimental evidence suggests that Tyr789 might be responsible for the changes observed in c- might displace the SH2 domain of c-Src, thereby Src kinase activity.

Oncogene Src localization and activation by PTPs JD Bjorge et al 5625 SHP-1 supporting a possible role of SHP-2 in the control of c-Src activity, possibly through a non-enzymatic SHP-1, also known as PTP1C, is a cytosolic, dual SH2 mechanism. domain-containing PTP that is expressed primarily in epithelial and hematopoietic cells. It has been found associated with c-Src in human platelets upon PTP1B and other PTPs that might target c-Src thrombin stimulation (Falet et al., 1996). In addition, Rather than screen identi®ed PTPs for activity against c- v-Src is capable of phosphorylating SHP-1 in SR-3Y1 Src, some laboratories, including our own, have adopted cells, a v-Src transformed rat ®broblast cell line a dierent approach, and have ®rst identi®ed cell types (Matozaki et al., 1994). where there are measurable elevations in c-Src speci®c Recently, evidence has been presented that also activity. This approach is of particular relevance because implicates SHP-1 in the dephosphorylation and activa- several dierent PTPs are known to target Src for tion of c-Src (Somani et al., 1997). Interestingly, the dephosphorylation and activation, and the expression authors found that although SHP-1 was capable of pattern of these PTPs varies according to cell type. Two dephosphorylating both Tyr(416/419) and Tyr(527/ cell types that have been identi®ed by our laboratory as 530), it appeared to exert a more pronounced eect possessing elevated Src activity are human melanocytes on Tyr(527/530), suggesting that SHP-1 may be more (O'Connor et al., 1992, 1995) and several human breast speci®c for Tyr(527/530). The authors also examined cancer cell lines (Egan et al., 1999). In both situations, we the status of c-Src in thymocytes derived from have detected elevated c-Src speci®c activity and motheaten and viable motheaten mice (motheaten mice hypophosphorylation of Tyr(527/530). Interestingly, express reduced SHP-1 phosphatase activity due to these cells also have elevated levels of PTP activity loss-of-function mutations within the SHP-1 gene). capable of dephosphorylating a synthetic peptide Although the c-Src protein levels were similar in both modeled against the carboxy-terminus of Src-family types of motheaten mice and normal mice, there was a members. We have hypothesized that this PTP activity markedly lower level of c-Src tyrosine kinase activity in may have an important role in controlling c-Src activity the motheaten and viable motheaten mice, accompa- and the cell phenotype. In the human breast cancer cell nied by increased levels of phosphorylation on lines in particular, high levels of PTP activity directed Tyr(527/530). In addition, dominant-negative SHP-1 against Tyr(527/530) may contribute to c-Src activation, expressed in HEY ovarian epithelial cancer cells and may be important in maintaining the transformed resulted in reduced dephosphorylation of SHP-1 phenotype. This activation of Src could occur indepen- substrates and reduced c-Src kinase activity (Somani dently, or in conjunction with activated receptor tyrosine et al., 1997). kinases, and might promote Src activation in either a direct, additive, or synergistic manner. We have puri®ed SHP-2 the major PTP activity from breast cancer cell lines that is capable of dephosphorylating the carboxy-terminal Another cytoplasmic SH2 domain-containing PTP that synthetic peptide and c-Src, and identi®ed it as PTP1B has been implicated in regulating c-Src activity is SHP- (Bjorge et al., submitted). PTP1B protein levels were 2. SHP-2 was initially demonstrated to become found to be elevated in several breast cancer cell lines and phosphorylated in v-Src transformed cells (Feng et PTP1B was found to be capable of both in vitro and in al., 1993). Subsequently, Peng and Cartwright demon- vivo activation of c-Src kinase activity as a result of its strated that SHP-2 could bind to c-Src, that an ability to dephosphorylate Tyr(527/530). These results activated mutant form of Src could phosphorylate suggest that PTP1B may have an important role to play SHP-2, and that Tyr(527/530) of c-Src could be in the regulation of Src activity and that it may also dephosphorylated by SHP-2 (Peng and Cartwright, contribute to Src activation in some forms of human 1995). SHP-2 appeared to be relatively speci®c for cancer. The ability of PTP1B to modulate Src activity Tyr(527/530) in vitro, where 50% dephosphorylation of has also been demonstrated in mouse L cell ®broblasts Tyr(527/530) by SHP-2 was accompanied by no (Arregui et al., 1998). A dominant negative form of signi®cant dephosphorylation of Tyr(416/419) (Peng PTP1B was shown to reduce Src activity and this was and Cartwright, 1995). SHP-2 was later demonstrated attributed to increased phosphorylation of Tyr(527/530). to be capable of activating c-Src activity in vivo by PTPs have also been implicated in Src activation that examining the eects of in vivo SHP-2 overexpression occurs as a result of activation of G-protein coupled (Walter et al., 1999). Interestingly, elevations in c-Src receptors. Src speci®c activity has been found to be activity were not accompanied by signi®cant changes in transiently elevated in platelets stimulated with throm- the phosphorylation status of Tyr(527/530) or Tyr(416/ bin, and this was accompanied by a reduction in the 419). In addition, a phosphatase-inactive mutant of phosphorylation of Tyr(527/530) (Clark and Brugge, SHP-2 was also capable of c-Src activation. This result 1993). Interestingly, SHP-1, a PTP that has been coupled with the ®nding that SHP-2 associates with c- implicated in the dephosphorylation of Tyr(527/530) Src by binding to its SH3 domain suggested that (see previous section on PTPs), has also been shown to perhaps most, or all, of the c-Src-activating activity of become activated upon thrombin stimulation of SHP-2 in vivo might be due to a mechanism not platelets (Li et al., 1995). Bombesin or vasopressin involving c-Src dephosphorylation. Instead, it may stimulation of Swiss 3T3 cells have been shown to involve binding to the c-Src SH3 domain, with the result in Src activation, and this activation was resulting allosteric activation of Src. The use of completely blocked by treatment of the cells with antisense oligonucleotides to reduce SHP-2 levels in vanadate, a PTP inhibitor (Rodriguez-Fernandez and 3T3 cells transfected with c-Src resulted in decreased c- Rozengurt, 1996). Recently, bradykinin has also been Src speci®c activity (Walter et al., 1999), further demonstrated to activate Src, and this was accompa-

Oncogene Src localization and activation by PTPs JD Bjorge et al 5626 nied by the activation of PTP activity that could ®nally puri®ed and cloned based upon its ability to dephosphorylate the synthetic phosphopeptide sub- bind to CSK (Kawabuchi et al., 2000) and its strate Raytide (Graness et al., 2000). Although further localization to glycosphingolipid-enriched membrane experimentation will be required to prove that the microdomains (GEMs) (Brdicka et al., 2000). Cbp/ elevated PTP activity is responsible for the observed PAG binds to and recruits CSK to the plasma increases in Src activity, this data certainly suggests membrane by virtue of a tyrosine phosphorylation site that the modulation of PTP activity may have an on Cbp/PAG and the SH2 domain in CSK. Binding of important role in controlling Src kinase activity in the Cbp/PAG to CSK also appears to activate CSK G-protein coupled receptor system. (Takeuchi et al., 2000). The tyrosine kinase responsible for the phosphorylation of Cbp/PAG is currently unknown. However, it has been proposed that a Src- Kinases that phosphorylate Tyr(527/530) family member itself may well be the kinase responsible Two tyrosine kinases, CSK (c-Src kinase) and CHK for this phosphorylation event. It is thought that this (CSK homologous kinase), have been identi®ed that may be a form of autoregulation in which activated c- are capable of phosphorylating Src on Tyr(527/530) Src phosphorylates Cbp/PAG, which in turn, recruits and inactivating its kinase activity. CSK was puri®ed CSK to the membrane, where it can then phosphor- on the basis of its ability to phosphorylate the carboxy- ylate and negatively regulate Src activity. Kawabuchi et terminal tyrosine of c-Src (Okada and Nakagawa, al. (Kawabuchi et al., 2000) have demonstrated the 1989). CSK is structurally related to Src-family kinases, ability of Cbp/PAG to regulate c-Src activity by but lacks a myristoylation signal and phosphorylation overexpressing Cbp/PAG in COS7 cells. Their results sites corresponding to Tyr(416/419) and Tyr(527/530) show that upon Cbp/PAG overexpression, CSK (Nada et al., 1991). CSK is expressed ubiquitiously, but becomes colocalized with Cbp/PAG, and c-Src kinase is most abundant in thymus, spleen, and neonatal brain activity is inhibited. Furthermore, Brdicka et al. tissue (Okada et al., 1991). This enzyme is capable of (Brdicka et al., 2000) have demonstrated that in resting phosphorylating Tyr(527/530) and inactivating c-Src T cells, Cbp/PAG is constitutively phosphorylated on both in vitro and in vivo (Nada et al., 1991). Mice tyrosine residues. Upon T cell activation, there is a lacking the enzyme (CSK 7/7) have neural tube rapid dephosphorylation of Cbp/PAG, dissociation of defects and die between days 9 and 10 of gestation CSK from Cpb/PAG, and activation of Src family (Nada et al., 1993; Imamoto and Soriano, 1993). Cells members. It will be interesting to identify the PTP derived from these embryos have highly elevated c-Src, responsible for dephosphorylation of Cbp/PAG. It c-Fyn, and c-Lyn kinase activities, accompanied by remains to be determined whether PTPs previously hypophosphorylation of Tyr(527/530) and hyperpho- identi®ed as being able to dephosphorylate Tyr(527/ sphorylation of Tyr(416/419) (Nada et al., 1993; 530) and activate c-Src, such as PTP-a, are also capable Imamoto and Soriano, 1993). of dephosphorylating Cbp/PAG. If so, this would Although Src-family kinases share many character- suggest the existence of an alternative regulatory istics with CSK (i.e. SH3, SH2, and kinase domain), the mechanism for reducing Tyr(527/530) phosphorylation, lack of an autophosphorylation site and a negative one that possibly utilizes reductions in CSK activity, regulatory carboxy-terminal tyrosine in CSK suggests rather than increases in Tyr(527/530) phosphatase that this protein has a very dierent mechanism of activity. regulation. CSK is phosphorylated on Tyr-184 in vivo, Other proteins that bind to CSK and may be but the identity of the kinase responsible for this important for Src regulation include two structurally phosphorylation and its potential role in CSK regulation related non-receptor PTPs, PEP (proline-enriched are presently unknown (Joukov et al., 1997). Ser/Thr protein tyrosine phosphatase) (Cloutier and Veillette, phosphorylation of CSK has also been proposed to be an 1996) and PTP-PEST (Davidson et al., 1997a), a non- important mechanism in regulating CSK activity. receptor PTP possessing features of PEST motifs which Phosphorylation by cAMP-dependent kinase has been are found in proteins with very short intracellular half- found to reduce CSK activity in vitro (Sun et al., 1997). lives (Yang et al., 1993). Although PEP is expressed CSK appears to translocate to subcellular sites where its exclusively in hematopoietic cells, PTP-PEST is ex- activity is probably required for normal regulation of pressed ubiquitously. PEP, the better characterized of Src-family kinases (Howell and Cooper, 1994). A the two proteins, has been shown to dephosphorylate number of CSK binding proteins have been identi®ed the autophosphorylation site of the Src-family member that may play an important role in membrane localiza- FynT (an isoform of Fyn that is expressed primarily in tion of CSK, and include GTPase-activating protein T-lymphocytes) when overexpressed, thereby inactivat- (GAP)-associated p62 (p62dok) (Neet and Hunter, 1995; ing FynT kinase activity (Coultier and Veillette, 1999). Yamanashi and Baltimore, 1997; Carpino et al., 1997) Overexpression of PEP also resulted in a general and the Src-family member Lck (Bougeret et al., 1996). reduction in the phosphorylation status of a number Recently, new insights into how CSK might be of proteins and the inhibition of T-cell receptor regulated have been obtained by two groups of signaling. PEP's ability to dephosphorylate the positive investigators who have discovered another protein regulatory site of Src-family members coupled with the capable of binding CSK. This protein, named CSK ability of CSK to phosphorylate the negative regula- binding protein (Cbp) or phosphoprotein associated tory site makes this PEP/CSK complex a potent with glycosphingolipid-enriched microdomains (PAG) negative regulator of Src-family function. Although (Kawabuchi et al., 2000; Brdicka et al., 2000), is a PTP-PEST has not yet been characterized with regard ubiquitously expressed transmembrane phosphoprotein to its ability to inactivate Src-family members, it is that is localized in the GM1 ganglioside-enriched interesting to speculate that due to its formation of a membrane domains. It was initially identi®ed and complex with CSK and its ubiquitous expression, that

Oncogene Src localization and activation by PTPs JD Bjorge et al 5627 it too may be a prime candidate for regulating the Src localization kinase activity of Src-family members such as Src. Recently, data suggesting that alterations in CSK Studies on the subcellular localization of Src have activity might play a role in human hepatocellular provided numerous clues as to its function. Although carcinoma have been reported (Masaki et al., 1999). the normal function of Src still remains largely unclear CSK activity has been found to be reduced in tumorous at present, it has been found to associate with cellular liver tissue when compared to normal control tissue. The membranes, in particular the plasma membrane, the investigators noted a slight upward shift in the molecular perinuclear membrane and endosomal membranes weight of CSK upon immunoblot analysis, and (Willingham et al., 1979; Courtneidge et al., 1980; suggested the possibility that an alternate isoform of Resh and Erikson, 1985; David Pfeuty and Nouvian- CSK is present in the tumorous tissue. These results Dooghe, 1990; Kaplan et al., 1992). At the plasma suggest a potential role for CSK in human cancers, and membrane Src is thought to be involved in signal might explain reports of elevations of Src kinase activity transduction events regulating cell growth and pro- in this type of cancer (Masaki et al., 1998). liferation via activated growth factor receptors. Initial A protein related to CSK has also been cloned by a studies on the localization of v-Src in Schmidt-Ruppin number of groups (Klages et al., 1994) and is also able avian sarcoma virus (ASV)-transformed NRK cells to regulate Src activity by phosphorylation of Tyr(527/ demonstrated the protein to be concentrated on the 530) (Chow et al., 1994; Sakano et al., 1994; Bennett et inner surface of the plasma membrane, particularly al., 1994; McVicar et al., 1994). Initially, some under rues (Willingham et al., 1979). Similarly, the confusion arose because it wasn't readily apparent cellular homologue of the viral transforming protein, c- whether all of these cloned proteins were derived from Src, has also been demonstrated to associate with the the same gene. It is now recognized that these proteins plasma membrane (Courtneidge et al., 1980). Subse- are all probably derived from the same gene and quent work has shown Src localization in focal dierences between the proteins are probably due to adhesion plaques and at adherens junctions between alternative splicing (Grgurevich et al., 1997). The name cells (Henderson and Rohrschneider, 1987; Schaller et CHK (CSK homologous kinase) has been adopted by al., 1994; Owens et al., 2000). This localization of Src most investigators. CHK is related to CSK, but tends to the cell periphery, in particular focal adhesions and to be expressed primarily in brain and hematopoietic cell-cell contacts has been demonstrated to be cells. In contrast to the rather dramatic phenotype dependent on the intact actin cytoskeleton (Fincham exhibited by CSK 7/7 mice, CHK 7/7 mice exhibit et al., 1996; Owens et al., 2000). no detectable phenotype, even in cells of hematopoietic In addition to being associated with plasma lineage (Hamaguchi et al., 1996; Samokhvalov et al., membranes in ®broblasts (typically at sites of focal 1997), suggesting that CHK probably has dierent adhesion (Kaplan et al., 1994)), both viral and cellular cellular functions than CSK. The similarities and Src proteins have also been detected in the cytoplasm dierences in function between CSK and CHK have and in the perinuclear Golgi region of the cell been highlighted in a paper by Davidson et al. (Willingham et al., 1979; Resh and Erikson, 1985). In (Davidson et al., 1997b). Both CSK and CHK were particular, 30 ± 40% of the total Src population is found to suppress Src-family kinase activity in CSK localized to the perinuclear region of the cell (Tanaka 7/7 ®broblasts, but only CSK could suppress and Kurth, 1984; Resh and Erikson, 1985). In antigen-induced signals in the T-cell line BI-141. This Schmidt-Ruppin ASV-transformed NRK cells, *60% dierence was attributed to the inability of membrane- of v-Src has been found to be associated with the targeting sequences added to CHK to eciently target cytosol, in comparison to *40% with the plasma CHK to cellular membranes, whereas this approach membrane (Willingham et al., 1979). The dierent could eciently target CSK to cellular membranes. It subcellular localizations of Src within cells are was postulated that other unidenti®ed targeting signals indicated in Table 1. within CHK might be more dominant than the Membrane association of Src is in part due to a membrane-targeting sequence added to CHK. postranslational modi®cation at its amino terminus ±

Table 1 Dierent subcellular localizations of Src Src localization Proposed function Reference*

Plasma membrane Mitogenic signaling via growth Willingham et al., 1979; Courtneidge et al., 1980; Focal adhesions; actin factor RTKs** and GPCRs; cell Resh and Erikson, 1985; Henderson and Rohrschneider, 1987; cytoskeleton; microtubule adhesion and spreading; cell Kaplan et al., 1992; Twamley-Stein et al., 1993; Kaplan et al., 1994; bundles at points of cell-cell migration; cell-cell contact Okamura and Resh, 1994; Fincham et al., 1996; Li et al., 1996; contact; adherens junctions; Luttrell et al., 1996; Schaller et al., 1999; Fincham et al., 2000; caveolae Owens et al., 2000

Cytoplasmic and perinuclear Protein trafficking; endosomal Willingham et al., 1979; Tanaka and Kurth, 1984; Resh and Erikson, Endosomes; microtubules membrane transport and regulation; 1985; Parsons and Creutz, 1986; Rendu et al., 1989; David-Pfeuty and (MTOC, spindle pole); cell cycle progression Noovian-Dooghe, 1990; Kaplan et al., 1992; Linstedt et al., 1992; Golgi; secretory organelles; Verbeek et al., 1996; Abu-Amer et al., 1997 synaptic vesicles

Nuclear Cell cycle regulation David-Pfeuty et al., 1993; David-Pfeuty and Noovian-Dooghe, 1995

*The references cited here are representative, and because of space limitations do not necessarily constitute a complete list of relevant references. **RTKs, receptor tyrosine kinases; GPCRs, G-protein-coupled receptors; MTOC, microtubule organizing center

Oncogene Src localization and activation by PTPs JD Bjorge et al 5628 the covalent attachment of a 14-carbon fatty acid the translocation of v-Src to peripheral focal adhesions moiety, myristate (Buss and Sefton, 1985; Schultz et requires the v-Src SH3 dependent association and al., 1985; Kaplan et al., 1988). However, myristoylation activation of phosphatidylinositol (PI) 3-kinase is not sucient for membrane anchorage (Kaplan et (Fincham et al., 2000). In contrast, c-Src requires its al., 1990b). Instead, other residues of Src in conjunc- myristoylation site as well as the SH3 domain for tion with myristoylation are required for mediating localization to focal adhesions (Kaplan et al., 1994). membrane association (Kaplan et al., 1990b). In Interestingly, activation of c-Src also appears to be particular, six basic residues at the amino terminus important in its subcellular localization (Kaplan et al., are essential for high anity binding to lipid bilayers 1994, 1995). Mutation of the regulatory Tyr(527/530) (Sigal et al., 1994). Moreover, three regions at the N- to a phenylalanine has been shown to dramatically terminus have been identi®ed that regulate the redistribute c-Src from endosomal membranes to focal subcellular localization of Src. The region containing adhesions, suggesting that the phosphorylation status amino acids 1 ± 14 of Src is sucient to mediate both of this tyrosine residue aects Src localization (Kaplan the myristoylation and the attachment of fusion et al., 1994). Moreover, the SH2 domain of c-Src, protein constructs to cytoplasmic granules (Kaplan et through its association with FAK, has also been al., 1990b). In contrast, amino acids 38 ± 111 of Src suggested to be an important mechanism in regulating cause proteins to associate with plasma membranes the protein's subcellular localization (Cobb et al., 1994; and perinuclear membranes, whereas amino acids 204 ± Schaller et al., 1994, 1999). 259 mediate association primarily with perinuclear Although much evidence has been acquired regard- membranes (Kaplan et al., 1990b). Although myristoy- ing the role of Src at the plasma membrane and its lation is not sucient for membrane anchorage, the interaction with growth factor receptors and integrin- myristate group is necessary for the perinuclear nucleated focal adhesion complexes (see reviews by retention of kinase inactive Src molecules (Fincham Abram and Courtneidge, 2000; Biscardi et al., 1999; and Frame, 1998). Interestingly, myristoylation of Src Thomas and Brugge, 1997), the functional signi®cance may also prevent its unregulated transport into the of Src at other subcellular locations such as cytoplas- nucleus, thus suggesting that a subpopulation of Src mic granules and perinuclear membranes has not been may exist that exerts speci®c functions at speci®c times well characterized. The punctate staining pattern Src of the cell cycle within the nucleus (David-Pfeuty et al., exhibits in ®broblasts is thought to represent the 1993). In cells overexpressing a nonmyristoylated protein's association with membrane vesicles. Further- mutant form of c-Src, starting in G2 phase and during more, analysis of c-Src in NIH c-src overexpresser cells mitosis, myristoylation-defective c-Src proteins translo- indicates a possible association of Src with endosomal cate to the nucleus and associate with a number of membranes (David-Pfeuty and Nouvian-Dooghe, components that participate in mitotic events, such as 1990). Analysis of indirect immuno¯uorescence by spindle poles and ®bers, and interchromosomal materi- three-dimensional optical sectioning microscopy re- al (David-Pfeuty et al., 1993). Furthermore, c-Src has vealed Src to be primarily associated with membranes been found to localize within both the nucleus and at the microtubule organizing center (MTOC) that nucleolus (David-Pfeuty and Nouvian-Dooghe, 1995). represent a late stage in the endocytic pathway (Kaplan Thus, it has been proposed that Src may regulate et al., 1992). This data contrasts with and extends progression through the cell cycle and entry into previous reports demonstrating Src localization at the mitosis by interacting with cell division regulatory plasma membrane. One explanation for this discre- components at the nuclear level (David-Pfeuty et al., pancy has been attributed to the fact that biochemical 1993; David-Pfeuty and Nouvian-Dooghe, 1995). fractionation techniques used in some prior studies did Interestingly, it has been shown that Src deletion not dierentiate between plasma membranes and mutants lacking the amino-terminal one-third of v-Src, endosomal membranes, which have similar densities including the membrane binding domain are still and are thus likely to cofractionate (Kaplan et al., capable of inducing cell proliferation, although they 1992). Moreover, c-Src has also been demonstrated to do not cause morphological transformation (Calothy et co-localize with a number of microtubule-related al., 1987). These results demonstrate that the target(s) structures, including microtubule bundles at points of involved in mitogenic activity is accessible to unmyr- cell-cell contact, the MTOC and a region associated istoylated Src and that the mitogenic response may be with the spindle poles during mitosis (Kaplan et al., dependent on a Src substrate(s) that is not localized at 1992). Interestingly, this association was dependent on the plasma membrane (Calothy et al., 1987). intact microtubules, suggesting that c-Src associates Studies have shown that v-Src stably interacts with a with the microtubule cytoskeleton via endocytic detergent-insoluble cytoskeleton matrix, whereas c-Src vesicles (Kaplan et al., 1992). This evidence highlights does not (Burr et al., 1980; Loeb et al., 1987). This a potential role for Src in regulating the transport or dierence in ability to interact with the cytoskeletal function of specialized secretory vesicles (Kaplan et al., matrix is modulated by the Src catalytic domain, which 1992). The presence of Src in secretory organelles of works in conjunction with its SH2 domain to chroman cells and platelets (Parsons and Creutz, determine the protein's ability to associate with the 1986; Rendu et al., 1989), its association with cytoskeleton (Okamura and Resh, 1994). However, the endosomally-derived synaptic vesicles in dierentiated assembly of v-Src into actin-associated focal adhesions PC-12 cells (Linstedt et al., 1992), and the development does not require its catalytic activity or its myristoyla- of osteopetrosis in mice that are null for Src (Soriano tion site (Fincham and Frame, 1998). Instead, the SH3 et al., 1991) further point to a possible role for Src in domain mediates the switch from a microtubule- protein tracking events. Osteoclasts that dissolve dependent perinuclear localization to actin-associated bone are highly specialized secretory cells that secrete focal adhesions (Fincham et al., 2000). Furthermore, lysosomal enzymes, and the absence of Src is thought

Oncogene Src localization and activation by PTPs JD Bjorge et al 5629 to disrupt the regulatory processes necessary for Sam68/p70 was initially cloned and described as the tracking lysosomal proteins (Kaplan et al., 1992). GAP-associated tyrosine phosphoprotein p62 that Inhibition of Src kinase activity by the Src tyrosine could associate with both mRNA and DNA (Wong kinase inhibitor PP1 supports this notion, as it has et al., 1992; Lock et al., 1996). been demonstrated to suppress collagenolytic protease Sam68 is a nuclear protein that consists of at least secretion in osteoclasts, and ultimately bone resorption six proline rich regions, an RNA binding region (Furuyama and Fujisawa, 2000). referred to as the hnRNP K homology (KH) domain The signi®cance of Src localization to endosomal (Gibson et al., 1993; Siomi et al., 1993) and a tyrosine- and perinuclear membranes remains to be determined. rich C-terminal region (Wong et al., 1992; Fumagalli et However, it is interesting to note that in breast cancer al., 1994; Chen et al., 1997; Ishidate et al., 1997). tissue, Src exhibits a predominantly perinuclear pattern Sam68 binds to poly(U) homopolymeric ribonucleo- in malignant cells, in contrast to a more evenly tides, in vitro (Taylor and Shalloway, 1994; Wang et distributed cytoplasmic pattern characteristic of normal al., 1995), and its ability to bind ribonucleotides can be breast epithelial cells (Verbeek et al., 1996). Also, at the negatively regulated by tyrosine phosphorylation, as onset of transformation, polyomavirus middle-T re- well as by interactions with the Src SH3 domain cruits Src to a perinuclear compartment coincident (Taylor et al., 1995; Wang et al., 1995). Both the SH3 with condensation of tubular endosomes (Zhu et al., and SH2 domains of Src are required for stable 1998). It has been suggested that this pronounced interactions with Sam68 during mitosis (Fumagalli et staining around the nucleus could re¯ect the localiza- al., 1994; Taylor and Shalloway, 1994; Taylor et al., tion of Src to endosomal membranes, where it might 1995). function to enhance the mitotic activity of cells by It has been suggested that Src phosphorylation of somehow mediating mitotic eects of Cdc2 or other Sam68 may be important in regulating events during eectors (Chackalaparampil and Shalloway, 1988; mitosis (Pillay et al., 1996). The drug radicicol (a Kaplan et al., 1992; Verbeek et al., 1996). potent inhibitor of Src kinase activity) has been demonstrated to reversibly inhibit Sam68 tyrosine phosphorylation by mitotic Src and the subsequent Nuclear and perinuclear targets of Src exit from mitosis of Src-transformed cells, whereas it has no eect on the ability of cells to enter mitosis, as The localization of Src to perinuclear membranes, determined by ¯ow cytometric analyses (Pillay et al., endosomes, and possibly even the nucleus suggests that 1996). This suggests that Src is important in the Src is involved in signal transduction events that do regulation of cell cycle function via Sam68, particularly not always involve the plasma membrane. However, during the later stages of mitosis (Pillay et al., 1996). limited information exists with respect to Src function The identi®cation of a naturally occurring isoform of at these intracellular locations, and in particular, the Sam68 with a deletion within the KH domain speci®c substrates with which Src may interact. Thus, (Sam68DKH) further supports a role for Sam68 in the identi®cation of Src targets at these subcellular regulating cell cycle progression (Barlat et al., 1997) domains is an essential starting point for the elucida- and its potential function in mediating cell cycle signals tion of the biological functions of Src kinase molecules generated by Src. Upon growth arrest of con¯uent localized to these regions. cultures of normal cells, expression of Sam68DKH is The tyrosine kinase activity of Src is increased in increased, whereas no expression of this isoform is mitotic cells arrested with the drug nocodazole observed in cells incapable of entering quiescence at (Chackalaparampil and Shalloway, 1988; Shenoy et con¯uency, such as Src-transformed cells (Barlat et al., al., 1989; Morgan et al., 1989; Bagrodia et al., 1991; 1997). Transfected Sam68DKH inhibits serum-induced Kaech et al., 1991; Shenoy et al., 1992; Taylor and DNA synthesis, whereas Sam68 overcomes this eect Shalloway, 1993). Although the function of this (Barlat et al., 1997). As a result, it has been proposed activation is not yet completely understood, there is that Sam68-isoforms, through their KH domain, are growing evidence which suggests that Src may play a involved in regulating cell proliferation, more precisely role in the regulation of the cell cycle (Taylor and at the G1/S transition (Barlat et al., 1997). Src activity Shalloway, 1993, 1996). Studies have been performed also appears to be required during this stage of the cell which have identi®ed a phosphorylated 68 kDa cycle and is necessary for committing cells to S phase protein that not only associates with, but is tyrosine (Wyke et al., 1993, 1995). Interestingly, constitutive phosphorylated by overexpressed wild-type or muta- tyrosine phosphorylation of Sam68 is thought to aect tionally activated forms of c-Src during mitosis in the cell cycle in T-cells (Fusaki et al., 1997), and mouse ®broblasts (Fumagalli et al., 1994; Taylor and constitutive tyrosine phosphorylation of Sam68 has Shalloway, 1994). This protein has been designated also been observed in leukemic T-cells (Vogel and Sam68 (Src-associated in mitosis). An identical Fujita, 1995). protein, called p70, was identi®ed as a tyrosine- Although Sam68 was initially identi®ed as a tyrosine phosphorylated protein that was capable of binding phosphorylated target of Src in cells overexpressing the Lck SH2 domain (Vogel and Fujita, 1995). This wild-type or mutationally activated forms of c-Src protein was found to be constitutively phosphorylated during mitosis (Fumagalli et al., 1994; Taylor and in leukemic T-cells, and became tyrosine phosphory- Shalloway, 1994), it is interesting to note that in a lated within minutes after stimulation of resting T- subsequent study, the metabolic labeling of untrans- cells with phytohemagglutinin or interleukin-2, sug- fected HeLa and NIH3T3 cells with [32P]orthophos- gesting that its tyrosine phosphorylation was not phate demonstrated that Sam68 was phosphorylated limited to mitosis (Vogel and Fujita, 1995). Initially, on threonine residues exclusively during mitosis, as well some confusion in nomenclature may have arisen as as on serine residues during both interphase and

Oncogene Src localization and activation by PTPs JD Bjorge et al 5630 mitosis (Resnick et al., 1997). No tyrosine phosphor- 1994) and its orthologue human breast tumor kinase ylation signals on Sam68 were observed under these (BRK) (Mitchell et al., 1994; Llor et al., 1999), two conditions (Resnick et al., 1997). Cdc2 was identi®ed as intracellular tyrosine kinases that are distantly related the kinase responsible for the threonine phosphoryla- to the Src kinase family, can associate with Sam68 tion of Sam68 during mitosis, with no eect on Sam68- (Derry et al., 2000). Both Sik and BRK colocalize RNA interactions (Resnick et al., 1997). with Sam68 in the nucleus (Derry et al., 2000). More It has been postulated that Src regulates gene speci®cally, BRK colocalizes with Sam68 in nuclear expression at the post-transcriptional level via Sam68 bodies called Sam68-SLM nuclear bodies (SNBs) in and evidence indicates that Src can act on the general both breast and colon tumor cell lines (Derry et al., mechanism of splicing and/or mRNA transport. Using 2000). Furthermore, Sik can tyrosine phosphorylate the murine tumor necrosis factor b (TNFb) gene as a Sam68 in vivo and negatively regulate its RNA reporter for pre-mRNA processing, co-transfection binding ability (Derry et al., 2000). Sik expression with an expression vector containing Src cDNA into in cells can also abolish the ability of Sam68 to act NIH3T3 cells was shown to induce a modi®cation of as a cellular homologue of Rev, a protein which RNA processing (Neel et al., 1995). Comparison of plays an essential role in the nuclear export of several modes of Src activation demonstrated that Src unspliced or partially spliced viral transcripts (Derry could either slow down the splicing rate or allow et al., 2000). Although Src has been demonstrated to export of partially spliced transcripts (Neel et al., interact with Sam68 during mitosis, Sik/BRK is the 1995). Interestingly, overexpression of an activated ®rst tyrosine kinase that has been shown to form of the Src family kinase Fyn in HEK293 cells phosphorylate Sam68 within the intact nucleus and has been observed to phosphorylate Sam68 and regulate its RNA binding ability. Interestingly, Sik negatively regulate its association with the splicing- has a very short unique amino terminus and, unlike associated factor YT521-B (Hartmann et al., 1999). Src, is not myristoylated (Vasioukhin et al., 1995), a However, a recent study has demonstrated that RNA modi®cation that is thought to prevent the transport transport and splicing of Lymphotoxin a transcripts of Src into the nucleus (David-Pfeuty et al., 1993). In are insensitive to the deletion of the Src SH3 domain, this respect, it would be interesting to speculate thus indirectly ruling out Sam68 as a potential whether a subpopulation of unmyristoylated Src regulator of these mRNA processing events via Src protein exists within cells that can translocate to (Gondran and Dautry, 1999). Mutation of the Src the nucleus to exert speci®c functions at speci®c times SH3 domain has been shown to disrupt its association of the cell cycle via Sam68. Lastly, it is also with Sam68 (Taylor and Shalloway, 1994). However, important to note that plasma membrane localization the Src SH3 mutant used in this study also contained of Fyn has also been demonstrated to be essential for an activating Src mutation (Gondran and Dautry, the constitutive tyrosine phosphorylation of nuclear 1999). This raises the possibility that Src may have Sam68 and that phosphorylation of Sam68 can occur still been able to phosphorylate Sam68 and interact outside the nucleus, suggesting that Sam68 molecules with the RNA binding protein through its SH2 can accumulate within the nucleus after they become domain. Interestingly, mutating the SH2 domain of phosphorylated (Lang et al., 1999). Src aected the RNA transport phenotype, and The identi®cation of nuclear targets of Src strength- disruption of the tyrosine kinase domain aected both ens its role in mediating cellular events that do not splicing and transport phenotypes (Gondran and necessarily involve its presence at the plasma mem- Dautry, 1999). brane. This further supports the notion that dierential Currently, Sam68 and Src are thought to interact localization patterns of Src serve a speci®c cellular only upon nuclear envelope breakdown during mitosis. function, which may be clari®ed by the identi®cation of However, there is evidence that suggests a constitutive its substrates at these particular subcellular locations. association between Src and Sam68 throughout the cell Attempts to identify additional targets of Src are thus cycle (Pillay et al., 1996). Furthermore, the early useful for the elucidation of the normal function of Src involvement of Sam68 in T-cell signaling suggests the within the cell. ASAP1, an Arf (ADP-ribosylation existence of a non-nuclear population of Sam68, whose factor) GTPase-activating protein was identi®ed in a possible function may be to serve as an adaptor screen for proteins that interact with the SH3 domain molecule (Vogel and Fujita, 1995; Fusaki et al., 1997; of Src (Brown et al., 1998). The protein is mostly found Lang et al., 1999). Studies in T-cells indicate that in the cytoplasm in a perinuclear, reticulate network in Sam68 may function as an adaptor linking the TCR- cells transfected with an ASAP1 expression vector coupled Src family kinases Fyn and Lck to downstream (Brown et al., 1998). It coimmunoprecipitates with Src eectors (Vogel and Fujita, 1995; Fusaki et al., 1997; and becomes tyrosine phosphorylated in cells expres- Lang et al., 1999). Numerous other partnerships with sing an activated mutant form of Src (Brown et al., dierent SH2 and SH3 domain-containing signaling 1998). The association of ASAP1 with Src, Arfs and molecules have also been documented including: PLCg- PIP2 is thought to be important in coordinating 1, Grb2, Nck, Jak3, SHP-1, Cbl, Grap (Grb2-like membrane tracking with actin cytoskeletal remodel- protein), the p21ras GTPase activating protein, the p85 ing (Brown et al., 1998; Randazzo et al., 2000). subunit of PI3-kinase, p47phox and Tec kinase family Interestingly, Src associates with and/or phosphorylates (Richard et al., 1995; Finan et al., 1996; Bunnell et al., various proteins implicated in vesicle transport, includ- 1996; Fusaki et al., 1997; Guinamard et al., 1997; ing synapsin I, dynamin, synaptophysin, synaptogyrin, Jabado et al., 1997, 1998; Lawe et al., 1997; Trub et al., and cellugyrin (Barnekow et al., 1990; Onofri et al., 1997; Guitard et al., 1998). 1997; Foster-Barber and Bishop, 1998; Janz and Recently, it has been demonstrated that murine Sudhof, 1998). Furthermore, a Golgi protein that Src-related intestinal kinase (Sik) (Siyanova et al., may be involved in vesicle docking/tethering, desig-

Oncogene Src localization and activation by PTPs JD Bjorge et al 5631 nated golgin-67, has also been identi®ed as a potential including: (i) binding of Src to activated growth factor Src target (Jakymiw et al., 2000). Puri®ed baculovirus receptor tyrosine kinases; (ii) dephosphorylation of the c-Src has been shown to associate with and tyrosine negative regulatory Tyr(527/530) residue by a PTP; and phosphorylate golgin-67 in vitro (Raharjo et al., (iii) mutation. Each of these mechanisms could unpublished data) but further studies will be required potentially activate Src activity, or in some cases may to provide information concerning the functional act in concert with others. consequences of Src interactions with golgin-67. Recently, a c-src mutation that results in an Cumulatively, this evidence suggest that Src might activated, truncated form of human c-Src, that ends have a role in regulating membrane tracking events, with Tyr530, has been identi®ed in highly metastatic with possible links to the Golgi apparatus (David- forms of colon carcinoma (Irby et al., 1999). This Pfeuty and Nouvian-Dooghe, 1990; Abu-Amer et al., mutation, which was not detected in less metastatic 1997). In osteoclasts, Src is localized to speci®c colon carcinomas, is the ®rst report of an activating intracellular fractions that contain Golgi-derived Src mutation in human cancer (see review by Yeatman membranes (Abu-Amer et al., 1997). It is thought that in this issue). Although studies in transfected cells have Src not only governs the formation of the rued revealed that the truncated, mutant c-Src protein is membrane in osteoclasts, but is itself packaged within phosphorylated in vivo on Tyr530, this protein has sub-Golgi vesicles that are transported to the bone- elevated kinase activity, presumably because it lacks at apposed plasma membrane (Abu-Amer et al., 1997). In least three amino acid residues carboxy-terminal to Rat-1 ®broblasts over-expressing chicken c-Src, Src is Tyr530 that are required for proper binding to the SH2 localized to late endosomal membranes (Kaplan et al., domain and the establishment of an inactive con- 1992). However, it is interesting to note that in this formation (Songyang et al., 1993; Cantley and study no direct attempt was made to distinguish Songyang, 1994). It is very interesting, and perhaps between endosomes and the trans-Golgi Network somewhat ®tting, that the ®rst activating mutation of (TGN), thus raising the possibility that Src may Src to be reported in a human cancer results in a Src regulate protein tracking events that may involve protein that is truncated in a location that is similar to the TGN. that of the v-Src protein of RSV, in a manner that probably abolishes the eectiveness of the carboxy- terminal negative regulatory region found in normal c- Involvement of c-Src human cancers Src (Irby et al., 1999).

This topic is discussed in more detail in the review article by Yeatman and colleagues in this issue of Conclusions and future directions Oncogene, and in other reviews (Biscardi et al., 1999), so our discussion of it will be limited to a very short Our understanding of Src structure, regulation, summary of a few relevant concepts and experimental localization, and function within the cell has increased ®ndings. dramatically since its ®rst discovery. Numerous modes For many years it was widely believed that Src of Src regulation have now been identi®ed. They would be found to be involved in the development or include alterations in the phosphorylation status of progression of at least some types of human cancers, Src mediated by kinases and phosphatases and/or Src's especially in light of the strong transforming activity of interaction with Src-binding proteins. Many studies, in the Rous sarcoma virus v-Src product in primary particular those that have identi®ed kinases and chicken embryo ®broblasts, and its potent tumorigeni- phosphatases that target Tyr(527/530) of Src, have city in chickens when introduced either by RSV often utilized overexpression or suppression systems to infection or by direct injection of v-src DNA (Fung characterize the regulatory protein's potential role in et al., 1983). In addition, mutationally activated or Src regulation. These studies have been extremely overexpressed forms of chicken, mouse, and human c- useful in identifying potentially important players in Src have been shown to transform cells in culture the Src regulation process, although other experiments under various conditions (Levy et al., 1986; Tanaka will be required to address how these factors are and Fujita, 1986; Lin et al., 1995), and mutationally utilized normally within the cell to modulate Src kinase activated human c-Src was found to be highly activity. Thus, further analyses addressing the regula- tumorigenic in chickens when introduced with a viral tion of these factors will be critical for the determina- vector (Tanaka et al., 1990). Taken together, these tion of the physiological regulatory processes of Src studies suggested that it was highly likely that aberrant kinase activation. activation of Src kinase activity by mutation or other It is becoming more evident that Src may be mechanisms could contribute to the development and involved in signaling events at various cellular progression of some human cancers. locations. In order to help clarify and elucidate the There is a large body of evidence that has normal functions of Src and fully comprehend its demonstrated that Src kinase activity, and sometimes transforming potential within the cell, Src interactions Src protein levels, are elevated in several cancers with speci®c targets or Src-binding proteins at the including colon and breast cancer, with a correlation dierent subcellular locations should be characterized often observed between increases in Src kinase activity as fully as possible. The speci®c localization of Src to and degree of malignancy (Bolen et al., 1987; Cart- the plasma membrane, cytoskeleton, endosomes, peri- wright et al., 1989; Ottenho-Kal et al., 1992; nuclear membranes and possibly even the nucleus Verbeek et al., 1996; Egan et al., 1999; Biscardi et illustrate that Src is a complex signaling molecule al., 1999). As mentioned previously, Src kinase activity with a number of potentially important cellular can be activated through a number of mechanisms functions.

Oncogene Src localization and activation by PTPs JD Bjorge et al 5632 Authors note Acknowledgments The present review is not meant to be comprehensive, and We thank the members of our laboratory and S Robbins will discuss only some of the broad range of topics relevant for their comments and suggestions regarding the manu- to Src kinase, especially some topics that have not been script. Experimental work in our laboratory was supported discussed extensively in other recent reviews on Src. by grants from the Medical Research Council of Canada, Several excellent reviews provide substantial coverage of the National Cancer Institute of Canada, the Canadian topics not dealt with extensively in this review, such as Src Breast Cancer Research Initiative, and the Alberta Breast interactions with growth factor receptor tyrosine kinases, Cancer Foundation. A Jakymiw is the recipient of an MRC and downstream signaling pathways e.g. (Abram and predoctoral studentship, DJ Fujita is a Scientist of the Courtneidge, 2000; Biscardi et al., 1999; Thomas and Alberta Heritage Foundation for Medical Research. Brugge, 1997; Taylor and Shalloway, 1996; Brown and Cooper, 1996). Also, we apologize that because of space limitations, we were not able to cite all relevant references in many instances.

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