The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P2 were determined to be Kd~12 μM and 0.4 μM, respectively, and Kd~50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P2 and an increased apparent affinity to PI(3,4,5)P3, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P2 and no synergy in its binding with PS and PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase “scoots" along the membrane surface (penetration <5 ？) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ~60 ？ away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN.
References
[1]
Di Cristofano A, Pandolfi PP (2000) The multiple roles of PTEN in tumor suppression. Cell 100: 387–390.
[2]
Mitra P, Zhang Y, Rameh LE, Ivshina MP, McCollum D, et al. (2004) A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast. J Cell Biol 166: 205–211.
[3]
Gupta R, Ting JTL, Sokolov LN, Johnson SA, Luan S (2002) A tumor suppressor homolog, AtPTEN1, is essential for pollen development in Arabidopsis. Plant Cell 14: 2495–2507.
[4]
Janetopoulos C, Borleis J, Vazquez F, Iijima M, Devreotes PN (2005) Temporal and spatial regulation of phosphoinositide signaling mediates cytokinesis. Dev Cell 8: 467–477.
[5]
Mutti NS, Wang Y, Kaftanoglu O, Amdam GV (2011) Honey bee PTEN – Description, developmental knockdown, and tissue-specific expression of splice-variants correlated with alternative social phenotypes. PLoS One 6: e22195.
[6]
Ogg S, Ruvkun G (1998) The C. elegans PTEN homolog, DAF-18, acts in the insulin receptor-like metabolic signaling pathway. Mol Cell 2: 887–893.
[7]
Groszer M, Erickson R, Scripture-Adams DD, Lesche R, Trumpp A, et al. (2001) Negative regulation of neural stem/progenitor cell proliferation by the Pten tumor suppressor gene in vivo. Science 294: 2186–2189.
[8]
Di Cristofano A, Kotsi P, Peng YF, Cordon-Cardo C, Elkon KB, et al. (1999) Impaired Fas response and autoimmunity in Pten+/？ mice. Science 285: 2122–2125.
[9]
Huang YH, Sauer K (2010) Lipid signaling in T-cell development and function. Cold Spring Harb Perspect Biol 2: a002428.
[10]
Stiles BL (2009) Phosphatase and tensin homologue deleted on chromosome 10: Extending its PTENtacles. Int J Biochem Cell Biol 41: 757–761.
[11]
Simpson L, Parsons R (2001) PTEN: Life as a tumor suppressor. Exp Cell Res 264: 29–41.
[12]
Li L, Ross AH (2007) Why is PTEN an important tumor suppressor? J Cell Biochem 102: 1368–1374.
[13]
Ji S-P, Zhang Y, Van Cleemput J, Jiang W, Liao M, et al. (2006) Disruption of PTEN coupling with 5-HT2C receptors suppresses behavioral responses induced by drugs of abuse. Nat Med 12: 324–329.
[14]
Goffin A, Hoefsloot LH, Bosgoed E, Swillen A, Fryns JP (2001) PTEN mutation in a family with Cowden syndrome and autism. Am J Med Genet 105: 521–524.
[15]
Butler MG, Dasouki MJ, Zhou X-P, Talebizadeh Z, Brown M, et al. (2005) Subset of individuals with autism spectrum disorders and extreme macrocephaly associated with germline PTEN tumour suppressor gene mutations. J Med Genet 42: 318–321.
[16]
Boccone L, Dessì V, Zappu A, Piga S, Piludu MB, et al. (2006) Bannayan-Riley-Ruvalcaba syndrome with reactive nodular lymphoid hyperplasia and autism and a PTEN mutation. Am J Med Genet A 140: 1965–1969.
[17]
Herman GE, Butter E, Enrile B, Pastore M, Prior TW, et al. (2007) Increasing knowledge of PTEN germline mutations: Two additional patients with autism and macrocephaly. Am J Med Genet A 143: 589–593.
[18]
Orrico A, Galli L, Buoni S, Orsi A, Vonella G, et al. (2009) Novel PTEN mutations in neurodevelopmental disorders and macrocephaly. Clin Genet 75: 195–198.
[19]
Stein MT, Elias ER, Saenz M, Pickler L, Reynolds A (2010) Autistic spectrum disorder in a 9-year-old girl with macrocephaly. J Dev Behav Pediatr 31: 632–634.
[20]
McBride KL, Varga EA, Pastore MT, Prior TW, Manickam K, et al. (2010) Confirmation study of PTEN mutations among individuals with autism or developmental delays/mental retardation and macrocephaly. Autism Res 3: 137–141.
[21]
Ross AH, Gericke A (2009) Phosphorylation keeps PTEN phosphatase closed for business. Proc Natl Acad Sci USA 106: 1297–1298.
[22]
Das S, Dixon J, Cho W (2003) Membrane-binding and activation mechanism of PTEN. Proc Natl Acad Sci USA 100: 7491–7496.
[23]
Rahdar M, Inoue T, Meyer T, Zhang J, Vazquez F, et al. (2009) A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN. Proc Natl Acad Sci USA 106: 480–485.
[24]
Lee JO, Yang H, Georgescu MM, Di Cristofano A, Maehama T, et al. (1999) Crystal structure of the PTEN tumor suppressor: Implications for its phosphoinositide phosphatase activity and membrane association. Cell 99: 323–334.
[25]
Wacklin HP (2010) Neutron reflection from supported lipid membranes. Curr Opin Colloid Interf Sci 15: 445–454.
[26]
McGillivray DJ, Valincius G, Vanderah DJ, Febo-Ayala W, Woodward JT, et al. (2007) Molecular-scale structural and functional characterization of sparsely tethered bilayer lipid membranes. Biointerphases 2: 21–33.
[27]
Valincius G, McGillivray DJ, Febo-Ayala W, Vanderah DJ, Kasianowicz JJ, et al. (2006) Enzyme activity to augment the characterization of tethered bilayer membranes. J Phys Chem B 110: 10213–10216.
[28]
Shenoy S, Moldovan R, Fitzpatrick J, Vanderah DJ, Deserno M, et al. (2010) In-plane homogeneity and lipid dynamics in tethered Bilayer Lipid Membranes (tBLMs). Soft Matter 6: 1263–1274.
[29]
Russell TP (1990) X-ray and neutron reflectivity for the investigation of polymers. Mater Sci Rep 5: 171–271.
[30]
Wiener MC, White SH (1991) Fluid bilayer structure determination by the combined use of x-ray and neutron diffraction. II. “Composition-space" refinement method. Biophys J 59: 174–185.
[31]
Vaknin D, Kjaer K, Als-Nielsen J, L？sche M (1991) Structural properties of phosphatidylcholine in a monolayer at the air/water interface. Neutron reflection study and reexamination of x-ray reflection experiments. Biophys J 59: 1325–1332.
[32]
Schalke M, L？sche M (2000) Structural models of lipid surface monolayers from x-ray and neutron reflectivity measurements. Adv Colloid Interf Sci 88: 243–274.
[33]
McGillivray DJ, Valincius G, Heinrich F, Robertson JWF, Vanderah DJ, et al. (2009) Structure of functional Staphylococcus aureus α-hemolysin channels in tethered bilayer lipid membranes. Biophys J 96: 1547–1553.
[34]
Nanda H, Datta SAK, Heinrich F, L？sche M, Rein A, et al. (2010) Electrostatic interactions and binding orientation of HIV-1 matrix, studied by neutron reflectivity. Biophys J 99: 2516–2524.
[35]
Redfern RE, Redfern DA, Furgason ML, Munson M, Ross AH, et al. (2008) PTEN phosphatase selectively binds phosphoinositides and undergoes structural changes. Biochemistry 47: 2162–2171.
[36]
Certain commercial materials, equipment, and instruments are identified in this manuscript in order to specify the experimental procedure as completely as possible In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials, equipment, or instruments identified are necessarily the best available for the purpose.
[37]
Maehama T, Dixon JE (1998) The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 273: 13375–13378.
[38]
Redfern RE, Daou M, Li L, Munson M, Gericke A, et al. (2010) A mutant form of PTEN linked to autism. Protein Sci 19: 1948–1956.
[39]
Heinrich F, Ng T, Vanderah DJ, Shekhar P, Mihailescu M, et al. (2009) A new lipid anchor for sparsely tethered bilayer lipid membranes. Langmuir 25: 4219–4229.
[40]
Cornell BA, Braach-Maksvytis VLB, King LB, Osman PDJ, Raguse B, et al. (1997) A biosensor that uses ion-channel switches. Nature 387: 580–583.
[41]
Schasfoort RBM, Tudos AJ, editors. (2008) Handbook of Surface Plasmon Resonance. Cambridge: Royal Society of Chemistry.
[42]
Dura JA, Pierce D, Majkrzak CF, Maliszewskyj N, McGillivray DJ, et al. (2006) AND/R: A neutron diffractometer/reflectometer for investigation of thin films and multilayers for the life sciences. Rev Sci Instrum 77: 074301.
[43]
Kirby BJ, Kienzle PA, Maranville BB, Berk NF, Krycka J, et al. (2012) Phase-sensitive specular neutron reflectometry for imaging the nanometer scale composition depth profile of thin-film materials. Curr Opin Colloid Interf Sci 17: 44–53.
[44]
Shekhar P, Nanda H, L？sche M, Heinrich F (2011) Continuous distribution model for the investigation of complex molecular architectures near interfaces with scattering techniques. J Appl Phys. in press.
[45]
Catmull E, Rom R (1974) A class of local interpolating splines. In: Barnhill RE, Reisenfeld RF, editors. Computer Aided Geometric Design. New York: Academic Press. pp. 317–326.
[46]
Connolly M (1983) Solvent-accessible surfaces of proteins and nucleic acids. Science 221: 709–713.
[47]
Lemmon MA (2008) Membrane recognition by phospholipid-binding domains. Nat Rev Mol Cell Bio 9: 99–111.
[48]
Because of the large amounts of protein required to determine Kd values above 10 μM with certainty, only one measurement was performed on this system using high protein concentrations All other measurements reported here were performed multiple times.
[49]
Golebiewska U, Gambhir A, Hangyas-Mihalyne G, Zaitseva I, R？dler J, et al. (2006) Membrane-bound basic peptides sequester multivalent (PIP2), but not monovalent (PS), acidic lipids. Biophys J 91: 588–599.
[50]
For this and other cases of PTEN binding to stBLMs with two anionic components, the binding curves could be well fitted with a one-component model with a two-component model not improving the fits significantly Conceivably, the protein binding affinities to the two anionic lipid components were too close to distinguish.
[51]
Narayan K, Lemmon MA (2006) Determining selectivity of phosphoinositide-binding domains. Methods 39: 122–133.
Campbell RB, Liu F, Ross AH (2003) Allosteric activation of PTEN phosphatase by phosphatidylinositol 4,5-bisphosphate. J Biol Chem 278: 33617–33620.
[54]
Wang J, Gambhir A, Hangyás-Mihályné G, Murray D, Golebiewska U, et al. (2002) Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions. J Biol Chem 277: 34401–34412.
[55]
Lingwood D, Ries J, Schwille P, Simons K (2008) Plasma membranes are poised for activation of raft phase coalescence at physiological temperature. Proc Natl Acad Sci USA 105: 10005–10010.
[56]
The ratios of integrated nSLD contributions of lipid headgroups and of protein–and hence also the ratios of the nSLD peaks near d = 50 ？ and 75 ？–vary from experiment to experiment due to different amounts of adsorbed protein (see Table 3) Note also that the amount of adsorbed protein varied in successive NR scans with different isotopic buffers because the buffer exchange removed some protein These variations were accounted for in the model, and thus the nSLD profiles for CM4 or D2O-based buffers show smaller amplitudes in the protein region although the overall shapes are identical to those shown for the H2O-based buffers in Fig 4.
[57]
Curtis JE, Raghunandran S, Nanda H, Krueger S (2012) SASSIE: A program to study intrinsically disordered biological molecules and macromolecular ensembles using experimental scattering constraints. Comp Phys Commun 183: 382–389.
