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pISSN 2288-6982 l eISSN 2288-7105 Biodesign MINI REVIEW P 59-66 The regulation of receptor protein tyrosine phosphatases by the dimerization of intracellular domains Ho-Chul Shin and Seung Jun Kim* Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea *Correspondence: [email protected] Protein tyrosine phosphatases (PTPs) are critical in many signal transduction pathways for cell regulation. The activity of PTPs is governed by interactions between various regulatory domains and their partners. Receptor-type PTPs (PTPRs) also have many extracellular regulatory domains. The intracellular domain of some PTPRs consists of D1 and D2 domains, similar to classical PTPs. The D1 domain is an active phosphatase domain, but the D2 domain has weak or no activity. The D2 domain regulates the phosphatase activity of PTPRs containing the D1–D2 domain. Many studies have shown that the dimerization of the D2 domain can inhibit the phosphatase activity of PTPRs. A few models have been proposed to explain how phosphatase activity is inhibited by dimerization, but the precise mechanism is still not established. In this review, we discuss the regulatory mechanism of the phosphatase activity of PTPRs via the intracellular domain. INTRODUCTION Protein phosphorylation is a well-known and common posttranslational modification. Phosphate can form an ester bond on the hydroxyl group of various amino acids, i.e., serine, threonine, and tyrosine. The phosphorylated residues carry a large negative charge on the protein surface. This charge transition on the surface induces changes in protein–protein interactions, and these changes by phosphorylation are reversible. Because phosphorylation is simple and reversible, but results in large changes, it is used as an “on-off switch” in almost all signaling pathways in eukaryotes. Phosphorylation of a protein residue was first observed in 1932 by Fritz A. Lipmann and P. A. Levene. They extracted phosphoserine from vitellin, a phosphoprotein in egg yolk (Lipmann and Levene, 1932). Phosphothreonine has been isolated from bovine casein, a phosphoprotein in milk (De Verdier, 1952). Since phosphoproteins and phosphorylated amino acids were introduced, they had been considered a good nutrient source for the young. Kinase and phosphatase, which mediate protein phosphorylation and dephosphorylation, were discovered around the same time. In 1946, Daniel L. Harris measured the dephosphorylation activity of the protein extracted from frog eggs (Harris, 1946). Kinase activity was measured in 1954 by George Burnett and Eugene P. Kennedy using proteins in the mitochondrial membranes of rat liver cells (Burnett and Kennedy, 1954). Since protein kinases (PKs) were discovered, their importance in many signal transduction pathways, e.g., cell migration, cell proliferation, and differentiation, has been established. Although it was previously thought that the role of protein phosphatases bdjn.org (PPs) is just the reverse of PKs, the importance of PPs is now clearly established as well as PKs (Hunter, 2000; Muratore and Cole, 2007; Tiganis and Bennett, 2007; Tonks, 2006). PKs and PPs can be largely classified into three types based on substrate specificity: serine/threonine-specific, tyrosine-specific, and dual-specific. Phosphotyrosine regulation is particularly important in many signaling pathways. Phosphotyrosine (pY) sites account for a small portion of the human phosphoproteome, i.e., only 1.8%; in contrast, phosphoserine (pS) and phosphothreonine (pT) sites account for 86.4% and 11.8% of the phosphoproteome, respectively (Olsen et al., 2006). Despite this small number of sites, protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP), which are regulatory proteins for phosphotyrosine, occupy 17.4% (90 PTKs and 518 PKs) and 54.3% (107 PTPs and 197 PPs) of total PKs and PPs in human cells (Sacco et al., 2012). Moreover, there are nearly equal numbers of PTKs and PTPs, indicating that the balance between PTKs and PTPs is important for signaling pathways. The first PTKs discovered were v-Src and c-Src from Rous sarcoma virus (RSV) and chickens (Oppermann et al., 1979; Stehelin et al., 1977). This finding led to the idea that external materials, like viruses, can promote cancer. Researchers attempted to find an counteracting enzyme of PTK after the discovery of Src, and measured the activity of PTPs from various sources (Chernoff and Li, 1983; Clari et al., 1986; Gallis et al., 1981; Horlein et al., 1982; Swarup et al., 1982). In 1988, N. T. Tonks and colleagues first purified PTP and measured the phosphatase activity of the protein PTP1B (Tonks et al., 1988). The discoveries of Src and PTP1B promoted research on the phosphorylation and dephosphorylation of tyrosine residues. Biodesign l Vol.4 l No.2 l Jun 30, 2016 © 2016 Biodesign 59 The regulation of receptor protein tyrosine phosphatases by the dimerization of intracellular domains The functions of PTKs are well studied, and several drugs for PTK inhibition have already been developed and used in clinical settings. However, the functions of PTPs are not as well-known as those of PTKs are. Drugs targeting PTPs have just recently been developed, even though many PTPs are considered important and potential therapeutic targets for cancer, diabetes, and degenerative brain disease (Das et al., 2015; Krishnan et al., 2014). PTPs can be classified into four classes: classes I to IV. Almost all PTPs belong to class I, and are further divided into classical PTPs and dual-specificity phosphatases. Classical PTPs, which commonly have a highly conserved phosphatase domain, are classified as cytosolic PTPs and receptor-type PTPs (PTPRs). The cytosolic PTPs have various regulatory domains or motifs, e.g., SH2, PDZ, KIMKIS, and the zinc-binding domain. The features of these cytosolic PTPs are various and they are part of several signaling pathways. However, PTPRs each have similar configurations. PTPRs have various regulatory domains outside of the cell membrane, e.g., the Iglike domain, fibronectin-like domain, and carboxylation site. These domains can interact with ligands and environmental stresses, and these interactions can induce signal transduction in cells. The PTPRs have only one transmembrane region and one or two phosphatase domains in the intracellular region after the extracellular domains. The PTPRs are classified into 8 subtypes according to their features (Table 1) (Andersen et al., 2001). Five of 8 subtypes (R1/R6, R2A, R2B, R4 and R5) have two classical PTP domains in tandem: the membrane proximal D1 domain and distal D2 domain. The D1 domain has strong phosphatase activity, but the D2 domain does not. Despite little or no activity, the D2 domain has a highly similar amino acid sequence and 3D structure to those of classical PTPs. Several experimental results in vivo as well as in vitro and structural analyses have shown that the D2 domain is a regulatory domain for phosphatase activity. However, the mechanism of phosphatase activity regulation by the D2 domain is still unclear. Here, we focus on the role and the mechanism of the regulatory functions of the D2 domain. TABLE I 1 Classification of PTPRs Subtype R1 / R6 Name of PTPR PTPRC R2A PTPRK, PTPRM, PTPRT, PTPRU R2B PTPRD, PTPRF, PTPRS R4 PTPRA, PTPRE R5 PTPRG, PTPRZ1 R3 PTPRB, PTPRH, PTPRJ, PTPRO, PTPRP R7 PTPRR R8 PTPRN, PTPRN2 D1-D2 in tandem D1 only A B STRUCTURAL FEATURES OF THE PHOSPHATASE DOMAIN OF CLASSICAL PTPS First, we will describe the chemistry and mechanism of classical PTPs, which have a highly conserved phosphatase domain, including the active site and the overall structure. Classical PTPs have four typical and important motifs: PTP signature motif, KNRY motif, WPD loop, and Q-loop (Figure 1A). A cysteine in the PTP signature motif, (I/V)HCXXGXXR(S/T), also known as a P-loop, is a catalytic residue, and nitrogen atoms of main chain and an 60 Biodesign l Vol.4 l No.2 l Jun 30, 2016 © 2016 Biodesign FIGURE I 1 Important motifs for phosphatase activity and phosphatase chemistry. (A) The left panel shows the D1 domain of PTPRS (PDB ID: 2FH7) and the right panel shows the D1 domain of PTPRC (CD45) and its substrate peptide (green) (PDB ID: 1YGR). The motifs for phosphatase activity are colored in the 3D structure. WPD loop: red, KNRY motif: cyan, PTP signature motif: blue, Q-loop: yellow. Important residues are displayed as sticks. (B) The hydrolysis mechanism of phosphotyrosine at active sites of classical PTPs. bdjn.org Ho-Chul Shin and Seung Jun Kim arginine of the PTP signature motif can stabilize the phosphate of phosphotyrosyl substrates. A tyrosine residue in the KNRY motif has specificity to the phosphotyrosine via a tyrosyl group. Dephosphorylation proceeds by a two-step displacement mechanism. The phosphoryl group of a substrate is transferred to a thiol group as a nucleophile of the catalytic cysteine. An aspartate in the WPD loop acts as both a general acid and a base in two catalytic steps. In the next step, glutamines in the Q-loop help activate a water molecule. The phosphate from the thiophosphate intermediate is hydrolyzed using this water molecule (Figure 1B). During dephosphorylation, the WPD loop shows unique large movements via substrate binding (Figure 1A). Without the substrate, the loop is located apart from the active site, but once the substrate is bound to the active site, the loop moves almost 6 Å and the aspartate of the WPD loop attaches near the phosphate of phosphotyrosine. The PTP D1 and D2 domains also have these motifs and the typical scaffold of classical PTPs. analysis of the amino acid sequence alignment (Figure 2). The four motifs of the D1 domain are highly conserved, including Tyr of the KNRY motif, Arg and Cys of the PTP signature motif, two Glns of the Q-loop, and Asp of the WPD loop. However, Tyr of the KNRY motif and Asp of the WPD loop of the D2 domains are not conserved. Divergence in these motifs resulted in low or no activity of the D2 domain. The D2 domain of PTPRA is also inactive and has Asp and Glu, instead of Tyr of the KNRY motif and Asp of the WPD loop. Substitutions from Asp and Glu to Tyr and Asp nearly restored the activity levels to those of the D1 domain (Buist et al., 1999; Lim et al., 1998). When these residues of the D2 domain of PTPRE were substituted to Tyr and Asp, COMPARISON BETWEEN THE PTP D1 AND PTP D2 DOMAINS The available D1 and D2 of the PTPR structures were superimposed and their RMSDs were calculated (Table 2). These data show a maximum RMSD of 1.951 Å and minimum of 0.556 Å. The average was 0.830 Å and the average excluding the row of the D2 domain of PTPRG was 0.6827 Å. All RMSDs were less than 1.000 Å, except that of the superimposed D1 domains with the D2 domain of PTPRG. These low RMSDs indicate that the D1 and D2 domains have almost the same scaffold as classical PTPs. Abnormally high, but generally low RMSDs between the D2 domain of PTPRG and D1 domains are due to flexible loops of the D2 domains of PTPRG, but the overall scaffold of the D2 domain of PTPRG is also the same as that of the classical PTPs. Despite these similarities, there are critical differences between the D1 domain and D2 domain based on an FIGURE I 2 The alignment of amino acid sequences among PTPRs containing D1 and D2 domains. ClustalX2 (Larkin et al., 2007) was used to align the amino acid sequences of 12 PTPRs containing D1 and D2 domains. Blue indicates the level of conservation. The residues in red boxes are important for PTP activity, displayed as sticks in Figure 1A. TABLE I 2 RMSDs among D1 domains and D2 domains of PTPRs with solved structures D1 domain D2 domain A E F G S A E F G S K M T Z1 0.780* (195)# 0.704 (200) 0.577 (195) 1.246 (142) 0.575 (205) 0.709 (189) 0.642 (208) 0.556 (198) 1.309 (151) 0.557 (206) 0.853 (197) 0.930 (215) 0.688 (205) 1.344 (166) 0.602 (203) 0.727 (190) 0.778 (211) 0.763 (217) 1.951 (185) 0.831 (226) 0.806 (187) 0.836 (193) 0.668 (216) 1.235 (166) 0.657 (207) 0.684 (190) 0.660 (202) 0.580 (182) 1.642 (181) 0.561 (181) 0.701 (194) 0.693 (184) 0.653 (204) 1.177 (166) 0.599 (204) 0.634 (192) 0.570 (204) 0.566 (266) 1.170 (158) 0.567 (205) 0.723 (186) 0.792 (213) 0.752 (218) 1.679 (171) 0.602 (195) *RMSDs (Å ) were calculated using PyMol (Schrodinger, 2010) #Numbers of superimposed amino acids. bdjn.org Biodesign l Vol.4 l No.2 l Jun 30, 2016 © 2016 Biodesign 61 The regulation of receptor protein tyrosine phosphatases by the dimerization of intracellular domains phosphatase activity of the D2 domain of PTPRE also increased, but less than the activity of D1 domain. PTPRC (CD45) has an additional substitution from Arg to Gln in the PTP signature motif. When these three residues were replaced with the conserved residues, phosphatase activity of the mutant D2 domain increased slightly (Lim et al., 1999). Divergence at these critical residues for catalytic activity leads to the inactivation of the D2 domain, despite the similarity in the 3D structural scaffolds of the D1 domain and D2 domain. PTPRs have a D2 domain, despite a lack of catalytic activity because the domain can play a role in the regulation of phosphatase activity in the intracellular domain. A RECEPTOR-TYPE PTP REGULATION VIA D2 DOMAIN DIMERIZATION B The PTPRs can be dimerized or oligomerized via ligand binding on extracellular domains (Fukada et al., 2006; Perez-Pinera et al., 2007). This interaction can change the structure of the intracellular domain and inactivate phosphatase activity. Several studies have proven that the dimerization of the intracellular domains of PTPR can decrease phosphatase activity. A homodimer of the D2 domain of PTPRE has been detected by immunoprecipitation, implying that the D2 domain can form a dimer in vivo (Toledano-Katchalski et al., 2003). The phosphatase activity of PTPRC is inhibited when the dimerization of a chimeric protein, in which the extracellular domain and transmembrane domain of PTPRC are replaced with EGFR (epithermal growth factor receptor), is induced by EGF (epithermal growth factor) (Desai et al., 1993). The chimeric protein of PTPRZ in which FKBP is inserted between the transmembrane domain and D1 domain is dimerized by AP20187. This artificial dimerization decreases the phosphatase activity of PTPRZ (Fukada et al., 2006). The dimerization of the D2 domain, irrespective of whether it is related to features of the extracellular domains or internal features of the intracellular domain, can inhibit the phosphatase activity of PTPRs. In 1996, the structure of the D1 domain of PTPRA was discovered. The structure displayed an antiparallel dimer of two PTPRA D1 domains and a unique structure was observed with respect to the location of the N-terminal wedge (N-terminal helix-turn-helix) of the D1 domain, which blocks the active site of another D1 molecule (Figure 3A). Furthermore, these wedge regions of D1 domains are generally conserved at the amino acid level (Figure 3B). Researchers have suggested the wedge inhibition model, in which the wedge domain can block the active site and inhibit the phosphatase activity of the D1 domain after dimerization (Bilwes et al., 1996). In 1999, the structure of the intracellular domain of PTPRF containing D1 and D2 domains was determined (Nam et al., 1999). However, there was no blockage of the active site by a wedge and accordingly the active site was still accessible in the structure. After the discovery of this structure, no blockage of the 62 Biodesign l Vol.4 l No.2 l Jun 30, 2016 © 2016 Biodesign C FIGURE 3 I The dimer structure of PTPRA and its wedge region. (A) The dimer structure of PTPRA (PDB ID: 1YFO). The wedge regions are indicated by a blue line. The red circles are the target residues for constitutive activation. Translucent red circles show the active sites of PTP. (B) The amino acid sequences of the wedge region of PTPRs are aligned. The red box shows the target residue for constitutive activation, labeled by a red circle in (A). (C) Two PTPRFs (PDB ID: 1LAR) are superimposed on the dimer of the PTPRA D1 domain on the right panel. The yellow box displays a steric clash between two D2 domains of the superimposed PTPRFs. active site by a wedge region was detected in the structure of the D1 domains and D1-D2 domains. Moreover, superimposing two D1-D2 domains of PTPRF onto two D1 domains of PTPRA suggested a wedge model with steric clashes between the D2 domains (Figure 3C) (Barr et al., 2009). The orientation of the D1 and D2 domains is highly conserved (Figure 4A) and a linker between D1 and D2 is too short and too tightly attached on the N-terminus of the D2 domain, making a short beta-strand to detach the D2 domain from the D1 domain (Barr et al., 2009). bdjn.org Ho-Chul Shin and Seung Jun Kim Additionally, the linker region between the D1 and D2 domain is highly conserved (GXTX(I/V)) (Barr et al., 2009). These analyses suggested that the conformational changes between D1 and D2 are unlikely. In addition, the highly conserved orientation of the D1-D2 domains suggests that the D1 and D2 domains interact (Figure 4A). The residues at the interface between the D1 and D2 domains are generally conserved (Figure 4B). Moreover, ∆iGs at the interface between the D1 and D2 domains of the structure of PTPRs are sufficient to interact with each other (for ∆ iG calculated using PISA (Krissinel and Henrick, 2007)), indicating that the interaction between D1 and D2 is an important feature (Figure 4C). Based on these problems, Stefan Knapp and colleagues suggested the inhibition model with a head-to-toe arrangement, rather than a wedge inhibition model (Barr et al., 2009). In the structure of PTPRG, the active site of the D1 domain is blocked by the D2 domain of another PTPRG in a head-to-toe arrangement (Barr et al., 2009). Their schematic model suggested that this orientation, like the PTPRG structure, could be formed under the membrane and could block the active site (Barr et al., 2009). However, from a different perspective, these conserved features indicate the possibility of mechanical conformational changes of the D1-D2 domain by dimerization to inhibit phosphatase activity. Interactions between D1 and D2 are not strong enough to stabilize the D1-D2 orientation. This means that some forces or changes in D1-D2 can induce changes in the D1-D2 orientation, and a steric clash between the D2 domains can be avoided. In fact, there is evidence supporting the wedge inhibition model in vivo (Tertoolen et al., 2001; ToledanoKatchalski et al., 2003). A mutation from Glu to Arg in the wedge region constitutively activates the phosphatase activity of PTPRC (CD45) (Majeti et al., 2000). This residue is in close proximity to the active site in the dimer structure of the PTPRA D1 domain, and a substitution of an arginine residue from a glutamate causes repulsion at the active site via a charge inversion and a structural clash owing to a large guanine group (Figure 3A, B). Phosphatase activation by the ER mutant on the wedge region of PTPRC decreases leukocyte recruitment and drives autoimmune diseases (Germena et al., 2015; Hermiston et al., 2005; Zikherman et al., 2013). Moreover, the activation of PTPRS with this mutation limits neuronal regeneration. Recently, several researches show the wedge peptide of PTPRs can inhibit the phosphatase activity of PTPRs. Proliferation of PC12 cells is increased by treatment of wedge peptide of PTPRF and the wedge peptide of PTPRM blocks outgrowth of retinal neurite (Xie et al., 2006). Jerry Silver and colleagues have shown that the wedge peptide of PTPRS causes a corn growth of neurons (Lang et al., 2015). This peptide also promotes regeneration of sympathetic axon in the cardiac scar by inhibition of PTPRS in vivo. R. T. Gardner and colleagues showed the possibility that wedge peptide of PTPRS could be used clinically to inhibit the activity of PTPRS (Gardner et al., 2015). Although dimerization of the intracellular domain, which may bdjn.org A B C FIGURE 4 I Conservation between the D1 domain and D2 domain. (A) The four PTPRs are superimposed to compare the orientations of the D1 and D2 domains. The conserved residues in the amino acid sequence alignments are presented in the inset. Blue areas indicate the level of conservation. (B) The interfaces between the D1 domain and the D2 domain are presented as a surface model. Blue areas indicate the level of conservation, similar to (A). The area in the red line is the contact region of the D1 domain and the D2 domain. (C) Estimated ∆iG and interface areas are shown for the structures for which the intracellular domain of PTPR are solved. be induced by ligand binding, can decrease the phosphatase activity of PTPRs, the inhibitory mechanism of phosphatase activity by dimerization is still unclear. RECEPTOR-TYPE PTP REGULATION BY OXIDATION Another potential inhibitory mechanism is the reversible oxidation of the intracellular domain. Oxidation is a well-known regulatory Biodesign l Vol.4 l No.2 l Jun 30, 2016 © 2016 Biodesign 63 The regulation of receptor protein tyrosine phosphatases by the dimerization of intracellular domains mechanism for enzymes that have a cysteine as the catalytic residue, and most PTPs have a cysteine as a catalytic residue. It had been reported that exogenous H2O2 stimulates the insulin receptor, which is a PTK, and increases the phosphorylated tyrosine levels of its substrates (Heffetz et al., 1990; Heffetz and Zick, 1989). These reports suggested that the inhibition of PTP activity may be mediated by oxidation. PTP is oxidized by exogenous H 2O 2 directly and reduced by GSH reversibly in vitro (Hecht and Zick, 1992). H2O2 increases endogenously via platelet-derived growth factor (PDGF) (Sundaresan et al., 1995). These results demonstrate the role of H2O2 as a signal transduction molecule. Subsequently, Rhee and colleagues discovered that the phosphatase activity of PTP1B decreases in response to endogenous H 2O 2 induced by EGF and this oxidation is reversible (Lee et al., 1998). Since these discoveries, signal transduction related to reversible oxidation of PTPs has been confirmed by several analyses, including structural evidence (Buhrman et al., 2005; Jeon et al., 2013; Lee et al., 2015; Salmeen et al., 2003; van Montfort et al., 2003). The reversible redox regulation occurs at a catalytic cysteine. This residue can be easily oxidized because the thiol group of cysteine, as a catalytic residue, is highly reactive in the cytosolic environment. The thiol group of cysteine can have several oxidized forms. In general, the thiol group is able to change sequentially to sulfenate, sulfinate, and sulfonate by oxidation. If another cysteine is located near the target cysteine, they can form a disulfide bond by oxidation. In special cases, the catalytic residue can make a sulfenyl-amide bond with a nitrogen atom between the thiol group in cysteine and the main chain nitrogen atom in the following residue (Figure 5). The sulfenic acid, disulfide, and sulfenyl-amide intermediate can be reversibly converted to a thiol group (Figure 5). These reversible oxidations are the key step for controllable redox regulatory signaling. PTPRs have this reversible redox regulation pathway because PTPRs also have cysteine as a catalytic residue. The various oxidized D2 domain structures of PTPRA have been solved and analyzed (Yang et al., 2007). One of the PTPRA structures has a sulfenyl- amide bond at Cys723 and Ser724, indicating that PTPRA can be regulated by reversible redox signaling. The redox regulation of PTPRs involves not only the direct inhibition of the catalytic cysteine by oxidation, but also two unique characteristics. First, the D2 dimerization is increased by oxidation of the intracellular domain. According to previous studies, the extent of homodimer formation of the intracellular domains of PTPRA and PTPRE was increased by H 2 O 2 (Blanchetot et al., 2002; Toledano-Katchalski et al., 2003). Second, the D2 domain is more sensitive to oxidation than the D1 domain (Groen et al., 2008; Persson et al., 2004). These two unique characteristics could be closely related to phosphatase activity regulation by the dimerization of D2 domains. According to limited proteolysis results for PTPRA, PTPRM, PTPRC, and PTPRS, it is possible that conformational changes of the intracellular domain can be induced by oxidation (Groen et al., 64 Biodesign l Vol.4 l No.2 l Jun 30, 2016 © 2016 Biodesign FIGURE 5 I The oxidation mechanism of a thiol group of a cysteine residue. Oxidation starts with a thiol group. ROS is a reactive oxygen species and RSH is a reductant containing a thiol group. 2008), and it is possible that these conformational changes induce the dimerization of the two intracellular domains. However, based on the recently solved structure of the oxidized intracellular domain of PTPRS, only the D1 domain is oxidized and there is no significant conformational change compared with the previously solved PTPRS (RMSD 0.197 Å). Analyses of the oxidized intracellular domain of PTPRS by using mass spectrometry have revealed that only D1 is oxidized (Jeon et al., 2013). Despite evidence for the induction of D2 dimerization by oxidation, the mechanism is still unclear and controversial. A SCHEMATIC REGULATORY MODEL FOR PTPR The inhibition of phosphatase activity by the dimerization of D2 domains is clearly established, but the mechanism is not yet known. The dimerization of D2 domains can be induced by two mechanisms. First, it is possible that the intracellular domains come close to each other via the dimer- or oligomerization of the extracellular domain. This could induce mechanical conformational changes between the two intracellular domains, potentially via a steric clash. Alternatively, the oxidation of the D2 domain could induce conformational changes at the interface between the D1 and D2 domains. These two possible conformational changes could facilitate the dimerization of the D2 domains. After D2 domains form a dimer, the active site of the D1 domain could be blocked by the wedge region or by head- bdjn.org Ho-Chul Shin and Seung Jun Kim to-toe dimer formation. There is no clear and direct evidence to support these mechanisms. Additional data, e.g., structural and biochemical data, are necessary to establish the mechanism of phosphatase inhibition by intracellular domains. ACKNOWLEDGEMENTS This work was supported by the Bio & Medical Technology Development Programs of the National Research Foundation, funded by the Korean Government (2011-0030027) and the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program. AUTHOR INFORMATION The authors declare no potential conflicts of interest. Original Submission: May 20, 2016 Revised Version Received: Jun 13, 2016 Accepted: Jun 14, 2016 REFERENCES Andersen, J.N., M ortensen, O.H., Peters, G.H., Drake, P.G., Iversen, L.F., Olsen, O.H., Jansen, P.G., Andersen, H.S., Tonks, N.K., and Moller, N.P. (2001). Structural and evolutionary relationships among protein tyrosine phosphatase domains. Mol Cell Biol 21, 7117-7136. Barr, A.J., Ugochukwu, E., Lee, W.H., King, O.N., Filippakopoulos, P., Alfano, I., Savitsky, P., Burgess-Brown, N.A., Muller, S., and Knapp, S. (2009). Large-scale structural analysis of the classical human protein tyrosine phosphatome. Cell 136, 352-363. 6693. 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