The Tumor Suppressor DAPK Is Reciprocally Regulated by Tyrosine Kinase Src and Phosphatase LAR

Molecular Cell

Article The Tumor Suppressor DAPK Is Reciprocally Regulated by Tyrosine Kinase Src and Phosphatase LAR Won-Jing Wang,1 Jean-Cheng Kuo,1,5 Wei Ku,2,5 Yu-Ru Lee,2,5 Feng-Chi Lin,1 Yih-Leong Chang,3 Yu-Min Lin,4 Chun-Hau Chen,1 Yuan-Ping Huang,1 Meng-Jung Chiang,1 Sheng-Wen Yeh,1 Pei-Rung Wu,2 Che-Hung Shen,2 Chen-Tu Wu,3 and Ruey-Hwa Chen1,2,* 1Institute

of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan of Molecular Medicine 3Department of Pathology, College of Medicine 4Institute of Biochemical Sciences National Taiwan University, Taipei 115, Taiwan 5These authors contributed equally to this work. *Correspondence: [email protected] DOI 10.1016/j.molcel.2007.06.037 2Institute

SUMMARY

Death-associated protein kinase (DAPK) is a calmodulin-regulated serine/threonine kinase and elicits tumor suppression function through inhibiting cell adhesion/migration and promoting apoptosis. Despite these biological functions, the signaling mechanisms through which DAPK is regulated remain largely elusive. Here, we show that the leukocyte common antigenrelated (LAR) tyrosine phosphatase dephosphorylates DAPK at pY491/492 to stimulate the catalytic, proapoptotic, and antiadhesion/ antimigration activities of DAPK. Conversely, Src phosphorylates DAPK at Y491/492, which induces DAPK intra-/intermolecular interaction and inactivation. Upon EGF stimulation, a rapid Src activation leads to subsequent LAR downregulation, and these two events act in synergism to inactivate DAPK, thereby facilitating tumor cell migration and invasion toward EGF. Finally, DAPK Y491/492 hyperphosphorylation is found in human cancers in which Src activity is aberrantly elevated. These results identify LAR and Src as a DAPK regulator through their reciprocal modification of DAPK Y491/492 residues and establish a functional link of this DAPK-regulatory circuit to tumor progression.

INTRODUCTION Reversible tyrosine phosphorylation plays crucial roles in many biological processes, such as growth, differentiation, migration, and death. Protein tyrosine phosphorylation level is determined by the balance between protein

tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), and thus a precise and coordinated regulation of these two classes of enzymatic activities ensures proper cellular responses to a variety of extracellular stimuli and intrinsic programs. Death-associated protein kinase (DAPK; also known as DAPK1) is a calmodulin (CaM)-regulated serine/threonine kinase and possesses multiple structural and functional domains, including a kinase domain, a CaM binding motif, eight ankyrin repeats (ARs), a cytoskeleton binding region, and a death domain (Deiss et al., 1995). Several lines of evidence indicate a role of DAPK in tumor suppression. DAPK expression is frequently reduced in various human tumor cell lines and tissues, and this DAPK downregulation correlates with the recurrence, metastatic progression, or unfavorable prognosis of several human cancers (Raveh and Kimchi, 2001; Bialik and Kimchi, 2004). Furthermore, DAPK is capable of suppressing oncogenic transformation in vitro (Raveh et al., 2001) and blocking tumor metastasis in vivo (Inbal et al., 1997). DAPK was identified based on its death-promoting effect (Deiss et al., 1995) and has been subsequently found to mediate apoptotic and autophagic death induced by a wide spectrum of stimuli (Cohen et al., 1997, 1999; Inbal et al., 1997, 2002; Raveh et al., 2001; Jang et al., 2002; Pelled et al., 2002). The proapoptotic function of DAPK is attributed in part to its effect on integrin inactivation, thereby suppressing adhesion-mediated survival signal (Wang et al., 2002). In addition to promoting cell death, DAPK elicits cytoskeleton remodeling effect through phosphorylating myosin light chain 2 (MLC) (Kuo et al., 2003; Bialik et al., 2004). Furthermore, DAPK functions as a potent inhibitor of cell migration (Kuo et al., 2006). It is believed that the proapoptotic and antimigratory activities of DAPK could act as a double safeguard mechanism to prevent malignancy. Despite its significance in tumor suppression, the molecular mechanism by which DAPK is regulated and its interplay with other tumor suppressors and oncoproteins have not been completely unraveled. Although primarily

Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc. 701

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

regulated by CaM binding (Cohen et al., 1997), DAPK activity can also be modulated by posttranslational modifications. Autophosphorylation at S308 inhibits DAPK catalytic activity (Shohat et al., 2001), and this phosphorylation is downregulated under certain death conditions (Shohat et al., 2001; Llambi et al., 2005). Phosphorylation of DAPK at S735 by ERK enhances its kinase activity (Chen et al., 2005), whereas phosphorylation at S289 by RSK attenuates its proapoptotic function (Anjum et al., 2005). The kinase activity of DAPK is required for all biological effects of DAPK, whereas the death domain regulates its proapoptotic function by interacting with ERK (Chen et al., 2005) and UNC5H2 (Llambi et al., 2005). In contrast to these two domains, the function of DAPK AR domain has not been well defined. Deletion of AR perturbs DAPK cytoskeletal function and subcellular localization (Bialik et al., 2004), whereas overexpressing a segment of AR (residues 451–498) interferes with the death-inducing function of DAPK (Raveh et al., 2000). Although these findings imply a role of AR-mediated protein-protein interaction in modulating DAPK activity, the interacting partner of DAPK AR has not been identified. Leukocyte common antigen-related protein (LAR, also known as PTPRF) is a receptor-like PTP (RPTP). Its extracellular region contains several immunoglobulin-like and fibronectin (FN) III-like domains, whereas the intracellular region consists of two phosphatase domains, termed D1 and D2. LAR undergoes proteolytic processing to form two noncovalently linked subunits: the extracelluar E subunit (LAR-E) and the phosphatase domain-containing P subunit (LAR-P) (Serra-Pages et al., 1994). Although genetic studies with LAR-deficient mice reveal a role of LAR in mammary gland development, postinjuring nerve regeneration, and cholinergic fiber innervation (Chagnon et al., 2004), the intracellular signaling and physiological substrates of LAR remain poorly characterized. In epithelial cells, LAR associates with cadherin-catenin complex and dephosphorylates b-catenin, which correlate with its abilities to inhibit cell migration and tumor formation (Muller et al., 1999). In addition, LAR interacts with focal adhesion-associated proteins a-liprin (Serra-Pages et al., 1995) and Trio (Debant et al., 1996), thus implicating a role in adhesion signaling and cytoskeleton remodeling. Here we report the reversible tyrosine phosphorylation as a regulatory mechanism for DAPK. The PTK Src phosphorylates DAPK at Y491/492 located in the AR domain, thereby suppressing DAPK catalytic and biological activities. Conversely, LAR dephosphorylates DAPK at the two residues to activate DAPK. Furthermore, we present evidence for the functional role of this DAPK phosphorylation in tumorigenesis. RESULTS LAR Interacts with DAPK The AR of DAPK is thought to mediate protein-protein interactions. In an attempt to dissect the signaling network of DAPK, we carried out a yeast two-hybrid screen using

this AR as bait. One of the positive clones identified from this screen encoded the C-terminal region of LAR (amino acids 1681–1897), encompassing most of its D2 domain. Yeast two-hybrid analysis revealed that both LAR (1681– 1897) and LAR D2 domain could specifically bind DAPK AR (Figure 1A). To assess the interaction between endogenous proteins in mammalian cells, we generated a DAPK-specific antibody that could immunoprecipitate endogenous DAPK (see Figure S1 in the Supplemental Data available with this article online). With this antibody, an interaction of endogenous DAPK with endogenous LAR was detected (Figure 1B). As D2 domains of several RPTPs are involved in substrate recruitment (Kashio et al., 1998; Zondag et al., 2000; Felberg et al., 2004), we investigated the affinity of DAPK to the substratetrapping mutant of LAR (LAR-D/A), in which the conserved D1506 residue in D1 was mutated to generate an inactive enzyme that locks substrate in its catalytic pocket (Flint et al., 1997). Remarkably, DAPK bound more strongly to LAR-D/A than to LAR (Figure 1C). As a control, the substrate-trapping mutant of TC45 did not interact with DAPK (Figure 1D). These data suggest DAPK as a substrate of LAR and predict an enzyme-substrate interaction between LAR-D1 and tyrosine-phosphorylated DAPK. We thus analyzed the binding mode of these two proteins in further detail. Deletion of D2 from wild-type (WT) LAR (DD2) abrogated its interaction with DAPK (Figure 1E, left). Interestingly, this D2-mediated interaction did not require DAPK tyrosine phosphorylation, as treatment of cell lysate with intestinal alkaline phosphatase or l phosphatase to globally abolish protein tyrosine phosphorylation (Figure S2A) did not affect this interaction (Figure 1E, left, and data not shown). Conversely, the substratetrapping mutant lacking D2 (DD2-D/A) could still bind DAPK (Figure 1E, right), presumably via the substratetrapping capability of mutated D1 domain. These results indicate that both catalytic D1 and noncatlytic D2 are involved in DAPK interaction and that the binding of D2 to DAPK is phosphorylation independent. LAR Dephosphorylates DAPK at pY491/492 We next tested whether DAPK is a substrate of LAR. Overexpression of LAR, but not its catalytically inactive mutant (LAR-C/S), led to a drastic reduction of DAPK tyrosine phosphorylation (Figure 1F). LAR overexpression, however, caused neither dephosphorylation of FAK (Figure S2B) nor a global tyrosine dephosphorylation of cellular proteins (Figure S2C). Furthermore, DAPK tyrosine phosphorylation was not affected by an intracellular PTP, PTP1B (Figure S2D). These results underscore the specificity of LAR-induced DAPK dephosphorylation. To map the dephosphorylation site(s) in DAPK, we generated several DAPK deletion mutants (Figure S3A). Examining the effect of LAR on tyrosine phosphorylation level of these mutants revealed that the phosphotyrosine residue or residues targeted by LAR are located in AR (Figures S3B–S3E). We thus mutated the eight tyrosine residues in AR. While all of the single mutants were efficiently

702 Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc.

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

Figure 1. Identification of DAPK as a Substrate of LAR (A) Yeast L40 cotransformed with indicated constructs was assayed for His3 phenotype (His) and b-galactosidase activity (b-gal). (B) Lysate of 293T cells was analyzed by immunoprecipitation (IP) followed by immunoblot (IB) with indicated antibodies. The anti-LAR antibody recognizes an epitope at N terminus, and thus LAR-E is revealed. (C) 293T cells cotransfected with various constructs were analyzed by IP and IB as indicated. Flag tag was added at the LAR C terminus, and thus both full-length LAR (LAR-F) and LAR-P are detected. (D and E) Lysates of 293T cells transfected as indicated were treated with alkaline phosphatase (PPase; [E], left panel) or untreated and then analyzed by IP and IB. In (D), lysate containing overexpressed DAPK was included (left lane) to reveal the position of DAPK. The positions of various forms of Flag-LAR are indicated (F, full-length LAR; P, LAR-P; FDD2, full-length LAR with D2 deletion; PDD2, LAR-P with D2 deletion). (F and G) LAR induces DAPK dephosphorylation. 293T transfectants as indicated were analyzed by IP and IB. (H) 293T transfectants were analyzed by IB with indicated antibodies. (I and J) LAR dephosphorylates DAPK in vitro. In (I), DAPK or mutant purified from transfected cells was incubated with the indicated amount of recombinant LAR-D1. In (J), baculovirus-derived DAPK bound on beads was incubated with purified Src. The beads were washed, incubated with LARD1 or PTP1B, and analyzed by IP, IB, or Coomassie blue staining.

Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc. 703

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

dephosphorylated upon LAR coexpression (Figure 1G and Figure S3F), the 491/492 double mutant (DAPK-DYF) was barely tyrosine phosphorylated in cells and could not be further dephosphorylated by LAR. To further confirm that both tyrosine residues were targeted by LAR, we generated two antibodies that specifically recognize DAPK phosphorylated at Y491 or Y492, respectively (see below). As shown in Figure 1H, DAPK phosphorylation at both residues was significantly reduced upon overexpression of LAR, but not LAR-C/S. Thus, our study indicates that LAR promotes DAPK dephosphorylation at pY491/492, the major tyrosine phosphorylation sites of DAPK. To determine whether LAR could dephosphorylate DAPK in vitro, we incubated the purified LAR-D1 with Flag-DAPK or Flag-DAPK-DYF immunoprecipitated from cell lysates. The tyrosine phosphorylation level of WT DAPK was gradually decreased by incubating with increasing amounts of LAR-D1 (Figure 1I). LAR-D1, however, could not dephosphorylate DAPK-DYF. To validate that LAR could directly dephosphorylate DAPK, we baculovirally expressed recombinant DAPK and purified it to near homogeneity (Figure S4). This purified DAPK was first phosphorylated in vitro by purified, recombinant Src (a DAPK Y491/492 kinase identified in this study; see below) and then incubated with purified LAR-D1. Again, LAR-D1 triggered a dose-dependent dephosphorylation of DAPK, whereas PTP1B did not dephosphorylate DAPK (Figure 1J). Furthermore, the extent of DAPK dephosphorylation by LAR was comparable to that of b-catenin (Figure S5), a known substrate of LAR (Muller et al., 1999; Dunah et al., 2005). These data collectively demonstrate that DAPK is a direct and efficient substrate of LAR. LAR Activates DAPK through a Y491/492-Dependent Mechanism Next, we investigated the functional interplay between DAPK and LAR. In vitro kinase assay using DAPK isolated from various 293T transfectants and MLC as the substrate revealed that DAPK kinase activity was elevated by coexpression of LAR, but not LAR-C/S. DAPK-DYF, which mimics tyrosine-dephosphorylated DAPK, exhibited a higher activity than did WT DAPK in the absence of LAR but could not be further stimulated by LAR (Figure 2A). Similar results were obtained by assaying the effect of LAR on DAPK catalytic activity in vivo using an antibody specifically recognizing T18/S19-phosphorylated MLC (Figure 2B). These results indicate that dephosphorylation of DAPK by LAR stimulates DAPK kinase activity, which predicts a similar effect on its biological functions. DAPK is known to promote apoptosis/anoikis and to suppress migration, both of which are ascribed in part to its inhibitory effect on integrin-mediated adhesion (Wang et al., 2002; Kuo et al., 2006). When assessed in 293T cells, the antiadhesion function of DAPK was potentiated by coexpression of LAR, but not LAR-C/S. Consistent with its higher kinase activity, the DAPK-DYF elicited a stronger antiadhesion effect than WT DAPK, and this effect could not be further enhanced by LAR (Figure 2C). To test the

regulation of DAPK apoptotic effect by LAR, we used NIH 3T3 cells, which can be induced to undergo apoptosis by DAPK overexpression (Wang et al., 2002). The apoptotic function of DAPK was stimulated by LAR, but not LARC/S, through a Y491/492-dependent manner (Figure 2D). Finally, we investigated the influence of LAR on the antimigratory effect of DAPK using a lung adenocarcinoma cell line CL1-5, as this cell is insensitive to the apoptotic effect but sensitive to the antimigratory function of DAPK (Kuo et al., 2006). Again, LAR enhanced the migration/ invasion-inhibitory activities of DAPK through a Y491/ 492-dependent manner (Figure 2E). Thus, our study indicates that LAR functions as an activator of DAPK through dephosphorylating DAPK at pY491/492. Knockdown of LAR Promotes DAPK Tyrosine Phosphorylation and Inactivation Having demonstrated DAPK dephosphorylation and activation in cells overexpressing LAR, we next investigated whether this DAPK regulation could be recapitulated by endogenous LAR. To this end, we generated two pools of 293T stable transfectants carrying LAR siRNA and control siRNA, respectively. The level of endogenous LAR was significantly reduced in cells expressing LAR siRNA, but not control siRNA (Figure 3A, top). This knockdown of LAR resulted in an elevation of DAPK tyrosine phosphorylation. Tyrosine phosphorylation of DAPK-DYF, however, was not affected by LAR siRNA (Figure 3B). Furthermore, tyrosine phosphorylation of endogenous DAPK was similarly enhanced by LAR depletion (Figure 3C). These observations collectively point out a physiological role of LAR in dephosphorylating DAPK, which suggests a functional interplay of these two proteins in physiological settings. Indeed, when DAPK was introduced to the three pools of 293T derivatives, the antiadhesion function of DAPK was compromised in cells carrying LAR siRNA, but not control siRNA (Figure 3D). To assess the influence of endogenous LAR on the antimigratory effect of DAPK, we utilized a cell line, CL1-0, the parental line of aforementioned CL1-5, as this cell line expressed a significant amount of endogenous LAR (Figure 3A, bottom). We generated two pools of CL1-0 stable transfectants carrying LAR siRNA and control siRNA, respectively. Expression of LAR was diminished only in cells expressing LAR siRNA (Figure 3A, bottom). This LAR depletion led to a great reduction of the antimigratory function of DAPK (Figure 3E). Importantly, the effects of LAR siRNA on DAPK tyrosine phosphorylation (Figure 3C), antiadhesion (Figure 3D), and antimigration (Figure 3E) functions were completely reversed by expressing an siRNA-resistant LAR cDNA (LARR), indicating that these effects are specific to LAR downregulation. Finally, we assessed the capability of endogenous LAR to regulate the function of endogenous DAPK. Knockdown of endogenous DAPK significantly promoted migration of CL1-0 cells or CL1-0 cells carrying control siRNA but only marginally affected that of LAR siRNA-expressing cells (Figure 3F), thus demonstrating a functional attenuation of endogenous DAPK

704 Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc.

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

Figure 2. LAR Promotes DAPK Catalytic and Biological Functions (A) DAPK isolated from indicated 293T transfectants was analyzed by in vitro kinase assay or by IB. Lysates were also analyzed by IB (bottom). (B) Various 293T transfectants were analyzed by IB as indicated. (C–E) 293T (C), NIH 3T3 (D), and CL1-5 (E) transfectants as indicated were assayed for adhesion (C), apoptosis (D), and migration/invasion (E). The incubation periods for migration and invasion assays were 15 and 24 hr, respectively. Data are represented as mean ± SEM (*p < 0.05; **p < 0.005; n R 3). The expression levels of various forms of DAPK and LAR are shown on the bottom.

Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc. 705

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

Figure 3. DAPK Dephosphorylation and Activation by Endogenous LAR (A) IB analysis showing the levels of LAR in parental cells, pools of stable transfectants carrying indicated siRNA as well as LAR-depleted cells transiently expessing siRNA-resistant LAR (LARR). (B and C) Lysates of 293T stable transfectants as in (A) were transiently transfected with DAPK or mutant (B) or untransfected (C) and analyzed by IP and IB as indicated. (D–F) 293T (D) or CL1-0 (E and F) stable transfectants as in (A) were transiently transfected with or without DAPK (D and E) or with DAPK siRNA or control siRNA () (F) and assayed for adhesion (D) or migration (E and F). The incubation time for migration assay was 60 (E) or 50 hr (F). Data are represented as mean ± SEM (*p < 0.05; **p < 0.005; n R 3). The expression level of DAPK is shown on the bottom.

706 Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc.

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

Figure 4. Src Phosphorylates DAPK at Y491/492 (A) 293T cells expressing Flag-DAPK were pretreated with or without 10 mM PP2, 1 mM SU6656, or 10 mM ST1571 and left in suspension for 30 min () or plated on FN for 60 min. Cells were lysed for IP and IB as indicated. (B and C) Src induces DAPK phosphorylation. 293T transfectants were analyzed by IP and/or IB as indicated. (D and E) Flag-DAPK (D) or endogenous DAPK (E) isolated from indicated MEF by IP was analyzed for its tyrosine phosphorylation. The expression level of Src is shown on the bottom. (F) Src and DAPK interact endogenously. 293T cell extract was analyzed by IP and IB as indicated. (G) Flag-DAPK or mutant isolated from transfected cells by IP was incubated with or without purified Src and then analyzed by autoradiography or IB. (H) GST-AR or mutant was phosphorylated by purified Src and then analyzed by autoradiography or Coomassie blue staining.

in LAR-depleted cells. These analyses provide compelling evidence for the role of endogenous LAR in regulating DAPK tyrosine phosphorylation and biological activities. Src Phosphorylates DAPK at Y491/492 Since studies described above have identified LAR as a DAPK activator through dephosphorylating DAPK at pY491/492, we reasoned that the PTK that phosphorylates DAPK at these two sites should function as a DAPK inhibitor. To search for such PTK, we examined

whether DAPK phosphorylation at Y491/492 could be stimulated by any extracellular signals. Notably, cell adhesion on FN induced tyrosine phosphorylation of DAPK, but not DAPK-DYF (Figure S6A), implying the involvement of an integrin-activating PTK in this event. Importantly, this induction of DAPK phosphorylation was blocked by Src family kinase (SFK) inhibitor PP2 or SU6656 (Figure 4A, left), but not by an inhibitor of Abl (Figure 4A, right), another PTK activated by adhesion signaling. Accordingly, expression of Src527F (Src active mutant) led to a robust

Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc. 707

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

708 Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc.

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

induction of DAPK tyrosine phosphorylation. DAPK491F or DAPK492F was only weakly phosphorylated, and DAPK-DYF was barely phosphorylated under the same conditions (Figure 4B). The function of Src in promoting DAPK Y491/492 phosphorylation was further confirmed by the two antibodies specifically recognizing Y491- and Y492-phosphorylated DAPK (Figure 4C). Notably, the specificities of these two antibodies were demonstrated by their inability to recognize DAPK single and double mutants that lost the corresponding tyrosine residue. Furthermore, in cells expressing Src527F, these two antibodies could detect endogenous Src-phosphorylated DAPK with a high specificity (Figure S7). To ascertain a physiological role of Src in DAPK phosphorylation, we made use of fibroblasts derived from Src/Yes/Fyn (SYF) null mouse embryos (Cary et al., 2002) and their WT counterparts. DAPK overexpressed in SYF cells exhibited a lower tyrosine phosphorylation level than that in the WT MEF. Reconstitution of SYF cells with Src restored the tyrosine phosphorylation level of DAPK (Figure 4D). Furthermore, endogenous DAPK isolated from WT MEF also displayed a higher tyrosine phosphorylation level compared with that from SYF cells (Figure 4E). Consistent with this regulation of endogenous DAPK by endogenous Src, the two proteins interacted endogenously (Figure 4F). These results collectively demonstrate the physiological role of Src in promoting DAPK phosphorylation at Y491/492. Previous studies have established a positive relationship between the activations of Src and FAK (Mitra and Schlaepfer, 2006). Accordingly, expression of an active FAK (CD2-FAK) also induced DAPK tyrosine phosphorylation (Figure S6B). To test whether Src is a direct kinase of DAPK Y491/492, we carried out an in vitro kinase assay using purified, bacterially expressed Src as the enzyme and kinase-defective DAPK42A isolated from transfected cells as the substrate (to avoid the detection of DAPK autophosphorylation). We found that Src could efficiently phosphorylate DAPK42A, but not its Y491/492 mutant (DAPK42A/DYF) (Figure 4G). Furthermore, when purified, bacterially expressed DAPK AR fused with GST (GST-AR) was used as the substrate, we found that GST-AR was phosphorylated by Src in a dosedependent manner, and again, mutation of the Y491/ 492 residues abrogated this phosphorylation (Figure 4H). These data indicate Src as a direct kinase for DAPK Y491/492 residues.

DAPK Phosphorylation by Src Inhibits Its Catalytic and Biological Activities The identification of Src as a DAPK Y491/492 kinase predicts its role in DAPK inactivation. Indeed, Src overexpression led to a reduction of the kinase activity of WT DAPK, but not DAPK-DYF (Figure 5A). Furthermore, the activity of endogenous DAPK was elevated in SYF cells compared with that in WT MEFs (Figure 5B), demonstrating a role of endogenous SFK in suppressing the activity of endogenous DAPK. Consistent with the inhibition of DAPK kinase activity by Src, Src overexpression attenuated the apoptotic function of DAPK, while the function of DAPKDYF was only marginally affected by Src (Figure 5C). Src is known to regulate cell migration and survival through activation of multiple signaling molecules and pathways (Playford and Schaller, 2004). To more precisely evaluate the modulation of DAPK biological functions by Src, we generated the Src phosphorylation-mimicking DAPKDYD mutant, in which the Y491/492 residues were both replaced by Asp. Importantly, this mutant displayed a reduced kinase activity compared with WT DAPK (Figure 5D). Consequently, the antiadhesion (Figure 5E), apoptotic (Figure 5F), and antimigration (Figure 5G) functions of DAPK were all attenuated by this phosphomimetic mutation. These results strongly suggest that Src inhibits DAPK catalytic and biological activities by phosphorylating DAPK at Y491/492. Next, we explored the mechanism by which Y491/492 phosphorylation inhibits DAPK activity. Modeling of DAPK AR revealed that the Y491/492 residues are located at junction between the ‘‘turn’’ and ‘‘helix 2’’ in the fourth AR, with the hydroxyl groups of two tyrosines pointing toward opposite side from the backbone structure (Figure S8, left). Given the exposure of these two tyrosines (Figure S8, right), we tested whether phosphorylated AR could interact with DAPK catalytic region (kinase domain and CaM binding motif). Strikingly, we found that Srcphosphorylated AR or AR-DYD, but not unphosphorylated AR or AR-DYF, could efficiently pull down DAPK(K-CaM) from cell lysate (Figure 5H). Reciprocally, immobilized DAPK(K-CaM) could pull down AR-DYD, whereas AR-DYF showed negligible interaction (Figure 5I, bottom). Furthermore, preincubation of purified DAPK(K-CaM) with AR-DYD, but not with AR-DYF, diminished its kinase activity (Figure 5I, top). These results indicate that Y491/ 492 phosphorylation induces an intra-/intermolecular

Figure 5. Src Suppresses DAPK Catalytic and Biological Activities (A and B) DAPK or mutant isolated from 293T transfectants (A) or endogenous DAPK isolated from indicated MEF (B) by IP was analyzed by in vitro kinase assay or by IB. In (A), Src expression was analyzed by IB. (C) NIH 3T3 transfectants were assayed for apoptosis or analyzed by IB as indicated. (D) Flag-DAPK or mutant isolated from transfected cells by IP was analyzed by in vitro kinase assay or by IB. (E–G) 293T (E), NIH 3T3 (F), or CL1-5 (G) transfectants were assayed for adhesion (E), apoptosis (F), or migration (G). The incubation time for migration assay was 36 hr. Data are represented as mean ± SEM (**p < 0.005; n R 3). DAPK (or mutant) expression was analyzed by IB. (H) Indicated GST fusion proteins were used to pull down Flag-DAPK(K-CaM) from cell lysate. The pull-down products and 5% amount of input lysate were analyzed by IB with anti-Flag. Equal input of GST fusion proteins is shown on the bottom. (I) Flag-DAPK(K-CaM) isolated from transfected cells was incubated with indicated amounts of GST fusion proteins and then analyzed by in vitro kinase assay or by IB with anti-Flag or anti-GST. The equal input of GST fusion proteins was revealed by Coomassie blue staining.

Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc. 709

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

interaction between DAPK AR and K-CaM to inhibit DAPK activity. Src and LAR Mediate EGF-Induced DAPK Inactivation Having uncovered a reciprocal role of Src and LAR in regulating DAPK, we next explored whether DAPK could be subject to both Src- and LAR-mediated regulation under a physiological stimulus. EGF signaling was reported to cause LAR downregulation (Ruhe et al., 2006) and is well known to induce Src activation. Accordingly, EGF treatment of A431 cells induced a rapid Src activation (Figure 6A) and Src/DAPK complex formation (Figure S9). Downregulation of LAR-E, however, occurred at a later stage, being evident at 60 min after EGF stimulation (Figure 6A). This was accompanied by a decrease of LAR-P and its phosphatase activity (Figures S10A and S10B). In correlating with the time course of Src activation and LAR downregulation, endogenous DAPK tyrosine phosphorylation was induced as early as 5 min after EGF treatment, and a further induction was seen at 60 min (Figure 6A, bottom). Furthermore, the activities of both exogenous (Figure 6B) and endogenous (Figure 6C, bottom) DAPK were downregulated at a similar kinetics. To validate the contribution of Src to the early phase of DAPK modulation induced by EGF, we utilized the inhibitor PP2. Surprisingly, PP2 restored both LAR protein level (Figure 6C, Figure S10A) and activity (Figure S10B) in EGF-treated cells, and a similar effect was observed with a Src-specific shRNA (Figure S10C). These findings thus demonstrate a requirement of Src in EGF-triggered LAR downregulation. Consistently, PP2 abrogated both early (5 min) and late (60 min) phases of DAPK tyrosine phosphorylation and inactivation induced by EGF (Figure 6C, bottom). Likewise, Src inhibitor SU6656 also blocked both early and late effects of EGF on DAPK (Figure 6D). Thus, our results indicate that EGF-induced Src activation triggers the initial and moderate attenuation of DAPK activity. At a later stage, the activated Src leads to LAR downregulation, and these opposing regulations of Src and LAR impinge together on DAPK to cause a further reduction of DAPK activity. Next, we investigated the functional significance of this LAR/Src-controlled DAPK regulation in EGF-induced migration of A431 cells. Remarkably, although WT DAPK could still elicit a modest inhibition of EGF-induced chemotactic migration, this effect resembled that of the phosphomimetic mutant DAPK-DYD (Figure 6E). On the contrary, the DAPK-DYF exhibited a greatly elevated antichemotactic activity. Similar results were obtained when assaying the ability of these DAPK derivatives to inhibit EGF-induced invasion (Figure 6E). The weak antimigration/anti-invasion effects of WT DAPK in A431 cells in response to EGF was most likely due to its high level of tyrosine phosphorylation. Indeed, DAPK expressed in EGF-treated A431 cells displayed an 10-fold higher level of Y491/492 phosphorylation than that in CL1-5 cells (Figure 6F) and thus elicited a much weaker antimigration effect in A431 than in CL1-5

(comparing Figure 6E with Figure 5G). Finally, the EGFinduced, Src/LAR-mediated DAPK Y491/492 phosphorylation was also observed in MCF7 cells (Figure 6G and Figure S10D), indicating that this mode of DAPK modulation is not restricted to cells with a high level of EGF receptor. Collectively, these results demonstrate the capability of Src and LAR in regulating DAPK under a physiological setting and suggest a functional role of this LAR/Src-mediated DAPK inactivation in EGF-induced cell migration.

DAPK Y491/492 Hyperphosphorylation in Human Tumors and Its Correlation with Elevated Src Activity Having demonstrated Src-mediated DAPK Y491/492 phosphorylation in a physiological setting, we next investigated whether this DAPK phosphorylation occurs in any pathological situation in which Src is aberrantly upregulated, such as in human tumors. We first evaluated the levels of pY416Src, pY491/492DAPK, and DAPK using a panel of human tumor cell lines that express a detectable level of DAPK. Importantly, this analysis revealed a statistically significant correlation between the level of pY416Src and pY491/492DAPK (p = 0.02, Figure 7A and Figure S11). The level of total DAPK, however, did not display drastic differences in these cell lines (Figure S11B). In cell lines with a high level of pY491/492DAPK, inhibition of Src significantly reduced this DAPK phosphorylation (Figure 7B). Among the 27 cell lines derived from six cancer types, elevated pY416Src and pY491/492DAPK levels were most frequently found in colon cancer cell lines (Figure S11B), which prompted us to examine colon tumor tissues by immunohistochemical (IHC) analysis. In six out of ten patient specimens, focal expression of pY416Src was observed in tumor cells (ranging from 5% to 50% in a given specimen). Importantly, in the areas showing positive in pY416Src signal, most of them exhibited positive staining for both pY491DAPK and pY492DAPK, and the strengths of pY491DAPK and pY492DAPK signals correlated with that of pY416Src signal (Figures 7C and 7D and Figure S12). Conversely, pY491DAPK and pY492DAPK signals were not observed in tumor areas that did not show pY416Src signal (data not shown). In addition, pY491DAPK, pY492DAPK, and pY416Src were not detected in normal colon mucosa in the same specimens (Figure 7C and Figure S12). Importantly, the pY416Src, pY491DAPK, and pY492DAPK signals were all enriched on cell membrane (Figure 7C, inset), consistent with the role of Src in phosphorylating these tyrosines and demonstrating the specificity of our IHC analysis. Together, these data indicate a positive correlation between elevated Src activity and DAPK Y491/492 hyperphosphorylation in human cancers and implicate a role of Src-mediated DAPK inactivation in tumorigenesis. Thus, Y491/492 phosphorylation represents a new mechanism through which DAPK is inactivated in tumors.

710 Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc.

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

Figure 6. LAR and Src Mediate EGF-Induced DAPK Inactivation (A) A431 cells were treated with EGF and analyzed by IP and IB as indicated. (B) Retrovirally expressed DAPK isolated from EGF-treated A431 cells was analyzed by in vitro kinase assay or by IB. (C and D) A431 cells were pretreated with or without 60 mM PP2 or 2 mM SU6656 and then exposed to EGF. Cells were lysed for IP, IB, and kinase assay as in (A) and (B). (E) A431 cells infected with retroviruses carrying DAPK or mutants were assayed for EGF-induced chemotactic migration and invasion. Data are represented as mean ± SEM (*p < 0.05; **p < 0.005; n R 4). DAPK expression was analyzed by IB (bottom). (F) Indicated amounts of lysates of CL1-5 cells overexpressing DAPK as in Figure 5G and A431 cells overexpressing DAPK and treated with EGF for 60 min as in Figure 6B were analyzed by IB. (G) MCF7 cells expressing indicated siRNA were treated with EGF and analyzed by IB with various antibodies (pDAPK, 1:1 mixture of pY491 and pY492 antibodies).

Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc. 711

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

Figure 7. Correlation of Src Activity with DAPK Y491/492 Phosphorylation in Tumors (A) Lysates of various tumor cell lines were analyzed by IB with indicated antibodies. (Top) Representative results from nine cell lines. (Bottom) Fisher’s test on the results derived from all 27 cell lines. (B) Indicated cells were treated with or without 1 mM SU6656 and then analyzed by IB. (C) Representative IHC results illustrate the brown membranous staining of pY491DAPK, pY492 DAPK, and pY416Src in colon cancer (right), but not in normal colon mucosa (left) in the same specimen. (D) Summary of the IHC data from ten colon cancer specimens. The percentage of tumor cells showing positive staining and the intensity of staining (S, strong; M, medium; W, weak) are indicated. (E) Scheme depicting the reciprocal regulation of DAPK by Src and LAR (left) and Src/LAR-mediated DAPK inactivation in response to EGF-induced cell migration/invasion (right).

712 Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc.

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

DISCUSSION Reciprocal Regulation of DAPK by Src and LAR In this study, we uncover the inhibitory phosphorylation at Y491/492 as a DAPK-regulatory mechanism and identify Src and LAR as DAPK upstream regulators through their reciprocal modification of these two tyrosines (Figure 7E, left). The Y491/492 residues are highly conserved among vertebrates (Figure S13), implying an evolutionary conservation of this DAPK-regulatory network. Even though the two residues are located in AR rather than in the DAPK catalytic region, their phosphorylation induces the interaction of AR with the K-CaM catalytic region, which is responsible for the inhibition of DAPK kinase activity. It is currently unclear where in the K-CaM region the phosphorylated AR binds and how this binding affects DAPK catalytic activity. However, the capability of AR-DYD to bind and inhibit KCaM suggests the involvement of an electrostatic interaction. It is worth noting that the crystal structure of DAPK kinase domain reveals a unique basic loop protruding from the surface of N-terminal lobe, thereby forming a caplike structure over the substrate binding domain (Tereshko et al., 2001). Future studies will be aimed at determining whether the phosphorylated AR binds this basic loop to interfere with substrate binding. We identify Src as an inhibitor of DAPK by phosphorylating DAPK at Y491/492. The physiological significance of this DAPK modulation is best evidenced by the elevated DAPK catalytic activity in SYF cells compared with that in WT MEF. Furthermore, the ability of bacterially purified Src to phosphorylate bacterially purified AR in vitro clearly demonstrates a direct role of Src in DAPK phosphorylation. Of note, time course analysis of the extent of phosphorylation on WT AR, AR-491F, and AR-492F indicates that Src phosphorylates Y491 and Y492 residues at a similar rate (Figure S14A). Consistent with the two phosphorylation sites, stoichiometry analysis reveals that DAPK catalyzes 1.7 mol of phosphate incorporation per mol of GST-AR (Figure S14B). Even though Src is capable of generating diphosphorylated DAPK in vitro, the structure of LAR catalytic pocket seems to disfavor its targeting of substrates that are doubly phosphorylated at adjacent sties (Tonks and Neel, 2001). This discrepancy might explain the inability of LAR to completely ablate tyrosine phosphorylation on Src-catalyzed AR in vitro (Figure 1J). However, under in vivo conditions, DAPK is unlikely phosphorylated at a saturating level, which predicts the existence of a portion of monophosphorylated DAPK to serve as the substrate of LAR. In line with the dephosphorylation of DAPK by LAR, we found that the catalytically active LAR D1 domain binds DAPK via an enzyme-substrate mechanism. However, the noncatalytic D2 also interacts with DAPK though a DAPK phosphorylation-independent manner. Thus, similar to several other RPTPs with tandem repeated PTP domains (Kashio et al., 1998; Zondag et al., 2000; Felberg et al., 2004), the D2 of LAR is responsible for docking substrate and may contribute to the determination of substrate specificity.

The Interplays among EGF, Src, LAR, and DAPK in Regulating Migration and Tumor Progression In addition to uncovering the Src/LAR-mediated DAPK regulation, our study also addresses the physiological significance of this DAPK-regulatory circuit. We found that EGF induces a concomitant Src activation and LAR downregulation and that these two events act in synergism to promote DAPK inactivation. This finding thus demonstrates the ability of both Src and LAR in regulating DAPK under a physiological stimulus. As our previous study identified an inhibitory role of DAPK in directed migration by interfering with cell polarization and directional persistence (Kuo et al., 2006), we propose that the EGFinitiated, Src/LAR-mediated DAPK inactivation would be important to restrain these antimigratory activities of DAPK, thereby facilitating a persistent cell locomotion toward EGF (Figure 7E, right). In support of this model, we found that WT DAPK only modestly inhibits EGF-induced migration and invasion. Moreover, this function of DAPK resembles that of DAPK-DYD but is in sharp contrast with the strong inhibitory effect elicited by DAPK-DYF. The ability of EGF signaling to inactivate DAPK seems to be inconsistent with our previous finding that DAPK activity can be upregulated by ERK (Chen et al., 2005), a downstream effector of EGF pathway. However, this inactivation of DAPK by EGF is mediated through a combinatory effect of Src activation and LAR downregulation. Perhaps the extent of DAPK inactivation caused by the LAR- and Src-mediated regulation exceeds by far that of DAPK activation induced by ERK so that the net effect of a reduced DAPK activity is observed upon EGF treatment. These observations also reveal a complex regulation of DAPK activity by growth-factor-elicited singling pathways. A recent study indicates that EGF induces the proteolytic processing of LAR-P, leading to the release of LAR-E from the cell surface and the reduction of LAR phosphatase activity (Ruhe et al., 2006). In our system, the proteolytic product of LAR-P could only be detected by overexposing the LAR-P immunoblot (data not shown), presumably due to the unstable nature of this cleavage product. Nevertheless, we extend the previous finding by showing a requirement of Src for this EGF-induced LAR modulation. However, the slower kinetics of LAR downregulation relative to that of Src activation implies an indirect role of Src in this modulation event. Alternatively, Src activation and an independent EGF downstream event occurring at a later stage may both be required to induce LAR downregulation. In line with the latter possibility, we found that the modulation of LAR seems to be specific to EGF signaling. Integrin-mediated cell adhesion, which also activates Src, did not affect LAR level (Figure S15). We demonstrate that EGF signaling orchestrates Src and LAR to inactivate the tumor suppressor DAPK. It is tempting to speculate that inactivation of the antimigratory and proapoptotic DAPK contributes in part to the tumorpromoting activity of EGF. Moreover, Src is itself a

Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc. 713

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

well-established proto-oncogene, whereas LAR has been implicated in tumor suppression (Wang et al., 2004). Therefore, our study has revealed a complex crosstalk of DAPK with several tumor-promoting and -suppressing factors. In addition, the proto-oncogene RSK was reported to antagonize the cell-death function of DAPK (Anjum et al., 2005). Thus, multiple oncoprotein/tumorsuppressor-elicited signaling pathways can impinge on DAPK to influence its activity and function, further highlighting the importance of DAPK in regulating malignancy. Inhibition of DAPK via Y491/492 Phosphorylation in Cancers By screening a panel of cell lines derived from human cancers, we detect DAPK hyperphosphorylation at Y491/492 in a number of cell lines, with a high incidence in colon cancer cell lines. This hyperphosphorylation of DAPK was also found in a subset of colon cancer tissues. Importantly, in both cell lines and tissues, the status of DAPK Y491/492 phosphorylation positively correlates with Src activity. These findings support not only a physiological role of Src in phosphorylating DAPK but also its pathological role in inactivating this tumor suppressor in human cancers. Previous studies have found loss/reduction of DAPK expression in a variety of human tumor cell lines and tissues, which is mainly caused by hypermethylation at 50 UTR of DAPK gene (Raveh and Kimchi, 2001; Bialik and Kimchi, 2004). In this study, we identify DAPK phosphorylation at Y491/492 as a mechanism for inhibiting this tumor suppressor in human cancers. Given that the aberrant upregulation of Src has been found in several types of human cancers, hyperphosphorylation of DAPK may not be restricted to colon cancer. Furthermore, missense or splice-site mutations in the coding region of LAR are found in a fraction of human tumors, including colon cancer (Wang et al., 2004). Future studies will be conducted to survey the status of DAPK Y491/492 phosphorylation in various cancer types and its correlation with clinicopathological features of patients. EXPERIMENTAL PROCEDURES Yeast Two-Hybrid Screen Yeast two-hybrid screens of a human placenta cDNA library (Clontech) using LexA-AR as bait and yeast two-hybrid analysis were performed as described (Chen et al., 2005).

as described in the Supplemental Experimental Procedures. 293T and NIH 3T3 were transfected using the calcium-phosphate method. WT MEF, SYF, CL1-0, and CL1-5 were transfected with the Lipofectamine 2000 Reagent (Invitrogen). Retroviral infection of A431 cells was performed as described (Kuo et al., 2006). Immunoprecipitation and DAPK Kinase Assay Cells were lysed and subjected to immunoprecipitation and immunoblot analyses as described (Chen et al., 2005). In some experiments, 500 mg cell lysate in 300 ml of reaction mixture was incubated with 100 units of calf intestinal alkaline phosphatase (New England BioLabs) for 1 hr at 37 C before immunoprecipitation. DAPK kinase assay with GST-MLC as the substrate was performed as described (Chen et al., 2005). Determination of the effect of GST-AR on kinase activity of DAPK(K-CaM) is described in the Supplemental Experimental Procedures. Phosphorylation and Dephosphorylation of DAPK In Vitro Dephosphorylation of DAPK by LAR was assayed in a 50 ml reaction containing 25 mM HEPES (pH 7.3), 5 mM EDTA, 10 mM DTT, 0–15 units of LAR-D1 (Sigma), and 2.5 mg of DAPK or DAPK-DYF and incubated at 37 C for 30 min. Phosphorylation of DAPK by Src was carried out in a 40 ml reaction mixture containing 25 mM Tris (pH 7.2), 31.25 mM MgCl2, 6.25 mM MnCl2, 0.5 mM EGTA, 62.5 mM Na3VO4, 0.5 mM DTT, 125 mM ATP, 10 mCi [g-32P]ATP, 0–15 units purified Src, and 2.5 mg DAPK or its mutants and incubated at 30 C for 10 min. In some reactions, 10 mg GST-AR or GST-AR-DYF was used as the substrate. To determine the time course and stoichiometry of phosphorylation of GST-AR by Src, 40 ng of GST-AR or its mutants immobilized on beads was incubated with 3 units of Src for 0–60 min. Incorporation of 32P to GST-AR was determined by scintillation counting. Protein Purification and Pull-Down Analysis Purification of DAPK from baculovirus and GST-AR or mutants from bacteria was described previously (Chen et al., 2005). GST-AR or its mutants (480 ng) immobilized on beads was phosphorylated by 36 units of purified Src for 1 hr at 37 C. The beads were washed and then incubated with 2.4 mg cell lysates for 2 hr at 4 C. Bound protein was analyzed by immunoblot. Adhesion, Apoptosis, Migration, and Invasion Assays Adhesion and apoptosis were assayed as described previously (Wang et al., 2002). Migration and invasion assays are described in the Supplemental Experimental Procedures. IHC Analysis Sections of tissues 4 mm thick were autoclaved in trilogy (Cell Marque) at 121 C for 10 min, treated with 3% H2O2-methanol, and incubated with Power Black Universal Blocking Reagent (BioGenex). Sections were incubated with antibodies for pY491DAPK, pY492DAPK, and pY416Src for 60, 60, and 30 min, respectively, at room temperature. Detection of the immunoreactive staining was carried out by the Super Sensitive Non-Biotin Polymer HRP Detection System (BioGenex).

Plasmids, siRNAs, and Reagents Plasmid constructions as well as the details of various siRNAs, antibodies, and reagents are included in the Supplemental Experimental Procedures. The pDest-b-catenin was provided by H.-M. Shih, whereas the PTP1B cDNA was from T.-C. Meng and cloned to pRK5M. pCDNA3-TC45-D/A was from T.-C. Meng, and CD2-FAK was from G. Whitney.

Supplemental Data Supplemental Data include Supplemental Experimental Procedures, Supplemental References, and 15 figures and can be found with this article online at http://www.molecule.org/cgi/content/full/27/5/ 701/DC1/.

Cell Culture, Transfection, and Retroviral Infection 293, 293T, NIH 3T3, A431, WT MEF, and SYF cells (from J. Cooper) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (FCS). CL1-0 and CL1-5 cells were cultured in RPMI1640 containing 10% FCS. Various tumor cell lines are cultured

We thank M. Streuli, J. Cooper, G. Whitney, H.-M. Shih, T.-C. Meng, Z.-F. Chang, W.-W. Lin, J.-Y. Chen, Y.-S. Jou, and the National RNAi Core Facility for reagents and cell lines; M. Tremblay for discussion; and H.-M. Shih and T.-C. Meng for critically reading the manuscript. This work was supported by National Science Council Frontier Grant

ACKNOWLEDGMENTS

714 Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc.

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

NSC95-2321-B-001-025 and Academia Sinica Thematic Project AS-95-TP-B02. W.-J.W. is supported by National Health Research Institute Postdoctoral Fellowship PD9501.

Inbal, B., Bialik, S., Sabanay, I., Shani, G., and Kimchi, A. (2002). DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. J. Cell Biol. 157, 455–468.

Received: October 9, 2006 Revised: March 5, 2007 Accepted: June 25, 2007 Published: September 6, 2007

Jang, C.W., Chen, C.H., Chen, C.C., Chen, J.Y., Su, Y.H., and Chen, R.H. (2002). TGF-beta induces apoptosis through Smad-mediated expression of DAP- kinase. Nat. Cell Biol. 4, 51–58.

REFERENCES Anjum, R., Roux, P.P., Ballif, B.A., Gygi, S.P., and Blenis, J. (2005). The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling. Curr. Biol. 15, 1762–1767. Bialik, S., and Kimchi, A. (2004). DAP-kinase as a target for drug design in cancer and diseases associated with accelerated cell death. Semin. Cancer Biol. 14, 283–294. Bialik, S., Bresnick, A.R., and Kimchi, A. (2004). DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation. Cell Death Differ. 11, 631–644. Cary, L.A., Klinghoffer, R.A., Sachsenmaier, C., and Cooper, J.A. (2002). SRC catalytic but not scaffolding function is needed for integrin-regulated tyrosine phosphorylation, cell migration, and cell spreading. Mol. Cell. Biol. 22, 2427–2440.

Kashio, N., Matsumoto, W., Parker, S., and Rothstein, D.M. (1998). The second domain of the CD45 protein tyrosine phosphatase is critical for interleukin-2 secretion and substrate recruitment of TCR-z in vivo. J. Biol. Chem. 273, 33856–33863. Kuo, J.C., Lin, J.R., Staddon, J.M., Hosoya, H., and Chen, R.H. (2003). Uncoordinated regulation of stress fibers and focal adhesions by DAPkinase. J. Cell Sci. 116, 4777–4790. Kuo, J.C., Wang, W.J., Yao, C.C., Wu, P.R., and Chen, R.H. (2006). The tumor suppressor DAPK inhibits cell motility by blocking the integrinmediated polarity pathway. J. Cell Biol. 172, 619–631. Llambi, F., Lourenco, F.C., Gozuacik, D., Guix, C., Pays, L., Del Rio, G., Kimchi, A., and Mehlen, P. (2005). The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase. EMBO J. 24, 1192–1201. Mitra, S.K., and Schlaepfer, D.D. (2006). Integrin-regulated FAK-Src signaling in normal and cancer cells. Curr. Opin. Cell Biol. 18, 516–523.

Chagnon, M.J., Uetani, N., and Tremblay, M.L. (2004). Functional significance of the LAR receptor protein tyrosine phosphatase family in development and diseases. Biochem. Cell Biol. 82, 664–675.

Muller, T., Choidas, A., Reichmann, E., and Ullrich, A. (1999). Phosphorylation and free pool of beta-catenin are regulated by tyrosine kinases and tyrosine phosphatases during epithelial cell migration. J. Biol. Chem. 274, 10173–10183.

Chen, C.H., Wang, W.J., Kuo, J.C., Tsai, H.C., Lin, J.R., Chang, Z.F., and Chen, R.H. (2005). Bidirectional signals transduced by DAPKERK interaction promote the apoptotic effect of DAPK. EMBO J. 24, 294–304.

Pelled, D., Raveh, T., Riebeling, C., Fridkin, M., Berissi, H., Futerman, A.H., and Kimchi, A. (2002). Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons. J. Biol. Chem. 277, 1957–1961.

Cohen, O., Feinstein, E., and Kimchi, A. (1997). DAP-kinase is a Ca2+/ calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO J. 16, 998–1008.

Playford, M.P., and Schaller, M.D. (2004). The interplay between Src and integrins in normal and tumor biology. Oncogene 23, 7928–7946.

Cohen, O., Inbal, B., Kissil, J.L., Raveh, T., Berissi, H., SpivakKroizaman, T., Feinstein, E., and Kimchi, A. (1999). DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain. J. Cell Biol. 146, 141–148. Debant, A., Serra-Pages, C., Seipel, K., O’Brien, S., Tang, M., Park, S.H., and Streuli, M. (1996). The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains. Proc. Natl. Acad. Sci. USA 93, 5466–5471. Deiss, L.P., Feinstein, E., Berissi, H., Cohen, O., and Kimchi, A. (1995). Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon-induced cell death. Genes Dev. 9, 15–30. Dunah, A.W., Hueske, E., Wyszynski, M., Hoogenraad, C.C., Jaworski, J., Pak, D.T., Simonetta, A., Liu, G., and Sheng, M. (2005). LAR receptor protein tyrosine phosphatases in the development and maintenance of excitatory synapses. Nat. Neurosci. 8, 458–467. Felberg, J., Lefebvre, D.C., Lam, M., Wang, Y., Ng, D.H.W., Birkenhead, D., Cross, J.L., and Johnson, P. (2004). Subdomain X of the kinase domain of Lck binds CD45 and facilitates dephosphorylation. J. Biol. Chem. 279, 3455–3462. Flint, A.J., Tiganis, T., Barford, D., and Tonks, N.K. (1997). Development of ‘‘substrate-trapping’’ mutants to identify physiological substrates of protein tyrosine phosphatases. Proc. Natl. Acad. Sci. USA 94, 1680–1685. Inbal, B., Cohen, O., Polak-Charcon, S., Kopolovic, J., Vadai, E., Eisenbach, L., and Kimchi, A. (1997). DAP kinase links the control of apoptosis to metastasis. Nature 390, 180–184.

Raveh, T., and Kimchi, A. (2001). DAP kinase—a proapoptotic gene that functions as a tumor suppressor. Exp. Cell Res. 264, 185–192. Raveh, T., Berissi, H., Eisenstein, M., Spivak, T., and Kimchi, A. (2000). A functional genetic screen identifies regions at the C-terminal tail and death-domain of death associated protein kinase that are critical for its proapoptotic activity. Proc. Natl. Acad. Sci. USA 97, 1572–1577. Raveh, T., Droguett, G., Horwitz, M.S., DePinho, R.A., and Kimchi, A. (2001). DAP kinase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation. Nat. Cell Biol. 3, 1–7. Ruhe, J.E., Streit, S., Hart, S., and Ullrich, A. (2006). EGFR signaling leads to downregulation of PTP-LAR via TACE-mediated proteolytic processing. Cell. Signal. 18, 1515–1527. Serra-Pages, C., Saito, H., and Streuli, M. (1994). Mutational analysis of proprotein processing, subunit association, and shedding of the LAR transmembrane protein tyrosine phosphatase. J. Biol. Chem. 269, 23632–23641. Serra-Pages, C., Kedersha, N.L., Fazikas, L., Medley, Q., Debant, A., and Streuli, M. (1995). The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions. EMBO J. 14, 2827–2838. Shohat, G., Spivak-Kroizman, T., Cohen, O., Bialik, S., Shani, G., Berrisi, H., Eisenstein, M., and Kimchi, A. (2001). The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism. J. Biol. Chem. 276, 47460–47467. Tereshko, V., Teplova, M., Brunzelle, J., Watterson, D.M., and Egli, M. (2001). Crystal structures of the catalytic domain of human protein kinase associated with apoptosis and tumor suppression. Nat. Struct. Biol. 8, 899–907.

Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc. 715

Molecular Cell Reciprocal Regulation of DAPK by Src and LAR

Tonks, N.K., and Neel, B.G. (2001). Combinatorial control of the specificity of protein tyrosine phosphatases. Curr. Opin. Cell Biol. 13, 182– 195.

Wang, Z., Shen, D., Parsons, D.W., Bardelli, A., Sager, J., Szabo, S., Ptak, J., Silliman, N., Peters, B.A., van der Heijden, M.S., et al. (2004). Mutational analysis of the tyrosine phosphatome in colorectal cancers. Science 304, 1164–1166.

Wang, W.J., Kuo, J.C., Yao, C.C., and Chen, R.H. (2002). DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals. J. Cell Biol. 159, 169–179.

Zondag, G.C.M., Reynolds, A.B., and Moolenaar, W.H. (2000). Receptor protein tyrosine-phosphatase RPTPm binds to and dephosphorylates the catenin p120ctn. J. Biol. Chem. 275, 11264–11269.

716 Molecular Cell 27, 701–716, September 7, 2007 ª2007 Elsevier Inc.