- Email: [email protected]
The Molecular Basis for the Substrate Specificity of Protein Tyrosine Phosphatase PTPN3
Structure
Previews ACKNOWLEDGMENTS
Deupi, X., Edwards, P., Singhal, A., Nickle, B., Oprian, D., Schertler, G., and Standfuss, J. (2012). Proc. Natl. Acad. Sci. USA 109, 119–124.
Krebs, A., Edwards, P.C., Villa, C., Li, J., and Schertler, G.F.X. (2003). J. Biol. Chem. 278, 50217–50225.
REFERENCES
Ernst, O.P., Gramse, V., Kolbe, M., Hofmann, K.P., and Heck, M. (2007). Proc. Natl. Acad. Sci. USA 104, 10859–10864.
Ostermaier, M.K., Peterhans, C., Jaussi, R., Deupi, X., and Standfuss, J. (2014). Proc. Natl. Acad. Sci. USA 111, 1825–1830.
Bayburt, T.H., Leitz, A.J., Xie, G., Oprian, D.D., and Sligar, S.G. (2007). J. Biol. Chem. 282, 14875– 14881.
Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D.A., Engel, A., and Palczewski, K. (2003). Nature 421, 127–128.
Schertler, G.F., and Hargrave, P.A. (1995). Proc. Natl. Acad. Sci. USA 92, 11578–11582.
I would like to thank Gregor Cicchetti for his help with the manuscript.
Buzhynskyy, N., Salesse, C., and Scheuring, S. (2011). J. Mol. Recognit. 24, 483–489. Chabre, M. (1975). Biochim. Biophys. Acta 382, 322–335. Corless, J.M., McCaslin, D.R., and Scott, B.L. (1982). Proc. Natl. Acad. Sci. USA 79, 1116–1120.
Gilliam, J.C., Chang, J.T., Sandoval, I.M., Zhang, Y., Li, T., Pittler, S.J., Chiu, W., and Wensel, T.G. (2012). Cell 151, 1029–1041. Gunkel, M., Scho¨neberg, J., Alkhaldi, W., Irsen, S., Noe´, F., Kaupp, U.B., and Al-Amoudi, A. (2015). Structure 23, this issue, 628–638.
Schertler, G.F., Villa, C., and Henderson, R. (1993). Nature 362, 770–772. Sommer, M.E., Hofmann, K.P., and Heck, M. (2012). Nat. Commun. 3, 995. Terrillon, S., and Bouvier, M. (2004). EMBO Rep. 5, 30–34.
The Molecular Basis for the Substrate Specificity of Protein Tyrosine Phosphatase PTPN3 Emily J. Parker1,* 1Maurice Wilkins Centre, Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, PO Box 4800, Christchurch 8140, New Zealand *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2015.03.005
In this issue of Structure, Chen et al. present structures of the FERM-containing protein tyrosine phosphatase PTPN3 in complex with a phosphopeptide fragment of susbtrate epidermal growth factor receptor pathway substrate, providing detailed information on substrate specificity. Protein tyrosine phosphatases (PTPs) play critical roles in cell signaling pathways. Together with the kinases, they control the balance of phosphorylated species, enabling specific and varied signaling responses. A large number of enzymes and substrates are involved in these pathways, and knowledge of the specific cellular substrates of the specific PTPs is important for a more complete understanding of the complex interactions that provide the signaling cascades. Dysfunction of PTPs has been associated with a number of human diseases including cancers, autoimmune disorders, diabetes, and neurological diseases. The work presented by Chen et al. (2015) describes studies that help provide a more coherent understanding of the molecular basis of substrate specificity in the FERM-containing subfamily of
nonreceptor PTPs (Chen et al., 2015). This subfamily contains PTPs N3, N4, N13, N14, and N21. These enzymes are characterized by the presence of an N-terminal FERM (4.1 protein [F], ezrin, radixin, and moesin) plasma membranelocalization domain and a C-terminal catalytic domain. Specifically, the work here examines the molecular interactions that govern the interaction of the PTP N3 (PTPN3) with substrate epidermal growth factor receptor (EGFR) pathway substrate (Eps15). Eps15 is a scaffolding adaptor that regulates endocytosis and trafficking of the EGFR that has recently been identified as a substrate for PTPN3 (Li et al., 2014). By taking a small phosphopeptide fragment of the substrate Eps15 and examining its interaction with a series of variants of the catalytic domain of PTPN3, the authors have revealed key
608 Structure 23, April 7, 2015 ª2015 Elsevier Ltd All rights reserved
interactions that likely determine both substrate recognition and the catalytic activity of the complex. Several structures are presented, which, combined with the kinetic assessment of the dephosphorylation reaction of the phosphopeptide substrate, provide interesting new insight into the molecular recognition processes involved. The structure of a catalytically inactive variant of the PTPN3 in combination with the phosphopeptide fragment of Eps15 (Figure 1) reveals an important interaction between H812 of the enzyme and a proline residue adjacent to the phosphotyrosine of the peptide substrate, which results in an atypical conformation of the C-terminal part of the peptide substrate. The importance of proline in delivering this conformation was evaluated by assessment of a variant synthetic phosphopeptide fragment in which the
Structure
Previews
Figure 1. Structure of PTPN3 Structure (PDB code 4RH5) of the catalytic domain of PTPN3 (D811A/C842S mutant, orange) in complex with phosphopeptide fragment of Eps15 (residues 846–854, cyan).
proline was replaced by valine; for this substrate-enzyme pairing, the atypical conformation was lost. Likewise the role of H812 was further investigated by the preparation of variant protein with the H812F substitution. This substitution enacts a natural occurrence of a phenylalanine in this position in the PTP 1B (PTP1B). Both this variant of PTPN3 and the PTP1B demonstrated higher Michaelis constants for the reaction with the synthetic phosphopeptide. A further set of kinetic studies and structures allow an examination of the molecular basis of the selectivity of the PTPN3 for the Eps15 substrate in contrast to the other subfamily phosphatases, all of which contain an equivalent to H812. Sequence comparisons led to mutagenesis studies that determined the importance of tyrosine (Y676) in the pTyr loop and methionine (M883 in PTPN3) in the WPD loop for substrate recognition. In addition, the importance of an aspartate in the WPD loop rather than a glutamate (as is found in PTP21) for catalytic activity was demonstrated and the molecular
basis for the low activity of this PTP and the PTPN3 D811E variant was revealed by a structure of this mutant in complex with the Eps15-phosphopeptide. These studies are elegantly complemented by in vivo experiments to test whether residues H812 and D811 of PTPN3 were required to regulate Eps15dependent EGFR signaling in cultured cells. Whereas wild-type PTPN3 significantly decreased EGF-induced phosphorylation of Eps15 in human embryonic kidney HEK293T cells following transfection, ectopic expression of D811E or H812F mutant form of PTPN3 did not reduce phosphorylation of Eps15. There is significant interest in gaining information on the structure of the PTPs to support an understanding of their biological roles (Hardy et al., 2015; Lountos et al., 2015; Ozek et al., 2014; Zhang et al., 2015). Recently, PTPN14 was reported to suppress metastasis by reducing intracellular protein trafficking through the secretory pathway, and RIN1 (Ras and Rab interactor 1) and PRKCD (protein kinase C-delta) were identified as binding partners and substrates of this phosphatase (Belle et al., 2015). Argonaute 2 was recently identified as a direct substrate of PTP1B, resulting in the regulation of gene silencing in oncogenic RAS-induced senescence (Yang et al., 2014). Additionally, structures of PTPN18 in complex with HER2 phosphopeptides have shown the molecular details of the interaction between PTPN18 and specific HER2 phosphorylation sites (Wang et al., 2014). PTPN3 has attracted considerable recent attention for its role in cancer, particularly the role it plays in partnership with the mitogen-activated kinase p38 gamma. Very recently, the same group determined the structure of this protein in combination with its kinase partner. These structures reveal extensive interac-
tions of p38 gamma across multiple domains of the phosphatase (Chen et al., 2014). PTPs play critical roles in complex signal transduction cascades, and understanding the molecular details that govern their substrate specificities may assist a complete description of these intricate pathways and provide opportunities for the development of therapeutics for the diseases with which these enzymes are associated. REFERENCES Belle, L., Ali, N., Lonic, A., Li, X., Paltridge, J.L., Roslan, S., Herrmann, D., W Conway, J.R., Gehling, F.K., Bert, A.G., et al. (2015). Sci. Signal. 8, ra18. Chen, K.E., Lin, S.Y., Wu, M.J., Ho, M.R., Santhanam, A., Chou, C.C., Meng, T.C., and Wang, A.H.J. (2014). Sci. Signal. 7, ra98. Chen, K.-E., Li, M.-Y., Chou, C.-C., Ho, M.-R., Chen, G.-C., Meng, T.-C., and Wang, A.H.J. (2015). Structure 23, this issue, 653–664. Hardy, S., Uetani, N., Wong, N., Kostantin, E., Labbe´, D.P., Be´gin, L.R., Mes-Masson, A., Miranda-Saavedra, D., and Tremblay, M.L. (2015). Oncogene 34, 986–995. Li, M.Y., Lai, P.L., Chou, Y.T., Chi, A.P., Mi, Y.Z., Khoo, K.H., Chang, G.D., Wu, C.W., Meng, T.C., and Chen, G.C. (2014). Oncogene. Published September 29, 2014. http://dx.doi.org/10.1038/ onc.2014.1312. Lountos, G.T., Cherry, S., Tropea, J.E., and Waugh, D.S. (2015). Acta Crystallogr. F Struct. Biol. Commun. 71, 199–205. Ozek, C., Kanoski, S.E., Zhang, Z.Y., Grill, H.J., and Bence, K.K. (2014). J. Biol. Chem. 289, 31682–31692. Wang, H.M., Xu, Y.F., Ning, S.L., Yang, D.X., Li, Y., Du, Y.J., Yang, F., Zhang, Y., Liang, N., Yao, W., et al. (2014). Cell Res. 24, 1067–1090. Yang, M., Haase, A.D., Huang, F.K., Coulis, G., Rivera, K.D., Dickinson, B.C., Chang, C.J., Pappin, D.J., Neubert, T.A., Hannon, G.J., et al. (2014). Mol. Cell 55, 782–790. Zhang, X., Belkina, N., Jacob, H.K.C., Maity, T., Biswas, R., Venugopalan, A., Shaw, P.G., Kim, M.S., Chaerkady, R., Pandey, A., and Guha, U. (2015). Proteomics 15, 340–355.
Structure 23, April 7, 2015 ª2015 Elsevier Ltd All rights reserved 609
Previews ACKNOWLEDGMENTS
Deupi, X., Edwards, P., Singhal, A., Nickle, B., Oprian, D., Schertler, G., and Standfuss, J. (2012). Proc. Natl. Acad. Sci. USA 109, 119–124.
Krebs, A., Edwards, P.C., Villa, C., Li, J., and Schertler, G.F.X. (2003). J. Biol. Chem. 278, 50217–50225.
REFERENCES
Ernst, O.P., Gramse, V., Kolbe, M., Hofmann, K.P., and Heck, M. (2007). Proc. Natl. Acad. Sci. USA 104, 10859–10864.
Ostermaier, M.K., Peterhans, C., Jaussi, R., Deupi, X., and Standfuss, J. (2014). Proc. Natl. Acad. Sci. USA 111, 1825–1830.
Bayburt, T.H., Leitz, A.J., Xie, G., Oprian, D.D., and Sligar, S.G. (2007). J. Biol. Chem. 282, 14875– 14881.
Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D.A., Engel, A., and Palczewski, K. (2003). Nature 421, 127–128.
Schertler, G.F., and Hargrave, P.A. (1995). Proc. Natl. Acad. Sci. USA 92, 11578–11582.
I would like to thank Gregor Cicchetti for his help with the manuscript.
Buzhynskyy, N., Salesse, C., and Scheuring, S. (2011). J. Mol. Recognit. 24, 483–489. Chabre, M. (1975). Biochim. Biophys. Acta 382, 322–335. Corless, J.M., McCaslin, D.R., and Scott, B.L. (1982). Proc. Natl. Acad. Sci. USA 79, 1116–1120.
Gilliam, J.C., Chang, J.T., Sandoval, I.M., Zhang, Y., Li, T., Pittler, S.J., Chiu, W., and Wensel, T.G. (2012). Cell 151, 1029–1041. Gunkel, M., Scho¨neberg, J., Alkhaldi, W., Irsen, S., Noe´, F., Kaupp, U.B., and Al-Amoudi, A. (2015). Structure 23, this issue, 628–638.
Schertler, G.F., Villa, C., and Henderson, R. (1993). Nature 362, 770–772. Sommer, M.E., Hofmann, K.P., and Heck, M. (2012). Nat. Commun. 3, 995. Terrillon, S., and Bouvier, M. (2004). EMBO Rep. 5, 30–34.
The Molecular Basis for the Substrate Specificity of Protein Tyrosine Phosphatase PTPN3 Emily J. Parker1,* 1Maurice Wilkins Centre, Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, PO Box 4800, Christchurch 8140, New Zealand *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2015.03.005
In this issue of Structure, Chen et al. present structures of the FERM-containing protein tyrosine phosphatase PTPN3 in complex with a phosphopeptide fragment of susbtrate epidermal growth factor receptor pathway substrate, providing detailed information on substrate specificity. Protein tyrosine phosphatases (PTPs) play critical roles in cell signaling pathways. Together with the kinases, they control the balance of phosphorylated species, enabling specific and varied signaling responses. A large number of enzymes and substrates are involved in these pathways, and knowledge of the specific cellular substrates of the specific PTPs is important for a more complete understanding of the complex interactions that provide the signaling cascades. Dysfunction of PTPs has been associated with a number of human diseases including cancers, autoimmune disorders, diabetes, and neurological diseases. The work presented by Chen et al. (2015) describes studies that help provide a more coherent understanding of the molecular basis of substrate specificity in the FERM-containing subfamily of
nonreceptor PTPs (Chen et al., 2015). This subfamily contains PTPs N3, N4, N13, N14, and N21. These enzymes are characterized by the presence of an N-terminal FERM (4.1 protein [F], ezrin, radixin, and moesin) plasma membranelocalization domain and a C-terminal catalytic domain. Specifically, the work here examines the molecular interactions that govern the interaction of the PTP N3 (PTPN3) with substrate epidermal growth factor receptor (EGFR) pathway substrate (Eps15). Eps15 is a scaffolding adaptor that regulates endocytosis and trafficking of the EGFR that has recently been identified as a substrate for PTPN3 (Li et al., 2014). By taking a small phosphopeptide fragment of the substrate Eps15 and examining its interaction with a series of variants of the catalytic domain of PTPN3, the authors have revealed key
608 Structure 23, April 7, 2015 ª2015 Elsevier Ltd All rights reserved
interactions that likely determine both substrate recognition and the catalytic activity of the complex. Several structures are presented, which, combined with the kinetic assessment of the dephosphorylation reaction of the phosphopeptide substrate, provide interesting new insight into the molecular recognition processes involved. The structure of a catalytically inactive variant of the PTPN3 in combination with the phosphopeptide fragment of Eps15 (Figure 1) reveals an important interaction between H812 of the enzyme and a proline residue adjacent to the phosphotyrosine of the peptide substrate, which results in an atypical conformation of the C-terminal part of the peptide substrate. The importance of proline in delivering this conformation was evaluated by assessment of a variant synthetic phosphopeptide fragment in which the
Structure
Previews
Figure 1. Structure of PTPN3 Structure (PDB code 4RH5) of the catalytic domain of PTPN3 (D811A/C842S mutant, orange) in complex with phosphopeptide fragment of Eps15 (residues 846–854, cyan).
proline was replaced by valine; for this substrate-enzyme pairing, the atypical conformation was lost. Likewise the role of H812 was further investigated by the preparation of variant protein with the H812F substitution. This substitution enacts a natural occurrence of a phenylalanine in this position in the PTP 1B (PTP1B). Both this variant of PTPN3 and the PTP1B demonstrated higher Michaelis constants for the reaction with the synthetic phosphopeptide. A further set of kinetic studies and structures allow an examination of the molecular basis of the selectivity of the PTPN3 for the Eps15 substrate in contrast to the other subfamily phosphatases, all of which contain an equivalent to H812. Sequence comparisons led to mutagenesis studies that determined the importance of tyrosine (Y676) in the pTyr loop and methionine (M883 in PTPN3) in the WPD loop for substrate recognition. In addition, the importance of an aspartate in the WPD loop rather than a glutamate (as is found in PTP21) for catalytic activity was demonstrated and the molecular
basis for the low activity of this PTP and the PTPN3 D811E variant was revealed by a structure of this mutant in complex with the Eps15-phosphopeptide. These studies are elegantly complemented by in vivo experiments to test whether residues H812 and D811 of PTPN3 were required to regulate Eps15dependent EGFR signaling in cultured cells. Whereas wild-type PTPN3 significantly decreased EGF-induced phosphorylation of Eps15 in human embryonic kidney HEK293T cells following transfection, ectopic expression of D811E or H812F mutant form of PTPN3 did not reduce phosphorylation of Eps15. There is significant interest in gaining information on the structure of the PTPs to support an understanding of their biological roles (Hardy et al., 2015; Lountos et al., 2015; Ozek et al., 2014; Zhang et al., 2015). Recently, PTPN14 was reported to suppress metastasis by reducing intracellular protein trafficking through the secretory pathway, and RIN1 (Ras and Rab interactor 1) and PRKCD (protein kinase C-delta) were identified as binding partners and substrates of this phosphatase (Belle et al., 2015). Argonaute 2 was recently identified as a direct substrate of PTP1B, resulting in the regulation of gene silencing in oncogenic RAS-induced senescence (Yang et al., 2014). Additionally, structures of PTPN18 in complex with HER2 phosphopeptides have shown the molecular details of the interaction between PTPN18 and specific HER2 phosphorylation sites (Wang et al., 2014). PTPN3 has attracted considerable recent attention for its role in cancer, particularly the role it plays in partnership with the mitogen-activated kinase p38 gamma. Very recently, the same group determined the structure of this protein in combination with its kinase partner. These structures reveal extensive interac-
tions of p38 gamma across multiple domains of the phosphatase (Chen et al., 2014). PTPs play critical roles in complex signal transduction cascades, and understanding the molecular details that govern their substrate specificities may assist a complete description of these intricate pathways and provide opportunities for the development of therapeutics for the diseases with which these enzymes are associated. REFERENCES Belle, L., Ali, N., Lonic, A., Li, X., Paltridge, J.L., Roslan, S., Herrmann, D., W Conway, J.R., Gehling, F.K., Bert, A.G., et al. (2015). Sci. Signal. 8, ra18. Chen, K.E., Lin, S.Y., Wu, M.J., Ho, M.R., Santhanam, A., Chou, C.C., Meng, T.C., and Wang, A.H.J. (2014). Sci. Signal. 7, ra98. Chen, K.-E., Li, M.-Y., Chou, C.-C., Ho, M.-R., Chen, G.-C., Meng, T.-C., and Wang, A.H.J. (2015). Structure 23, this issue, 653–664. Hardy, S., Uetani, N., Wong, N., Kostantin, E., Labbe´, D.P., Be´gin, L.R., Mes-Masson, A., Miranda-Saavedra, D., and Tremblay, M.L. (2015). Oncogene 34, 986–995. Li, M.Y., Lai, P.L., Chou, Y.T., Chi, A.P., Mi, Y.Z., Khoo, K.H., Chang, G.D., Wu, C.W., Meng, T.C., and Chen, G.C. (2014). Oncogene. Published September 29, 2014. http://dx.doi.org/10.1038/ onc.2014.1312. Lountos, G.T., Cherry, S., Tropea, J.E., and Waugh, D.S. (2015). Acta Crystallogr. F Struct. Biol. Commun. 71, 199–205. Ozek, C., Kanoski, S.E., Zhang, Z.Y., Grill, H.J., and Bence, K.K. (2014). J. Biol. Chem. 289, 31682–31692. Wang, H.M., Xu, Y.F., Ning, S.L., Yang, D.X., Li, Y., Du, Y.J., Yang, F., Zhang, Y., Liang, N., Yao, W., et al. (2014). Cell Res. 24, 1067–1090. Yang, M., Haase, A.D., Huang, F.K., Coulis, G., Rivera, K.D., Dickinson, B.C., Chang, C.J., Pappin, D.J., Neubert, T.A., Hannon, G.J., et al. (2014). Mol. Cell 55, 782–790. Zhang, X., Belkina, N., Jacob, H.K.C., Maity, T., Biswas, R., Venugopalan, A., Shaw, P.G., Kim, M.S., Chaerkady, R., Pandey, A., and Guha, U. (2015). Proteomics 15, 340–355.
Structure 23, April 7, 2015 ª2015 Elsevier Ltd All rights reserved 609